RU2821911C2 - Methods for detecting nucleic acid variants - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to molecular biology. Described is a method of detecting the presence of a variant sequence in a nucleic acid target sequence consisting of two strands and comprising a nucleotide(s) of interest. Kit and a reporter oligonucleotide for realizing the method are also described. Methods for predicting clinical effectiveness and predicting the presence of the clinical condition are also described.

EFFECT: providing analysis for evaluation/identification of microsatellites of ≥15 nucleotides with very high sensitivity.

24 cl, 18 dwg, 13 ex

Description

Field to which the invention relates

The present invention relates to methods for detecting the presence of a sequence variant in a target nucleic acid sequence comprising nucleotides of interest, in particular for detecting germline and somatic mutations, as well as microsatellite instability. Also described are methods for predicting the effectiveness of a drug and detecting the presence of a clinical disorder in a subject, as well as reporter oligonucleotides and kits for implementing the methods.

BACKGROUND OF THE INVENTION

Inherited (germline) or non-hereditary (somatic) mutational events, such as single nucleotide substitutions, deletions, insertions, transfers, and duplications, have instrumental effects on human biology. Genetic profiling provides information related to responses to external factors such as exercise and diet, and is also used to inform disease diagnosis, prognosis and treatment regimens. Genetic analysis is usually carried out using tools such as DNA sequencing or allele-specific PCR. Methods involving variable binding of molecular probes can be used in the context of germline variations, but when combined with technologies such as HRM can also be used in the context of somatic mutations with low allele frequencies.

Repetitive nucleotide sequences, such as direct or reverse repeats, are observed in many organisms. As an example, hundreds of thousands of microsatellite loci are distributed throughout the human genome and thus occur statistically approximately once every 100,000 base pairs. A microsatellite locus is a region of genomic DNA that contains short tandem repeats, in which the shortest repeat units are typically one to five nucleotides in length. Accordingly, the repeat unit of a particular microsatellite locus is usually designated as a mono-, di-, tri-, tetra-, or pentanuceotide repeat locus, respectively. A single microsatellite locus typically comprises approximately 10 to 40 of these repeat units arranged in tandem. Further, each microsatellite locus of normal genomic DNA of most diploid species, such as mammalian genomic DNA, includes two alleles at each locus. The two alleles may be identical or different from each other in terms of length and may vary between individuals.

Microsatellite instability (MSI) or replication error (RER) is an example of genomic instability that occurs in certain human neoplasms in which the tumor cells have decreased the ability to mediate accurate DNA replication. MSI is a common marker of underlying functional inactivation of the human DNA mismatch repair (MMR) gene. Functional loss of the MMR gene is thought to occur due to biallelic deactivation through coding region mutations, loss of heterozygosity (LOH), and/or promoter methylation. Further, germline mutations of the MMR genes are known to represent an autosomal dominant genetic defect in most relatives of patients with hereditary nonpolyposis colon cancer (HNPCC). Other mutations caused by tumor cells in subjects with HNPCC lead to biallelic deactivation of a specific MMR gene, causing loss of accurate microsatellite DNA replication in tumors. Thus, MSI represents a marker of an underlying defect in DNA mismatch repair and is also associated with increased rates of mutations in coding DNA. This mutator phenotype, which results from a defect in MMR, causes both coding region base substitutions and frameshift mutations in direct repeats at equal frequencies, in addition to causing MSI. The production of MMR defects and the resulting mutator phenotype are thought to represent an early event in tumorigenesis.

MSI is not only associated with a germline defect in families with HNPCC, but is also present in approximately 15 to 20% of sporadic colon cancers, with this finding also reflecting an overall increase in genomic instability (also measured as tumor-associated mutation burden) . Detection of MSI-related defects in tumors was also associated with better prognosis for tumors of the same stage. Thus, it is clinically important to identify tumors with MSI not only for germline MMR defects (in families with HNPCC) but also for prognostic stratification. While clinical (Bethesda guidelines (Rodriguez-Bigas et al., 1997)) and histopathological features may raise suspicion that a colon tumor is associated with microsatellite instability and likely arose in a family with HNPCC, clinicopathological features are often insufficient to diagnose the presence of MSI. Accordingly, molecular testing can be used to determine the MSI-related status of a clinically suspicious tumor (Boland et al., 1998).

In addition to colon tumors, MSI has also been associated with other types of cancer and other genetic diseases. By way of illustration, these include, but are not limited to, pancreatic carcinomas, gastric carcinomas, bladder cancers, prostate carcinomas, lung cancers, uterine carcinomas, and breast cancers. Other examples of genetic diseases that are thought to be associated with microsatellite instability include, for example, Huntington's disease (HD), dentatorubropallidoluis atrophy (DRPLA), spinobulbar and muscular atrophy (SBMA), myotonic dystrophy (DM), fragile X syndrome -chromosomes, FRAXE mental retardation and spinocerebellar ataxia (SCA), X-linked agammaglobulinemia (Bruton's disease), Bloom's syndrome (BS), craniofrontonasal syndrome (CFNS), and idiopathic pulmonary fibrosis (IPF).

In view of the above, it is apparent that the analysis of nucleotide sequence variants, such as repetitive nucleotide sequences such as microsatellites, has many diagnostic and prognostic uses, among other applications. Thus, there is a need for sensitive, objective and reliable assays.

Brief description of the invention

The invention corresponds to that described in the claims. The methods described herein provide a rapid, easy, objective and sensitive method for the analysis of germline and somatic mutations, as well as microsatellite instability.

Provided herein is a method for detecting the presence of a sequence variant in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), preferably including repeats, wherein said target nucleic acid sequence consists of a sequence variant or a control sequence, this method includes the following steps:

a) Obtaining a first sample comprising nucleic acids believed to include the specified sequence variant;

b) Obtaining a second sample comprising nucleic acids comprising said control sequence, wherein the second sample is a control sample;

c) Preparation of a reporter oligonucleotide;

d) Providing a primer set consisting of a first primer and a second primer, wherein the primers of the set together are capable of amplifying a target nucleic acid sequence including an NOI;

e) Amplifying a target nucleic acid sequence in the presence of said first sample, said first primer and said second primer, thereby producing a first amplicon comprising nucleic acids believed to comprise the sequence variant; and amplifying a target nucleic acid sequence in the presence of said second sample, said first primer and said second primer, thereby providing a second amplicon including a control sequence, wherein the second amplicon is a control amplicon;

f) Performing a melting analysis, such as high sensitivity melting curve analysis (HRM), on the first amplicon, thereby obtaining a first profile characterized by the first melting curve, and performing a melting analysis, such as HRM analysis, on the second amplicon, thereby obtaining a second profile characterized by a second melting curve, wherein the second profile is a control profile characterized by a control melting curve; wherein each amplicon includes a first strand and a second strand, wherein the melting assay includes hybridizing a reporter oligonucleotide to one strand of each amplicon, detecting a signal emitted by a fluorophore, and obtaining a first and second melting curve;

wherein the reporter oligonucleotide is a sequence of 10 to 50, preferably 15 to 50 nucleotides in length, into which 2 to 10 hydrophobic nucleotides have been inserted, wherein the reporter oligonucleotide includes a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' end, and wherein the reporter oligonucleotide includes a hybridization sequence H,

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence,

and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeat sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize with the first and second amplicons when the hybridization sequence hybridizes with them; And

g) Comparing the first profile with a control profile, with the difference between the first profile and the control profile indicating that the first sample contains a sequence variant.

Provided herein is a method for detecting the presence of a sequence variant in a two-strand nucleic acid target sequence comprising a nucleotide(s) of interest (NOI), wherein said target nucleic acid sequence consists of a sequence variant or a control sequence, wherein said method the following includes the following steps:

a) Obtaining a first sample comprising nucleic acids believed to include the specified sequence variant;

b) Obtaining a second sample comprising nucleic acids comprising said control sequence, wherein the second sample is a control sample;

c) Preparation of a reporter oligonucleotide;

d) Providing a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid sequence;

e) Amplifying a target nucleic acid sequence in the presence of said first sample, said first primer and said second primer, thereby producing a first amplicon comprising nucleic acids believed to comprise the sequence variant; and amplifying a target nucleic acid sequence in the presence of said second sample, said first primer and said second primer, thereby providing a second amplicon including a control sequence, wherein the second amplicon is a control amplicon;

f) Performing a high-sensitivity melting curve analysis (HRM) of the first amplicon, thereby obtaining a first HRM profile characterized by a first melting curve, and performing an HRM analysis of a second amplicon, thereby obtaining a second HRM profile characterized by a second melting curve, wherein the second HRM the profile is a control profile characterized by a control melting curve; wherein each amplicon includes a first strand and a second strand, wherein the HRM analysis includes hybridizing a reporter oligonucleotide to one strand of each amplicon, detecting a signal emitted by a fluorophore, and obtaining a first and second melting curve; wherein the reporter oligonucleotide is a sequence from 10 to 50 in length, into which from 2 to 10 hydrophobic nucleotides were inserted,

wherein the reporter oligonucleotide includes a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' end end, and

wherein the reporter oligonucleotide includes a hybridization sequence H, wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; and/or

in this case, the hydrophobic nucleotide has the structure X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue; Q is an intercalator that does not participate in a Watson-Crick hydrogen bond; and Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence; and e) Comparing the first HRM profile with a control HRM profile, wherein the difference between the first HRM profile and the control HRM profile indicates that the first sample contains a sequence variant.

Also provided herein is a kit for detecting the presence of a sequence variant in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), preferably including repeats, wherein said target nucleic acid sequence consists of a variant sequence or a control sequences, wherein the specified set includes:

a) a reporter oligonucleotide comprising a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from 3' - end, wherein the reporter oligonucleotide is a sequence of 10 to 50 nucleotides in length, preferably 15 to 50 nucleotides, into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence H, and

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence; and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeat sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize with the first and second amplicons when the hybridization sequence hybridizes with them; and b) a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid sequence.

Also provided herein is a kit for detecting the presence of a sequence variant in a two-strand nucleic acid target sequence comprising a nucleotide(s) of interest (NOI), wherein said target nucleic acid sequence consists of a sequence variant or a control sequence, wherein This set includes:

a) a reporter oligonucleotide comprising a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' -end, wherein the reporter oligonucleotide is a sequence 10 to 50 in length into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence H,

wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And

in this case, the hydrophobic nucleotide has the structure X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence including the control sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence; and b) a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid.

Also provided herein is a reporter oligonucleotide that can hybridize to a single strand of a target nucleic acid consisting of two strands and comprising the nucleotide(s) of interest (NOI), preferably comprising repeats, wherein said reporter oligonucleotide includes a first fluorophore, preferably at its 5 '-end or at a distance of no more than 4 nucleotides from the 5'-end, and the first quencher, preferably at its 3'-end or at a distance of no more than 4 nucleotides from the 3'-end, wherein the reporter oligonucleotide is a sequence of length from 10 to 50, preferably from 15 to 50 nucleotides, into which from 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence M,

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid, and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeating sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize to the second strand of the first and second amplicons when the hybridization sequence hybridizes thereto; And

Also provided herein is a reporter oligonucleotide comprising a first fluorophore, preferably at its 5' end or no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or no more than 4 nucleotides from the 3 '-end, wherein the reporter oligonucleotide is a sequence 10 to 50 in length into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence H, wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And

in this case, the hydrophobic nucleotide has the structure X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid, and wherein the hybridization sequence is complementary to a sequential fragment of the second strand sequence of the target nucleic acid.

Also provided herein are methods for predicting the effectiveness of a treatment for a clinical condition in a subject, which may include the following steps:

a. obtaining a sample from a specified subject;

b. implementing a method for detecting the presence of a sequence variant, as described herein, to determine whether a sample includes a sequence variant

wherein the presence of said sequence variant indicates whether said drug is effective in treating said clinical condition in said subject.

Also provided herein are methods that can be used to predict or even diagnose the presence of any clinical condition associated with a particular mutation in a subject, comprising the following steps:

a) obtaining a sample from a specified subject;

b) detecting the presence of a mutation associated with a clinical condition in a sample by performing the sequence variant detection methods described herein;

wherein the presence of said sequence variant indicates that said subject has said clinical condition.

Description of the figures

Fig. 1 Principle of a method for detecting a variant of a nucleic acid sequence including an NOI. (A) Target nucleic acid (black chains). The second chain includes NOI (marked in light gray). The first strand is complementary (black line). Arrows indicate primers. The first primer hybridizes to the second strand and primer amplification occurs (dashed line). The second primer hybridizes to the first strand and primer amplification occurs (dashed line). For simplicity, only the amplified portion of the target nucleic acid is shown. The second primer is present here in excess compared to the first primer (asymmetric PCR). This ensures that more DNA is produced that includes the NOI sequence (dashed black line and dotted gray line). (B) The amplified DNA consists of a mixture of double-stranded target nucleic acids and single-stranded target nucleic acids, including NOI. Shown here is the single-stranded sequence including the NOI (in light gray). A reporter oligonucleotide (RO) hybridizes to this strand. It includes here the fluorophore F (circle) and quenchers Q (squares). A melting curve is obtained (X-axis: T indicates temperature; Y-axis: F indicates fluorescence).

Fig. 2 Microsatellite instability does not necessarily lead to significant changes in melting temperature. BAT25 analysis using asymmetric PCR. The X-axis shows temperature. (A) Melting curves where bilinear normalization and temperature shift were used with an intensity threshold of 0.1 RFU (relative fluorescence units). The difference between the curves, measured as the maximum amplitude difference between the curves, was -0.07 RFU. (B) First negative derivative curves without applying a temperature shift, T _m of normal tissue was 57.31°C, T _m of tumor tissue was 57.41°C.

Fig. 3 Bilinear normalization shifts the melting curves closer to 0 before and after the actual melting phase. Microsatellite NR22 is analyzed using asymmetric PCR and reporter oligonucleotide. The X-axis shows temperature. HRM curves were obtained using: (A) standard normalization without temperature shift; (B) bilinear normalization without temperature shift.

Fig. 4 Bilinear normalization shifts the melting curves closer to 0 before and after the actual melting. HRM difference curves based on melting curves FIG. 3, where the reference HRM curve is used as a baseline, which is subtracted from another (here, first) melting curve. The X-axis shows temperature. (A) without bilinear normalization without temperature shift; (B) with bilinear normalization without temperature shift.

Fig. 5In theory, there should be no difference in terms of melting temperature between normal and tumor tissue samples obtained from a person with microsatellite stability. However, differences in quantity, concentration of salts, impurities and other parameters between samples can lead to small differences in terms of melting point. However, bilinear normalization and temperature shift can reduce the difference between normal and microsatellite-stable tumor tissue from a patient. NR24 analysis using asymmetric PCR and reporter oligonucleotide. The X-axis shows temperature. (A) HRM profiles where standard normalization was applied. The difference between the curves, measured as the maximum difference in fluorescence at a certain temperature, between the normalized areas in the difference plots was ∼0.04 RFU. The X-axis shows temperature. (B) HRM profiles where bilinear normalization was used but no temperature shift threshold was used. The difference between the curves was ~0.02 RFU. (C) HRM curves where bilinear normalization and temperature shift were used with an intensity threshold of 0.1 RFU. The difference between the curves was ~0.01 RFU.

Fig. 6 The temperature shift can counteract the change in melting temperature caused by different salt concentrations in DNA buffers. NR24 analysis using asymmetric PCR and reporter oligonucleotide. The X-axis shows temperature. (A) HRM profiles where bilinear normalization was used but no temperature shift was used. The difference between the curves, measured as the maximum fluorescence difference at a given temperature, between the normalized areas in the difference plots was ∼0.07 RFU. (B) HRM curves where bilinear normalization and temperature shift were used with an intensity threshold of 0.1 RFU. The difference between the curves was ~0.01 RFU.

Fig. 7 Asymmetric PCR assay for microsatellite MON027 on 16 normal tissue DNA samples. HRM curves were obtained using: (A) bilinear normalization without temperature shift; (B) bilinear normalization and temperature shift with an intensity threshold of 0.1 RFU. The X-axis shows temperature. The use of a temperature shift makes it possible to use one sample as a universal control.

Fig. 8 Graphs of the difference between HRM curves Fig. 7. (A) Bilinear normalization without temperature shift; (B) Bilinear normalization and temperature shift with an intensity threshold of 0.1 RFU. The X-axis shows temperature.

Fig. 9 Asymmetric PCR produces more single-stranded target amplicon. NR22 assays using symmetric and asymmetric PCR. The X-axis shows the number of PCR cycles, and the Y-axis shows fluorescence.

Fig. 10 Asymmetric PCR produces a higher signal-to-noise ratio and a sharper melting curve (i.e., narrower melting peaks). NR22 analysis using symmetric and asymmetric PCR. (A) melting curves; (B): Melting curves normalized using standard normalization. The X-axis shows temperature.

Fig. 11 Asymmetric PCR facilitates the discrimination between patients with MSS and MSI. Data obtained using NR22 analysis are shown. HRM curves were obtained after applying standard normalization with asymmetric PCR (A) or symmetric PCR (B). The X-axis shows temperature.

Fig. 12 Asymmetric PCR facilitates the distinction between patients with MSS and MSI. Data obtained using NR22 analysis are shown. Graphs of the difference between HRM curves Fig. 11. (A) asymmetric PCR; (B): symmetric PCR. The X-axis shows temperature.

Fig. 13 A one-way overhang in a reporter oligonucleotide can lead to an increase in melting temperature. Shown are data from an NR22 assay using an asymmetric reporter oligonucleotide PCR with two different reporter oligonucleotides. (A) HRM curves. (B) HRM first negative derivative curves. The X-axis shows temperature.

Fig. 14 Double quenching of the reporter oligonucleotide increases the signal-to-noise ratio. Shown are data obtained using the BAT26 assay using asymmetric PCR. HRM curves (A) and first negative derivative curves (B) using single-quenched (“non-DQ probe”) and double-quenched (“DQ probe”) reporter oligonucleotides. The X-axis shows temperature.

Fig. 15 Additional nucleotide repeats in the reporter oligonucleotide hybridization sequence provide the ability to detect longer microsatellites. Data obtained from the NR21 assay using asymmetric PCR are shown. The X-axis shows temperature.

Fig. 16 A point mutation results in a change in melting temperature of several degrees. The experiment used an analysis based on exon 13 of the KIT gene. PCR was performed as asymmetric PCR. The X-axis shows temperature. Dashed lines indicate results against wild-type target nucleic acid, and solid lines indicate results against target nucleic acid including mutation. (A) Normalized HTM curves; the Y axis shows fluorescence. (B) Melting peaks (first negative derivatives of melting curves). Y axis represents -dF/dT.

Fig. 17 A strong helper sequence helps discriminate between wild type and mutant. As part of the experiment, NR24 analysis was used. PCR was performed as asymmetric PCR. The X-axis shows temperature and the Y-axis shows fluorescence. Dashed lines indicate results against wild-type target nucleic acid, and solid lines indicate results against target nucleic acid including mutation. (A) Normalized HRM curves with the first reporter oligonucleotide including the first helper sequence. (B) Normalized HTM curves with a second reporter oligonucleotide including a second helper sequence that is stronger than the first helper sequence.

Fig. 18 A strong helper sequence helps discriminate between wild type and mutant. As part of the experiment, NR24 analysis was used. PCR was performed as asymmetric PCR. The X-axis shows temperature and the Y-axis shows fluorescence. Dashed lines indicate results against wild-type target nucleic acid, and solid lines indicate results against target nucleic acid including mutation. (A) Difference plots with the first reporter oligonucleotide including the first helper sequence. (B) Difference plots with a second reporter oligonucleotide including a second helper sequence that is stronger than the first helper sequence.

Detailed Description of the Invention

Definitions

Amplicon The term "amplicon" refers to a molecule made by copying or transcribing another molecule. Examples of processes by which amplicons can be generated include transcription, cloning, and/or polymerase chain reaction (PCR) or other nucleic acid amplification technique (e.g., strand displacement amplification (SDA) PCR, duplex PCR amplification, and etc). Typically, an amplicon is a copy of or complementary to a selected nucleic acid (eg, template or target nucleic acid).

Bilinear Normalization The term "Bilinear Normalization" is used here to refer to a mathematical transformation applied to a curve(s) by scaling the fluorescence-related data of the various curves to normalized curves. This allows comparison of different curves while eliminating other factors that may affect the fluorescence signal (eg, different fluorescence strengths at different positions in the instrument, different plastic translucencies, and other factors that introduce variability in fluorescence measurements). Bilinear normalization forces the curve(s) to have the same value (within the average value of the normalization region) and to have the curve run as horizontally (flat) as possible within the selected normalization regions. Several algorithms are known in the art that can be applied to a curve to transform the curve(s) into curve(s) that allow comparison of different melting curves. Normalization also reduces the impact of various optical and mechanical differences between wells.

Hydrophobic Nucleotide The term “hydrophobic nucleotide” as used herein refers to the hydrophobic nucleotides described in detail herein below under the heading “hydrophobic nucleotide”. In particular, the hydrophobic nucleotide according to the invention contains an intercalator attached to the nucleotide/nucleotide analogue/framework monomer unit via a linker.

Melting Point The term "melting point" as used herein refers to the temperature in degrees Celsius at which 50% of the helical (hybridized) relative to the coiled (non-hybridized) forms are present. The melting point can also be denoted as ( _Tm ). Melting of nucleic acids and nucleic acid analogs refers to the thermal separation of the two strands of a double-stranded nucleic acid molecule.

Microsatellite The terms "microsatellite", "microsatellite marker" or "microsatellite locus" refers to a region of genomic DNA that includes tandem nucleotide repeats. These repeats or “repeating units” typically range in length from about one to about seven base pairs. Microsatellite loci typically comprise approximately 10 to 40 similar repeat units arranged in tandem. In addition to mononucleotide repeats, which are repeats of a single nucleotide, other examples of repeated nucleotide sequences include dinucleotide repeats, such as AT repeats and GC repeats, trinucleotide repeats, such as CGG repeats, CGC repeats, TAT repeats, ATT repeats, tetranucleotide repeats, pentanucleotide repeats and/ or their complementary repeats.

Nucleotide analog The term “nucleotide analog” includes all nucleotide analogs capable of being incorporated into a nucleic acid framework and capable of performing specific base pairings substantially like naturally occurring nucleotides. Nucleotide analogues in accordance with the present invention include, but are not limited to, nucleotide analogues selected from the group consisting of PNA (peptide nucleic acid), 1~HK (HNA, hexitol nucleic acid), MNA (morpholino nucleic acid acid), ANA (ANA, arabino-nucleic acid), LNA (LNA, blocked nucleic acid), CNA (XNA, xeno-nucleic acid), MHK (INA, intercalator nucleic acid), CAN (CAN, cell-free nucleic acid), CNA (CeNA, cyclohexene nucleic acid), TNA (TNA, threose nucleic acid), (2'-NH)-THK, (3'-NH)-THK, α-L-ribo-LNA, α-L- xylo-LNA, β-D-xylo-LNA, α-D-ribo-LNA, [3.2.1]-LNA, bicyclo-DNA, 6-amino-bicyclo-DNA, 5-epi-bicyclo-DNA, α- bicyclo-DNA, tricyclo-DNA, bicyclo[4.3.0]-DNA, bicyclo[3.2.1]-DNA, bicyclo[4.3.0]amide-DNA, β-D-ribopyranosyl-nucleic acid, α-L-lyxopyranosyl -nucleic acid, 2'-R ₁ -RNA, 2'-OR ₁ RNA (R ₁ represents a substituent), α-L-RNA, α-D-RNA, and β-D-RNA.

Nucleotide of Interest The term "nucleotide(s) of interest" as used herein refers to the nucleotide(s) within the target nucleic acid sequence, which may be present in two different variations. The term "nucleotide(s) of interest" may also be referred to herein as "NOI". Thus, the NOT may consist of a sequence variant or may consist of a control sequence, also referred to herein as a “control sequence.” In some embodiments, the control sequence may be a wild-type sequence.

Nucleotide The term "nucleotide" as used

here, the meaning refers to naturally occurring nucleotides, such as naturally occurring ribonucleotides or deoxyribonucleotides or naturally occurring derivatives of ribonucleotides or deoxyribonucleotides. Naturally occurring nucleotides include deoxyribonucleotides, which have one of four nitrogenous bases: adenine (A), thymine (T), guanine (G), or cytosine (C), and ribonucleotides, which have one of four nitrogenous bases: adenine (A), uracil (U), guanine (G), or cytosine (C).

Oligonucleotides The term "oligonucleotide" as used herein refers to nucleotide oligomers and/or nucleotide analogues and/or hydrophobic nucleotides. Preferably, the oligonucleotide is an oligomer of nucleotides, optionally including one or more hydrophobic nucleotides.

Target control sequence The term “target control sequence” refers to a fragment of a target nucleic acid sequence including a control sequence.

Standard Normalization The term "standard normalization" refers to the scaling of fluorescence-related data from different curves to normalized curves and allows comparison of different curves while eliminating other factors that may affect the fluorescence signal (eg, different fluorescence signal strengths at different positions in the instrument, different plastic transparencies and other factors that introduce variability in fluorescence measurements).

Target Nucleic Acid Sequence The term “target nucleic acid sequence” as used herein refers to a nucleic acid sequence comprising the nucleotide(s) of interest (NOI). The target nucleic acid sequence can be amplified using a set of primers.

Temperature Shift The term "temperature shift" as used herein refers to a mathematical tool that is applied to two or more normalized curves to shift them in the temperature direction to produce the same T _m value at a certain fluorescence cutoff (intensity threshold). Applying a similar transformation algorithm to a data set reduces the influence due to, for example, different salt concentrations in different samples.

Sequence Variant The term “sequence variant” as used herein refers to the nucleotide(s) within the target nucleic acid sequence that differ from the reference sequence. Thus, the target nucleic acid sequence may contain an NOI that is a sequence variant, or the target nucleic acid sequence may contain an NOI that is a control sequence, or the NOI may even be another sequence variant. The sequence variant may be, for example, a mutation, such as a single nucleotide mutation, or may be an insertion or deletion. The sequence variant and control sequence may overlap, such that the sequence of one includes the entire sequence of the other. In such cases, the sequence variant and the reference sequence may differ in terms of the number of nucleotides comprising the sequence.

Methods for detecting the presence of a sequence variant in a target nucleic acid sequence

The invention relates to methods for detecting the presence of a sequence variant in a target nucleic acid sequence. Thus, these methods are particularly useful in detecting the presence of a particular sequence in a target nucleic acid sequence as two or more different sequences. These methods can be used, for example, to distinguish between wild-type and mutant sequences and to distinguish between different polymorphic sequences. These methods are particularly useful for detecting sequence variants resulting from microsatellite instability by detecting the presence of sequence variants having a length different from the length of the wild-type or control sequence. Thus, these methods can be usefully used to detect a target nucleic acid sequence including repeats, especially microsatellites.

Provided herein is a method for detecting the presence of a sequence variant in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), preferably including repeats, wherein said target nucleic acid sequence consists of a sequence variant or a control sequence, this method includes the following steps:

a) Obtaining a first sample comprising nucleic acids believed to include the specified sequence variant;

b) Obtaining a second sample comprising nucleic acids comprising said control sequence, wherein the second sample is a control sample;

c) Preparation of a reporter oligonucleotide;

d) Providing a primer set consisting of a first primer and a second primer, wherein the primers of the set together are capable of amplifying a target nucleic acid sequence including an NOI;

e) Amplifying a target nucleic acid sequence in the presence of said first sample, said first primer and said second primer, thereby producing a first amplicon comprising nucleic acids believed to comprise the sequence variant; and amplifying a target nucleic acid sequence in the presence of said second sample, said first primer and said second primer, thereby providing a second amplicon including a control sequence, wherein the second amplicon is a control amplicon;

f) Performing a melting analysis, such as high sensitivity melting curve analysis (HRM), on the first amplicon, thereby obtaining a first profile characterized by the first melting curve, and performing a melting analysis, such as HRM analysis, on the second amplicon, thereby obtaining a second profile characterized by a second melting curve, wherein the second profile is a control profile characterized by a control melting curve; wherein each amplicon includes a first strand and a second strand, wherein the melting assay includes hybridizing a reporter oligonucleotide to one strand of each amplicon, detecting a signal emitted by a fluorophore, and obtaining a first and second melting curve;

wherein the reporter oligonucleotide is a sequence of 10 to 50, preferably 15 to 50 nucleotides in length, into which 2 to 10 hydrophobic nucleotides have been inserted,

wherein the reporter oligonucleotide includes a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' end end, and wherein the reporter oligonucleotide includes the hybridization sequence M,

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence,

and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeat sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize with the first and second amplicons when the hybridization sequence hybridizes with them; And

g) Comparing the first profile with a control profile, with the difference between the first profile and the control profile indicating that the first sample contains a sequence variant.

Provided herein is a method for detecting the presence of a sequence variant in a two-strand nucleic acid target sequence comprising a nucleotide(s) of interest (NOI), wherein said target nucleic acid sequence consists of a sequence variant or a control sequence, wherein said method includes the following steps:

a) Obtaining a first sample comprising nucleic acids believed to include the specified sequence variant;

b) Obtaining a second sample comprising nucleic acids comprising said control sequence, wherein the second sample is a control sample;

c) Preparation of a reporter oligonucleotide;

d) Providing a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid sequence;

e) Amplifying a target nucleic acid sequence in the presence of said first sample, said first primer and said second primer, thereby producing a first amplicon comprising nucleic acids believed to comprise the sequence variant; and amplifying a target nucleic acid sequence in the presence of said second sample, said first primer and said second primer, thereby providing a second amplicon including a control sequence, wherein the second amplicon is a control amplicon;

f) Performing a high-sensitivity melting curve analysis (HRM) of the first amplicon, thereby obtaining a first HRM profile characterized by a first melting curve, and performing an HRM analysis of a second amplicon, thereby obtaining a second HRM profile characterized by a second melting curve, wherein the second HRM the profile is a control profile characterized by a control melting curve; wherein each amplicon includes a first strand and a second strand, wherein the HRM analysis includes hybridizing a reporter oligonucleotide to one strand of each amplicon, detecting a signal emitted by a fluorophore, and obtaining a first and second melting curve;

wherein the reporter oligonucleotide is a sequence from 10 to 50 in length, into which from 2 to 10 hydrophobic nucleotides were inserted,

wherein the reporter oligonucleotide includes a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' end end, and wherein the reporter oligonucleotide includes a hybridization sequence H,

wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And

in this case, the hydrophobic nucleotide has the structure

X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence; And

g) Comparing the first HRM profile with a control HRM profile, with the difference between the first HRM profile and the control HRM profile indicating that the first sample contains a sequence variant.

Accordingly, in the presence of two or more different target nucleic acid sequences, these methods can be used to distinguish between at least two different target nucleic acid sequences, that is, a target nucleic acid sequence including a sequence variant and a target nucleic acid sequence , which does not include a sequence variant. The latter may also be referred to as a "control sequence". The methods may optionally be used to distinguish target nucleic acid sequences, including other sequence variants. The general principle of the method is shown as an example in Fig. 1. Briefly, the difference between the first and second profile, in particular the difference between the analyzed melting profile and the control melting profile, indicates that the tested sample (first sample) contains a sequence variant; It will be appreciated that, as used herein, a difference preferably means a significant difference.

In embodiments of the invention, when using these methods to distinguish between a wild-type sequence and a mutant sequence, the mutant sequence may be a control sequence. However, often the wild-type sequence will be a control sequence. The methods of the invention can also be used to distinguish between a wild-type sequence and several different mutant sequences, in which case the wild-type sequence will typically be a control sequence and the several different mutant sequences will be variant sequences. In some embodiments of the methods, the control sequence is a microsatellite sequence. The sequence variant in this case may be a microsatellite sequence consisting of a number of tandem repeats with a different number of tandem repeats than the number of tandem repeats of the reference sequence. For example, a sequence variant may be the result of microsatellite instability—in these cases, the sequence variant may be a plurality of sequence variants having varying numbers of tandem repeats. On the other hand, in this case the control sequence will have a constant number of tandem repeats.

In some embodiments, a sequence variant may represent a mutation that is indicative of a disease-associated condition, or may provide a prediction of the effectiveness of a particular treatment.

Nucleotide(s) of Interest (NOI), Sequence Variant, and Reference Sequence A sequence variant can be any sequence that is different from another sequence, in particular a reference sequence. Often, a sequence variant is a mutant sequence, for example a sequence that differs from the wild type sequence, for example due to the presence of mutations causing the substitution of one nucleotide for another, and/or due to the insertion and/or deletion of nucleotides compared to the reference sequence. However, a sequence variant may also be a polymorphic sequence or any other sequence that differs from the reference sequence. Preferably, the sequence variant is a microsatellite variant having a length different from the normal microsatellite length. The present methods are indeed particularly useful in detecting microsatellite instability. They can be used to detect instability of short microsatellites (less than 15 nucleotides in length) as well as longer microsatellites (more than 15 nucleotides in length). In a subject with microsatellite instability, longer microsatellites will mutate faster than shorter microsatellites—therefore, it may be beneficial to use a target nucleic acid that is a longer microsatellite that is 15 nucleotides or more in length.

The sequence variant may consist of at least one, for example 1, for example 2, for example 3, for example 4, for example 5, for example 6, for example 7, for example 8, for example 9, for example 10, for example 10 to 20, for example from 20 to 50, for example more than 50 nucleotides. Preferably, the sequence variant consists of 10 nucleotides or more, such as 15 nucleotides or more. Thus, in some embodiments, the sequence variant consists of 11, 12, 13, 14, 15, 16, 17, 19, 20 nucleotides or more, such as 25, 30, 35, 40, 45, or 50 nucleotides or more. For example, a sequence variant consists of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or more.

In some embodiments, the sequence variant is based on a single nucleotide mutation or single nucleotide polymorphism (SNP).

A sequence variant may be based on a change of one or more nucleotides by one or more different nucleotides compared to the reference target sequence. Moreover, the term “sequence variant” can refer to a deletion or insertion of nucleotides within a nucleic acid, such as a deletion or insertion of nucleotides relative to a reference target sequence.

The target nucleic acid sequence may include a polymorphism site (see more detail below) and thus the control target sequence may include one polymorphism, while the “sequence variant” may correspond to another polymorphism.

In one embodiment, the target control sequence is a wild-type sequence, that is, the most commonly occurring sequence in nature, while the sequence variant includes one or more mutations, insertions or deletions compared to the wild-type sequence. Accordingly, a sequence variant according to the present invention may, in one embodiment, be based on a polymorphism, such as a single nucleotide polymorphism (SNP). For example, a polymorphism may indicate a specific DNA profile. Knowing a particular DNA profile can be used, for example, to identify a subject. For example, a specific DNA profile can be used to identify a criminal or potential criminal or to identify a corpse or part of a corpse. Moreover, a specific DNA profile can be used to determine relatedness between subjects, such as parent-child or more distant relationships. Kinship can also be the relationship between different species or different populations of a particular species.

In some embodiments, the control sequence is or includes a microsatellite having a certain number of repeats. The sequence variant may then be one of the sequence variants having a different number of repeats than that observed in the control or wild type sequence.

In one embodiment, a sequence variant may indicate a clinical condition, or a mutation may indicate an increased risk of a clinical condition. In particular, the sequence variant may represent a microsatellite, and the clinical condition may be associated with microsatellite instability.

Said clinical condition may be selected, for example, from the group consisting of neoplastic diseases, neurodegenerative diseases, cardiovascular diseases, and metabolic disorders, including diabetes.

Moreover, a sequence variant may indicate a particular response to treatment with a predetermined drug. For example, the presence of a sequence variant may indicate whether a subject will respond favorably to treatment with a specified drug or the ability/inability of a subject to tolerate treatment with a specific drug. For example, detection of MSI can be used in the decision-making process to prescribe the use of checkpoint inhibitors, such as, for example, Keytruda® (pembrolizumab, Merck & Co. Inc.) or Opdivo (nivolumab, Bristol-Myers Squibb).

The sequence variant may be located in a specific gene, gene segment, microsatellite, or any other DNA sequence. Moreover, the sequence variant may be located in an mRNA, microRNA, or any other RNA sequence. The methods described herein provide the ability to detect certain DNA, which may be of eukaryotic, prokaryotic, archaeal, or viral origin. For example, the invention may facilitate diagnosis and/or genotyping of various infectious diseases by analyzing specific sequences known to be associated with a particular microorganism.

Nucleic acid target sequence

The present methods are designed to detect the presence of a sequence variant in a target nucleic acid sequence consisting of two strands and including the nucleotide(s) of interest (NOI). While the target nucleic acid sequence consists of two strands and is typically DNA, it is apparent that the present methods can be readily adapted to detect the presence of a sequence variant in the target nucleic acid by amplifying one strand of the target nucleic acid sequence acids - for example if the amplification is RNA amplification. Thus, the present methods can also be used to detect a sequence variant in a target RNA that arises, for example, from transcription of a double-stranded target nucleic acid sequence.

Thus, a target nucleic acid is a sequence of interest that is believed to include a sequence variant. In other words, it often corresponds to a specific locus.

The target nucleic acid sequence may consist of at least one, for example 1, for example 2, for example 3, for example 4, for example 5, for example 6, for example 7, for example 8, for example 9, for example 10, for example 10 to 20 , for example from 20 to 50, for example more than 50 nucleotides. Preferably, the target nucleic acid sequence consists of 15 nucleotides or more. Thus, in some embodiments, the target nucleic acid sequence consists of 11, 12, 13, 14, 15, 16, 17, 19, 20 nucleotides or more, such as 25, 30, 35, 40, 45, or 50 nucleotides or more . For example, the target nucleic acid sequence consists of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or more. Because the present methods are particularly useful for detecting microsatellite sequence variants, especially microsatellites having a length of 15 nucleotides or more, wherein the target nucleic acid sequence is a control sequence, its length is preferably 15 nucleotides or more.

In some embodiments, the target nucleic acid is a microsatellite comprising a series of tandem repeats. For the reference sequence, the number of tandem repeats is M, with M being an integer. For the control sequence, the total length is n nucleotides. The sequence variant has M' tandem repeats and a total length of n' nucleotides. M and M' are distinct integers, n and n' are distinct integers. However, it is possible that some of the sequence variants have M tandem repeats and a total length of n nucleotides, but in this case, some or most of the sequence variants will still have a different number of repeats and a different total length compared to the reference length, which will provide the ability of the present methods for detecting the presence of sequence variants that differ from a reference sequence.

In some embodiments, n and/or n' is 1, such as 1, such as 2, such as 3, such as 4, such as 5, such as 6, such as 7, such as 8, such as 9, such as 10, such as 10 to 20, for example from 20 to 50, for example more than 50 nucleotides. Preferably n and/or n' is 15 nucleotides or more; in some embodiments, n is at least 15 nucleotides or more. Thus, in some embodiments, n and/or n' consists of 11, 12, 13, 14, 15, 16, 17, 19, 20 nucleotides or more, such as 25, 30, 35, 40, 45 or 50 nucleotides or more. For example, p and/or p' consists of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides or more. Preferably, n consists of at least 15, 16, 17, 18, 19, 20 nucleotides or more, for example 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides or more , for example 25, 30, 35, 40, 45 or 50 nucleotides or more. In some embodiments, n' is 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22,23, 24, 25, 26, 27, 28, 29 or 30 nucleotides or more, such as 25, 30, 35, 40, 45 or 50 nucleotides or more.

The present methods can be used to detect a sequence variant that may be indicative of a particular disorder. For example, the disorder may be associated with microsatellite instability.

It is often useful to analyze a sample for the presence of several different sequence variants. Accordingly, the present methods can be adapted so that multiple sequence variants are detected simultaneously. Sometimes it is sufficient to determine the presence of only at least one of several sequence variants.

In some embodiments, the target nucleic acid sequence is a plurality of target nucleic acid sequences including one or more of BAT25, BAT26, NR21, NR22, NR24, and MON027, which are microsatellite markers comprising mononucleotide repeats. The methods provide the ability to detect sequence variants having a different number of repeats than the number of data target nucleic acid sequences of the control sequence, and thus different overall lengths.

The control or wild-type sequence of BAT25 corresponds to that specified in SEQ ID NO: 1. The mononucleotide repeats of BAT25 correspond to positions 147 to 171 of SEQ ID NO: 1. Thus, the control sequence has M mononucleotide repeats, with M=25.

The control or wild-type sequence of BAT26 corresponds to that specified in SEQ ID NO: 2. The mononucleotide repeats of BAT26 correspond to positions 222 to 248 of SEQ ID NO: 2. Thus, the control sequence has M mononucleotide repeats, with M=27.

The control or wild-type sequence of NR21 corresponds to that specified in SEQ ID NO: 3. The mononucleotide repeats of NR21 correspond to positions 189 to 209 of SEQ ID NO: 3. Thus, the control sequence has M mononucleotide repeats, with M=21.

The control or wild-type sequence of NR22 corresponds to that specified in SEQ ID NO: 4. The mononucleotide repeats of NR22 correspond to positions 152 to 172 of SEQ ID NO: 4. Thus, the control sequence has M mononucleotide repeats, with M=21.

The control or wild-type sequence of NR24 corresponds to that specified in SEQ ID NO: 5. The mononucleotide repeats of NR24 correspond to positions 165 to 187 of SEQ ID NO: 5. Thus, the control sequence has M mononucleotide repeats, with M=23.

The control or wild-type sequence of MON027 corresponds to that specified in SEQ ID NO: 6. The mononucleotide repeats of MON027 correspond to positions 300 to 327 of SEQ ID NO: 6. Thus, the control sequence has M mononucleotide repeats, with M=28.

In some embodiments, the target nucleic acid sequence is BAT25, as defined in SEQ ID NO: 1, and the NOI corresponds to the sequence defined by positions 147 to 171 of SEQ ID NO: 1. In other embodiments, the target nucleic acid sequence is BAT26, as defined in SEQ ID NO: 2, and the NOI corresponds to the sequence defined by positions 222 to 248 of SEQ ID NO: 2. In other embodiments, the target nucleic acid sequence is NR21, as defined in SEQ ID NO: 3, a NOI corresponds to the sequence defined by positions 189 to 209 of SEQ ID NO: 3. In other embodiments, the target nucleic acid sequence is NR22, as defined in SEQ ID NO: 4, and the NOI corresponds to the sequence defined by positions 152 to 172 SEQ ID NO: 4. In other embodiments, the target nucleic acid sequence is NR24, as defined in SEQ ID NO: 5, and the NOI corresponds to the sequence defined by positions 165 to 187 of SEQ ID NO: 5. In other embodiments, the sequence The target nucleic acid is MON027 as defined in SEQ ID NO: 6, and the NOI corresponds to the sequence defined by positions 300 to 327 of SEQ ID NO: 6.

In other embodiments, the target nucleic acid sequence is two target nucleic acid sequences. For example, two target nucleic acid sequences are BAT25 and BAT26; BAT25 and NR21; BAT25 and NR22; BAT25 and NR24; BAT25 and MON027; BAT26 and NR21; BAT26 and NR22; BAT26 and NR24; BAT26 and MON027; NR21 and NR22; NR21 and NR24; NR21 and MON027; NR22 and NR24; NR22 and MON027; NR24 and MON027, where NOIs are defined as described above.

In other embodiments, the target nucleic acid sequence is three target nucleic acid sequences. For example, three target nucleic acid sequences are BAT25, BAT26 and NR21; BAT25, BAT26 and NR22; BAT25, BAT26 and NR24; VAT25, VAT26 and MON027; BAT25, NR21 and NR22; BAT25, NR21 and NR24; BAT25, NR21 and MON027; BAT25, NR22 and NR24; BAT25, NR22 and MON027; BAT25, NR24 and MON027; BAT26, NR21 and NR22; BAT26, NR21 and NR24; BAT26, NR21 and MON027; BAT26, NR22 and NR24; BAT26, NR22 and MON027; BAT26, NR24 and MON027; NR21, NR22 and NR24; NR21, NR22 and MON027; NR21, NR24 and MON027; NR22, NR24 and MON027, where NOIs are defined as described above.

In other embodiments, the target nucleic acid sequence is four target nucleic acid sequences. For example, the four target nucleic acid sequences are BAT25, BAT26, NR21 and NR22; BAT25, BAT26, NR21 and NR24; VAT25, VAT26, NR21 and MON027; BAT25, BAT26, NR22 and NR24; VAT25, VAT26, NR22 and MON027; BAT26, NR21, NR22 and NR24; BAT26, NR21, NR22 and MON027; BAT26, NR21, NR24 and MON027; NR21, NR22, NR24 and MON027, where NOIs are defined as described above.

In other embodiments, the target nucleic acid sequence is five target nucleic acid sequences. For example, the five target nucleic acid sequences are BAT25, BAT26, NR21, NR22 and NR24; VAT25, VAT26, NR21, NR22 and MON027; VAT25, VAT26, NR22, NR24 and MON027; BAT26, NR21, NR22, NR24 and MON027, where NOIs are defined as described above.

In other embodiments, the target nucleic acid sequence is six target nucleic acid sequences. For example, the six target nucleic acid sequences are BAT25, BAT26, NR21, NR22, NR24 and MON027, where the NOIs are defined as described above.

In preferred embodiments, the target nucleic acid is five target nucleic acids, preferably BAT25, BAT26, NR21, NR22 and NR24; or BAT25, BAT26, NR22, NR24 and MON027, where the NOIs are defined as described above. In other preferred embodiments, the target nucleic acid is six target nucleic acids, preferably BAT25, BAT26, NR21, NR22, NR24 and MON027, where the NOIs are defined as described above.

Samples

In the first step of the method, a first sample is obtained, which includes nucleic acids that are believed to include a detectable sequence variant. In the second step of the method, a second sample is obtained, which includes a control sequence. Thus, the second sample is a control sample, and these terms will be used interchangeably here.

The sample may include cells comprising the specified nucleic acids. The cells may be, for example, prokaryotic cells or eukaryotic cells, such as plant cells or mammalian cells.

The sample may be, for example, a synthetically prepared sample that may or may not have been further processed in vitro, but most often the sample will be a sample obtained from a subject.

In some embodiments, the first sample is a sample obtained from a subject suffering or suspected of suffering from a disease or disorder characterized by the presence of a sequence variant, and the second sample is a sample obtained from a healthy subject. It is also possible to use a first sample comprising cells, for example, of tissue affected by the disease, for example cells suspected of containing a sequence variant characteristic of the disease, from one subject suspected of having the disease, and a second sample comprising cells of healthy tissue, i.e. cells that do not contain a sequence variant from the same subject. The present methods, in some embodiments, provide the ability to use a universal control sample.

Thus, it is often desirable to test the DNA or RNA of a subject, such as a mammal, such as a human. In this case, the sample is a sample obtained from the specified subject. Thus, the sample may, for example, include nucleic acids selected from the group consisting of DNA, mRNA, microRNA, or any other RNA sequence. The sample may be obtained from a sample of a biological fluid, such as a blood sample, a biopsy, a sample of hair, nails, or the like, or any other suitable sample. In embodiments of the invention in which the subject has cancer, the sample may be a sample of a cancer tumor surgically removed from the subject, or a biopsy of the tumor. However, within embodiments of the invention in which the subject has cancer, the sample may also be a blood sample, which typically may contain CTCs (circulating tumor cells, CTCs) and cfDNA (cell-free DNA, cfDNA).

The sample may be processed in vitro before detecting the presence of the sequence variant. For example, the sample may be subjected to one or more purification steps, which may remove all or part of the nucleic acids from the sample. Moreover, the sample can then be reverse transcribed.

The sample may include a complex biological mixture of nucleic acid (RNA or DNA) and non-nucleic acids, such as an intact cell or crude cell extract.

If the target DNA is double-stranded or otherwise has a secondary and/or tertiary structure that may make it difficult to detect, it may require heating before implementing the methods of the invention. Also, in some cases, it may be desirable to extract nucleic acids from complex biological samples before performing amplification using any methods known in the art.

The sample may include a wide range of eukaryotic and prokaryotic cells, including protoplasts; or other biological materials that may carry target deoxyribonucleic acids. Thus, these methods can be applied to animal tissue culture cells, animal cells (eg, blood, serum, plasma, reticulocytes, lymphocytes, urine, bone marrow tissue, cerebrospinal fluid or any product derived from blood or lymph) or any type of biopsy tissue (eg, muscle biopsy, liver biopsy, kidney biopsy, bladder biopsy, bone biopsy, cartilage biopsy, skin biopsy, pancreatic biopsy, intestinal biopsy, thymus biopsy, mammalian biopsy, uterine biopsy, testicular biopsy, eye biopsy, or biopsy brain homogenized in lysis buffer), plant cells or other cells sensitive to osmotic shock, and cells of bacteria, yeast, viruses, mycoplasmas, protozoa, rickettsia, fungi and other small microbial cells and the like. The assay and isolation procedures of the present invention can be used, for example, to detect non-pathogenic or pathogenic microorganisms of interest. By detecting the presence of a sequence variant in a biological sample, the presence of microorganisms can be determined.

In some embodiments, at least the first sample is obtained from a subject suffering or suspected of suffering from a disease or disorder that is characterized by the presence of a sequence variant as detailed above, in particular the presence of a microsatellite sequence variant or the presence of a mutant sequence. In some embodiments, the disease is a cancer or genetic disease, such as pancreatic carcinoma, gastric carcinoma, bladder cancer, prostate carcinoma, lung cancer, uterine carcinoma, breast cancer, hereditary nonpolyposis colon cancer. In some embodiments, the disease is a genetic disorder such as Lynch syndrome, Huntington's disease (HD), dentatorubro-pallidoluis atrophy (DRPLA), spinobulbar and muscular atrophy (SBMA), myotonic dystrophy (DM), fragile X syndrome, mental FRAXE retardation and spinocerebellar ataxia (SCA), X-linked agammaglobulinemia (Bruton's disease), Bloom's syndrome (BS), craniofrontonasal syndrome (CFNS), and idiopathic pulmonary fibrosis (IPF).

Amplification

The present methods require amplification of a target nucleic acid sequence, including NOI, from a first sample and a second sample. This requires obtaining a set of primers. The primer set consists of a first primer and a second primer, which together are capable of amplifying a target nucleic acid sequence including NOI, as is known in the art.

The primer set is capable of priming amplification of a target nucleic acid sequence when used in, for example, PCR, such as real-time PCR, in the presence of said target nucleic acid sequence and with reagents as known in the art. Such reagents are well known to one skilled in the art and have been described, for example, in Sambrook J et al. 2000. Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Habor Laboratory Press. The reaction then produces an amplicon. The amplicon obtained by performing amplification in the presence of the first sample is referred to as the "first amplicon". The amplicon obtained by performing amplification in the presence of a second (or control) sample is referred to as the “second amplicon” or “control amplicon”.

To provide a set of primers with specificity for a target nucleic acid sequence, the primers include a sequence that is identical to a portion of the target nucleic acid sequence. In particular, the 3' end of the first and second primers may include a sequence of at least 15 nucleotides that is identical to a portion of the target nucleic acid sequence including the control sequence or sequence variant, except for no more than one mismatch.

The exact length of the primer sequence that is identical to the target nucleic acid sequence fragment can be adjusted to match a primer having a melting temperature that can be used in PCR amplification. The melting temperature of a primer depends on several factors, but particularly on GC content and length. Because primers should preferably be identical to a fragment of a target nucleic acid sequence including a sequence variant (or a sequence complementary to a sequence variant), there are limitations in terms of the specific primer sequence. Accordingly, the melting temperature can be adjusted in a certain manner by adjusting the length of the primer. One skilled in the art will be able to design a primer with a suitable melting temperature, and software capable of being used for this purpose is publicly available.

Thus, the 3' end of the primers may include a sequence of at least 15 nucleotides, for example at least 20 nucleotides, for example at least 25 nucleotides, for example in the range of 15 to 50 nucleotides, for example in the range 20 to 40 nucleotides that is identical to the target nucleic acid sequence including the sequence variant except for no more than one mismatch.

In one embodiment of the invention, the first and/or second primer consists of a sequence of at least 15 nucleotides, for example at least 20 nucleotides, for example at least 25 nucleotides, for example in the range of 15 to 50 nucleotides, for example, in the range of 20 to 40 nucleotides, which is identical to the target nucleic acid sequence including the sequence variant except for no more than one mismatch.

As stated above, the primer sequence includes or consists of a sequence identical to (or complementary to) the target nucleic acid sequence, including the sequence variant except for no more than one mismatch. In some embodiments, there is one mismatch in one or both of the first and second primers, as this may further increase the specificity of the assay. In particular, said mismatch may be located at position 2, 3, or 4 from the 3' end of the primers.

In some embodiments, it may be useful to design primers that hybridize to the target nucleic acid sequence outside the region corresponding to the sequence variant. For example, if the target nucleic acid sequence is a microsatellite, and the sequence variant differs from the reference sequence in the number of nucleotide repeats, then the primers preferably hybridize above and below the tandem repeat region, since otherwise the amplification may be nonspecific and result in a series amplicons with different lengths.

Primers may include at least one hydrophobic nucleotide, as described here.

The amplification step may be PCR, such as real-time PCR. In some embodiments, the amplification reaction (eg, PCR or real-time PCR) is an asymmetric reaction. The term "asymmetric" means that the reaction is directed toward amplifying a greater length of one template strand than the other. This is accomplished by using different numbers of primers within a primer pair.

The first primer hybridizes to the first strand of the target nucleic acid sequence, which includes the NOI sequence. The second primer hybridizes to the second strand of the target nucleic acid sequence, which is complementary to the sequence with the NOI. At a later stage, the presence of a sequence variant is detected by analyzing the melting profile of the amplicons, which includes a sequence identical to the sequence with NOI, in the presence of a reporter probe, as described below. As shown in the examples, melting profile analysis is performed when the amplicon strand to which the reporter oligonucleotide can hybridize is present in greater abundance than the amplicon strand that is complementary to the reporter oligonucleotide. Thus, in preferred embodiments, the amplification step is asymmetric, that is, performed with more second primer than first primer. This directs the amplification to produce more amplicon strand to which the reporter oligonucleotide hybridizes.

Melting analysis (including HRM analysis)

Melting analysis, in particular HRM analysis (high-sensitivity melting curve analysis), is based on the analysis of the melting-related properties (in particular the double-stranded to single-stranded phase transition profile) of the generated heteroduplex amplicons. The melting profile of amplicons depends on guanine-cytosine content, length, sequence, and heterozygosity. Changes in the nucleotide sequence lead to the formation of heteroduplexes, which change the shape of the melting curve compared to the wild-type melting profile.

After obtaining the first amplicon and the second amplicon, for example by using an asymmetric PCR reaction, the method includes the step of performing a melting analysis, in particular an HRM analysis, to obtain melting profiles (eg HRM profiles) for each amplicon. A first melting profile (or first HRM profile) is obtained for the first amplicon, and a second melting profile (or second HRM profile), also referred to as a control profile (or reference HRM profile), is obtained for the second amplicon. The melting profiles (or HRM profiles) are then compared as detailed below.

Each melting profile (eg HRM) is characterized by a melting curve. A first melting profile (eg, HRM) of a first amplicon is characterized by a first melting curve, and a second melting profile (eg, HRM) of a second amplicon is characterized by a second (or control) melting curve.

A melting assay (eg, HRM) of each amplicon involves hybridizing a reporter oligonucleotide, including at least one fluorophore and one quencher, to one strand of each amplicon (having a sequence identical to that of the NOI), such as a second strand. The reporter oligonucleotide is described in more detail below. After hybridization, the signal emitted by the fluorophore is detected to produce a first and second melting curve, as is known in the art. The method may further include the step of transforming the melting curves prior to further analysis. For example, melting curves can be transformed into first negative derivative curves, which are then compared.

The first melt profile (eg HRM) and the second melt profile (eg HRM) are then compared. If the first sample contains a sequence variant, then the first melting curve (and optionally the difference curves and derivatives of the melting curves) will differ from the second melting curve (and optionally its derivatives) corresponding to the control sample. Thus, detecting a difference between the first and second melting profile (eg, HRM) indicates that the first sample contains a sequence variant.

The difference between melting profiles (eg HRM) may be a difference in terms of the shape of the melting curves or difference curves or the shape of derivative melting curves. For example, in some embodiments, the first melt curve has a different shape than the control melt curve. Typically, derivatives of melting curves are normal curves (or bell-shaped curves) with a maximum value. The slope of the first melt curve may be less "sharp" than the slope of the second curve, causing a change in the shape of the first melt curve compared to the second melt curve. The slope of the derivative of the first melting curve may be steeper than the slope of the derivative of the second melting curve, e.g., the absolute value of the slope is greater for the derivative of the first melting curve than the absolute value of the derivative slope for the second melting curve, at least at point(s) thereof inflection To determine the difference between melting profiles (eg HRM), the analysis may include the step of obtaining a difference curve, where one of the melting curves is taken as a control and subtracted from the other melting curve, preferably where the melting curve of a control sample is taken as a control and subtracted from the melting curve of the sample with option.

Two curves (or difference curves or derivatives) at each temperature T can be located at a distance D _T from each other. The distance D _T is determined for each temperature, with the absolute value of the largest distance maxD _t indicating the difference between the two curves if it exceeds a predefined threshold value.

One skilled in the art will know how to determine an appropriate value for a given threshold. The threshold value is the D _T value that allows discrimination between a subject exhibiting microsatellite instability and normal subjects. The Dt value is the difference, eg, a negative difference or a positive difference, between the melting profile obtained for the control (or wild-type sequence) and the melting profile obtained for the sequence variant(s). To determine an appropriate threshold value, the melting profiles of a number of subjects (usually 100-500 patients) known to have microsatellite instability are determined. Using these melting profiles, a threshold is determined as a value that allows the desired or appropriate discrimination between wild-type and variant sequences.

Thus, the difference between the analyzed melting profiles is calculated and compared with a threshold value; thus, the difference may represent a quantitative difference that can be compared with a quantitative threshold value. If this difference between the analyzed melting profiles, eg HRM profiles, exceeds a threshold value, then the sample for which the first melting profile is determined is classified as including a sequence variant. Thus, when the target nucleic acid sequence is a microsatellite, the subject from which the sample was obtained is considered to have microsatellite instability. If this difference between the analyzed melting profiles, eg HRM profiles, is below a threshold value, then the sample is classified as containing a wild type sequence. Thus, when the target nucleic acid sequence is a microsatellite, the subject from which the sample was obtained is considered to not have microsatellite instability.

An alternative is to determine the area between the curves. If this area exceeds a predetermined threshold value, then the first and second melting curves are considered different. In this case, one skilled in the art will also know how to determine the value of this threshold value.

Other ways of characterizing the difference between two melting curves or their difference curves or derivatives thereof will be apparent to one skilled in the art.

Any of the above differences between the first and second melt curves or between their difference curves or derivatives of the first and second melt curves indicate a difference between the first and second HRM profile and thus indicate the presence of a sequence variant in the first sample.

In some embodiments, the first and second melting curves and/or derivatives thereof and/or difference curves thereof are normalized. For example, in the vicinity of the point or at the lowest temperature point for which the melting was measured, for example in the range from 0.5 to 20°C from the lowest temperature point the curve is normalized to the first value, and in the vicinity or at the highest temperature point the curve normalized to the second value. For example, the first value is 1 and the second value is 0.

To make differences between curves more visible, a melting analysis, such as an HRM analysis, may include a standard normalization step that aligns the curves along the Y axis based on the average staining intensity at the initial and final normalization sites.

It is also possible to apply bilinear normalization to each curve (melting curve, difference plot or derivatives). The first linear function corresponds to the initial staining intensity and is used as the upper limit of the final adjusted scale. The second linear function corresponds to the final staining intensity and is used as the lower limit of the adjusted scale.

A melting analysis, such as an HRM analysis, may further or alternatively include the step of applying a temperature correction (temperature shift) to the curves (melt curves or difference curves) at a certain fluorescence intensity. Such steps can also reduce the risk of false-positive results. In preferred embodiments, the melting analysis includes at least the step of applying a temperature shift to the melting curves at a certain fluorescence intensity.

Accordingly, in some embodiments, the step of comparing the first profile with a reference profile includes or consists of the following steps:

i) aligning the first and second melting curves at a specified fluorescence intensity along a temperature axis, thereby eliminating differences in terms of melting temperature between the first and second melting curves at said fluorescence intensity;

ii) determining the difference in signal emitted by the fluorophore between the first and second melting curve; And

iii) comparing the difference determined in step ii) with a threshold value, wherein a difference exceeding the threshold value indicates that the first sample includes a sequence variant, and a difference not exceeding the threshold value indicates that the first sample includes a reference sequence.

The threshold value is determined as described above. The difference is a quantitative difference that is compared to a quantitative threshold as described above. Thus, in step iii), if the threshold value is a positive threshold value, then a difference greater than the specified threshold value indicates that the first sample includes a sequence variant. If the threshold value is a negative threshold value, then a difference less than or equal to the specified threshold value indicates that the first sample includes a sequence variant.

In some embodiments, the methods described herein do not include the step of determining the exact length and/or sequence of the sequence variant. This is because it is often not necessary to determine the exact length and/or sequence of sequence variants to determine that a subject in whom a sequence variant has been detected has a disease or disorder as described herein. Instead, it is often sufficient to determine whether a difference exists, but the exact nature of the difference is not always significant. In preferred embodiments, the methods described herein do not include the step of determining the exact length and/or sequence of the sequence variant. This is because detection of a sequence variant may be sufficient to select samples for further analysis; it is usually not necessary to determine the exact length and/or sequence of sequence variants.

Reporter oligonucleotide

The present methods require a reporter oligonucleotide to perform HRM analysis. The reporter oligonucleotide includes at least a first fluorophore and at least a first quencher. They are useful in terms of melting analysis such as HRM analysis. Preferably, the reporter oligonucleotide includes a first fluorophore at least at its 5' end or at least at its 3' end, or at a distance of no more than 4 nucleotides from the 5' end or 3' end. The reporter oligonucleotide preferably includes a first quencher at at least its 5' end or at least its 3' end, or at a distance of no more than 4 nucleotides from the 5' end or 3' end. Thus, the fluorophore and quencher may be located at the 5' end or 3' end, or at a distance of no more than 4 nucleotides from the 5' end or 3' end, but are not necessarily located at the last nucleotide of the reporter oligonucleotide. Preferably, if the first fluorophore is located at the 5' end or at a distance of no more than 4 nucleotides from the 5' end, the first quencher is not located at the 5' end or at a distance of no more than 4 nucleotides from the 5' end. Instead, it is located at the 3' end or at a distance of no more than 4 nucleotides from the 3' end or in the internal region of the reporter. Conversely, if the first fluorophore is located at the 3' end or at a distance of no more than 4 nucleotides from the 3' end, then the first quencher is not located at the 3' end or at a distance of no more than 4 nucleotides from the 3' end. Instead, it is located at the 5' end or at a distance of no more than 4 nucleotides from the 5' end or in the internal region of the reporter. The term "internal region" refers here to a region of a reporter oligonucleotide that does not include the 5 terminal nucleotides at each end. Preferably, the first fluorophore and the first quencher are not located close to each other.

Suitable fluorophores and quenchers are readily available to those skilled in the art to select them for use in the present methods.

A reporter oligonucleotide is used for a melting assay (or HRM assay). Thus, the reporter nucleotide sequence is subject to certain restrictions that depend on the sequence that it hybridizes and detects. The reporter oligonucleotide includes a hybridization sequence H that, for example, is identical to the NOI and that hybridizes to a strand that is complementary to the NOI. In other words, the reporter oligonucleotide hybridizes to the first and second amplicon strand that includes the NOI. Thus, within embodiments in which the NOI includes repeats, the reporter oligonucleotide also includes repeats, that is, the H hybridization sequence also includes repeats, as described in more detail below.

The reporter oligonucleotide may, in addition to a sequence that is identical to the NOI, also include additional nucleotides in the hybridization sequence. For example, as shown in the examples below, this may be particularly relevant in cases where the target nucleic acid is a microsatellite, or where the sequence variant is expected to involve an insertion. If the microsatellite has M tandem repeats having a total length of n nucleotides in the control sequence, then the reporter oligonucleotide hybridization sequence preferably has M"tandem repeats, with M"≥M+1, preferably M"≥M+1 or M"≥M+ 2. If the tandem repeat is a mononucleotide repeat, then the hybridization sequence is n" nucleotides long, with n"≥n+1, preferably n"≥n+1 or n"≥n+2. In other words, the hybridization sequence may include at least one or two additional nucleotides. This provides more sensitive discrimination between the longer sequence variant and the reference sequence.

In some embodiments, particularly where the NOI is a microsatellite, the reporter oligonucleotide hybridization sequence preferably includes a sequence that consists of repeats that are identical or complementary to the NOI repeats, and may usefully also include a terminal sequence, referred to herein as a helper sequence, which hybridizes to the first and second amplicons immediately upstream or immediately downstream of these repeats when the hybridization sequence hybridizes to them. In other words, the reporter oligonucleotide hybridization sequence preferably includes repeats and further includes from 1 to 10 nucleotides, for example 1 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, which hybridize to the NOI or a complementary strand thereof immediately upstream or immediately below the repeat sequence. Thus, the helper sequence, when hybridized to the first and second amplicons, causes the hybridization sequence to hybridize to said repeat sequence. A helper sequence will often have a higher affinity for its complementary sequence than a hybridization sequence for its complementary sequence. In other words, the helper sequence will often hybridize faster to its complementary sequence than the hybridization sequence will hybridize to its complementary sequence.

The helper sequence can be 3'-terminal or 5'-terminal. Preferably, the reporter oligonucleotide includes a 5'-terminal helper sequence that hybridizes to the first and second amplicons immediately upstream or immediately downstream of these repeats when the hybridization sequence hybridizes to them. The helper sequence provides discrimination of melting profiles, as shown in Example 13. A helper sequence is designated as strong if it results in a high increase in the T _m of the hybridization sequence when the helper sequence hybridizes to the first or second amplicon. It is well known to one skilled in the art that hybridization between two sequences is stronger if the sequences comprise a higher G/C ratio compared to two hybridized sequences comprising a higher A/T ratio; thus, a helper sequence comprising a higher G/C ratio than A/T will be more potent than a helper sequence comprising a lower G/C ratio than A/T. It is also known that longer sequences provide stronger hybridization than shorter sequences, so the strength of the helper sequence can be adjusted by increasing the length that hybridizes to the first and/or second amplicon. Increasing the strength of a helper sequence may be a good way to discriminate between a sequence variant and a control sequence. Preferably, the helper sequence does not include repeats, even in embodiments where the NOI includes repeats.

In some embodiments, particularly where the target nucleic acid sequence is a microsatellite, the helper sequence increases the T _m of the hybridization sequence by at least 5°C, such as by at least 6°C, such as by at least , by 7°C, for example, by at least 8°C, for example, by at least 9°C, for example, by at least 10°C, for example, by at least 11°C, for example, at least 12°C, for example, at least 13°C, for example, at least 14°C, for example, at least 15°C or more compared to T _m the same hybridization sequence without the helper sequence. Thus, the T _m of a hybridization sequence including a helper sequence may be 5-25°C higher, for example 10-20°C higher, for example 12.5-17.5°C higher, than the T _m of a hybridization sequence without a helper sequence.

The reporter oligonucleotide may include a second quencher. Preferably, the second quencher is a non-terminal region, that is, located in an internal region of the reporter oligonucleotide. In other words, the second quencher is not located at the 5' end or at the 3' end or within 4 nucleotides of the 5' end or 3' end. In some embodiments, inclusion of a second quencher may allow discrimination between a sequence variant and a reference sequence.

While any reporter oligonucleotide that can provide melting assay capability, that is, any reporter oligonucleotide with the properties described above, can be used in a given HRM assay, certain reporter oligonucleotides are particularly useful. The inclusion of hydrophobic nucleotides at certain positions of the reporter oligonucleotide is of particular interest. As can be seen from the examples, reporter oligonucleotides including such hydrophobic nucleotides increase the sensitivity of the method.

Thus, in some embodiments, in addition to the features described above, the reporter oligonucleotide includes at least one hydrophobic nucleotide located at its 5' end or at a distance of no more than 10 nucleotides from the 5' end, and/or the reporter oligonucleotide includes, at least one hydrophobic nucleotide located at its 3' end or at a distance of not more than 10 nucleotides from the 3' end.

A hydrophobic nucleotide has the structure

X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into a nucleic acid framework,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or scaffold monomer unit and said intercalator.

The framework monomer unit X may be any of the framework monomer units described herein below in the "Scaffold Monomer Unit" section.

Intercalator Q may be any of the intercalators described herein below in the "Intercalator" section.

Hydrophobic nucleotides that can be used in the context of the present invention are described in detail in international application WO 2017/045689, in particular in the section entitled “Hydrophobic nucleotides” on page 30, lines 2-25.

In some embodiments, the reporter oligonucleotide has the following general structure

5'-(N) _a -Z-(N) _d- Z-(N) _e -Z-(N) _b -3'

wherein

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

the total number of nucleotides or nucleotide analogues is at least 10; And

a and b individually represent integers ranging from 0 to 4; And

d and e individually represent integers ranging from 1 to 19; And

a+b+d+e is at least 10;

and (N) _a -(N) _d -(N) _e -(N) _b is identical to the control sequence.

In other embodiments, the reporter oligonucleotide has the following general structure

5'-(N) _a -Z-(N) _f- Z-(N) _g -Z-(N) _h -Z-(N) _b -3'

wherein

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

a and b individually represent integers in the range from 0 to 4; And

f, g and h individually represent integers ranging from 1 to 18; And

a+b+f+g+h is at least 10 and does not exceed 50;

and (N) _a -(N) _f -(N) _g -(N) _h -(N) _b is identical to a fragment of a target nucleic acid sequence including a control sequence; or

has the following general structure

5'-(N) _a -Z-(N) _i -Z-(N) _j -Z-(N) _k -Z-(N) _i -Z-(N) _b -3'

wherein

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

a and b individually represent integers in the range from 0 to 4; And

i, j, k and I individually represent integers ranging from 1 to 17; And

a+b+i+j+k+I is at least 10 and does not exceed 50;

and (N) _a -(N) _i -(N) _j -(N) _k -(N) _I -(N) _b is identical to a fragment of the target nucleic acid sequence including the control sequence; or

has the following general structure

5'-(N) _a -Z-(N) _m -Z-(N) _n -Z-(N) _o -Z-(N) _p -Z-(N) _q -Z-(N) _b - 3'

wherein

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

a and b individually represent integers in the range from 0 to 4; And

m, n, o, p and q are individually integers ranging from 1 to 16; And

a+b+m+n+o+p+q is at least 10 and does not exceed 50; and (N) _a -(N) _m -(N) _n -(N) _o -(N) _p -Z-(N) _q -(N) _b is identical to a portion of the target nucleic acid sequence including the control sequence.

In some embodiments, the reporter oligonucleotide is designed to detect a microsatellite. In such embodiments, the hybridization sequence spans at least tandem peptides. Preferably, the hybridization sequence also includes a helper sequence consisting of 1 to 20 nucleotides immediately upstream or downstream of the tandem repeats, for example 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides immediately upstream or downstream of tandem repeats, preferably into which at least one hydrophobic nucleotide has been inserted, as described herein. The helper sequence may consist of at least one, for example at least 1, for example 2, for example 3, for example 4, for example 5, for example 6, for example 7, for example 8, for example 9, for example 10, for example 10 up to 20, for example from 20 to 50, for example more than 50 nucleotides. For example, a helper sequence consists of 15 nucleotides or more. Thus, in some embodiments, the helper sequence consists of 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 19, 20 nucleotides or more, such as 25, 30, 35 , 40, 45 or 50 nucleotides or more. For example, a helper sequence consists of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or more. For example, a helper sequence consists of 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides or more, such as 9 nucleotides.

In embodiments wherein the reporter oligonucleotide is intended for use in detecting a microsatellite, such as BAT25, BAT26, NR21, NR22, NR24, or MON027, as described herein.

Intercalator

The term "intercalator" in the context of the present methods means any molecular moiety comprising at least one substantially planar conjugated system that is capable of cofolding with nucleic acid nitrogenous bases. The intercalator preferably consists essentially of at least one substantially planar conjugated system that is capable of cofolding with nucleic acid nitrogenous bases.

The intercalator includes at least one tg electron system, which, according to the present invention, can interact with other molecules including the tg electron system. These interactions can contribute positively or negatively to the hydrophobic interactions of these intercalators. Hunter and Sanders (1990) J, Am Chem. Soc. 112:5525-5534 have proposed a number of different orientations and conditions under which two systems of electrons can interact positively with each other.

The intercalator preferably includes a chemical group selected from the group consisting of polyaromatic and heteropolyaromatic compounds, and even more preferably the intercalator consists essentially of a polyaromatic or heteropolyaromatic compound. Most preferably, the intercalator is selected from the group consisting of polyaromatic and heteropolyaromatic compounds.

The polyaromatic and heteropolyaromatic compounds of the present invention may consist of any suitable number of rings, for example 1, for example 2, for example 3, for example 4, for example 5, for example 6, for example 7, for example 8, for example more than 8. Moreover, polyaromatic and heteropolyaromatic the compounds may be substituted with one or more substituents selected from the group consisting of hydroxyl, bromo-, fluoro-, chloro-, iodo-, mercapto-, thio-, cyano-, alkylthio-, heterocycle, aryl, heteroaryl, carboxyl, carboalkyl , alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl, carbonyl and amido.

Accordingly, intercalator Q may be, for example, an intercalator selected from the group consisting of phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, naphthene, phenanthrene, pikene, chrysene, naphthacene, acridones, benzanthracenes, stilbenes, oxalo-pyridocarbazoles, azidobenzenes, porphyrins, psoralens, and any of the above intercalators substituted with one or more substituents selected from the group consisting of hydroxyl, bromo-, fluoro-, chloro-, iodo-, mercapto-, thio-, cyano-, alkylthio-, heterocycle, aryl, heteroaryl, carboxyl, carboalkyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl, carbonyl and/or amido.

Preferably, the intercalator is selected from the group consisting of phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, naphthene, phenanthrene, picene, chrysene, naphthacene, acridones, benzanthracenes, stilbenes, oxalo-pyridocarbazoles, azidobenzenes, porphyrins and psoralens.

In a preferred embodiment, the intercalator is selected from the group consisting of phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, phenanthrene, chrysene, naphthacene, naphthacene, benzanthracenes, stilbenes, and porphyrins.

In another preferred embodiment, the intercalator includes pyrene or pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4(1H)-one or 7,9-dimethylpyrido[3', 2',4,5]thieno[3,2-d]pyrimidin-4(3H)-one. The intercalator may also consist of pyrene or pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4(1H)-one or 7,9-dimethylpyrido[3',2' ,4,5]thieno[3,2-d]pyrimidin-4(3H)-one.

The hydrophobic nucleotide may include other intercalators, in particular those described in WO 2017/045689 in the section entitled "intercalator", on page 30, line 26 to page 40, line 4, or in international application WO 03/052132 in the section "intercalator" on page 46, line 10 to page 54, line 13.

Framework monomer unit

X may also be a framework monomer unit that can be incorporated into a nucleic acid or nucleic acid analog framework. The term "scaffold monomer unit" of a nucleotide or nucleotide analog means herein the portion of the nucleotide that participates in the incorporation into the framework of a nucleic acid or nucleic acid analog. The framework monomer unit (X) is preferably covalently linked to a linker (Y), which is covalently linked to an intercalator. Any suitable scaffold monomer unit can be used to incorporate the intercalator into oligonucleotide analogues. Any type of linker linking said backbone monomer unit and said intercalator may also be used. In addition, a scaffold monomer unit may include one or more leaving groups, protecting groups, and/or reactive groups that may be removed or altered in any way during or after the synthesis of an oligonucleotide or oligonucleotide analogue comprising said scaffold monomer unit.

The framework monomer unit may be any suitable framework monomer unit. In one embodiment, the scaffold monomer unit may be selected, for example, from the group consisting of scaffold monomer units DNA, RNA, PNA, HNA, KHK(XNA), nucleic acid), MNA (MNA, morpholino-nucleic acid), ANA (ANA, arabino-nucleic acid), 3HK (LNA, blocked nucleic acid), CAN (CAN, cell-free nucleic acid), CNA (CeNA, cyclohexene-nucleic acid ), TNA (TNA, threose nucleic acid), (2'-NH)-TNA, (3'-NH)-TNA, α-L-ribo-LNA, α-L-xylo-LNA, β-D- xylo-LNA, α-D-ribo-LNA, [3.2.1]-LNA, bicyclo-DNA, 6-amino-bicyclo-DNA, 5-epi-bicyclo-DNA, α-bicyclo-DNA, tricyclo-DNA, bicyclo[4.3.0]-DNA, bicyclo[3.2.1]-DNA, bicyclo[4.3.0]amide-DNA, β-D-ribopyranosyl-nucleic acid, α-L-lyxopyranosyl-nucleic acid, 2'-R -PHK, α-L-PHK or α-D-PHK, β-D-RNA and mixtures thereof and hybrids thereof, as well as phosphorus atom modifications thereof, such as, but not limited to, phosphorothioates, methylphospholates, phosphoramidiates, phosphorodithiates, phosphoselenoates , phosphotriesters and phosphoborates. In addition, non-phosphorus compounds, such as, but not limited to, methyliminomethyl, formacetate, thioformacetate, and linking groups including amides, can be used to couple nucleotides.

A number of scaffold monomer units are described in international application WO 2017/045689 in the section entitled "Scaffold monomer unit", on page 40, line 5 to page 56, line 3 and in international application WO 03/052132 in the section entitled "Unit Scaffold Monomer" on page 24, line 27 to page 43, line 14. They also describe a number of different nucleotide scaffold monomer units and nucleotide analogues that can be used with the present invention, and how they are linked to nitrogenous bases by linkers that are attached at one or two positions of the backbone monomer unit.

Linker

An intercalator nucleotide linker is a moiety connecting an intercalator and a framework monomer of said hydrophobic nucleotide, preferably covalently connecting said intercalator and a framework monomer unit. The linker may include one or more atoms or bond(s) between atoms.

Based on the above definitions of framework and intercalator, a linker corresponds to the shortest distance connecting the framework and intercalators. If the intercalator is connected directly to the framework, then the linker is a bond.

The linker usually consists of a chain of atoms or a branched chain of atoms. The chains can be saturated or unsaturated. The linker may also be a ring structure with or without conjugated bonds.

Linkers that can be used are described in detail in the international application WO 2017/045689, in particular in the section entitled "Linker" on page 56, line 5 to page 59, line 10 and in WO 03/052132 in the section "Linker" on page 54, line 15 to page 58, line 7.

Kit

Also provided herein is a kit for detecting the presence of a sequence variant in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), preferably including repeats, wherein said target nucleic acid sequence consists of a variant sequence or a control sequences, wherein the specified set includes:

a) a reporter oligonucleotide comprising a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' -end,

wherein the reporter oligonucleotide is a sequence of 10 to 50 nucleotides in length, preferably 15 to 50 nucleotides, into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence H, and

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence; and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeat sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize with the first and second amplicons when the hybridization sequence hybridizes with them; And

b) a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid sequence.

There is also a set including:

a) a reporter oligonucleotide, which may be any of the reporter oligonucleotides described herein below under "Reporter Oligonucleotide"

b) a primer set consisting of a first primer and a second primer, which may be any of the primer sets described herein above in the "Amplification" section.

The kit is particularly useful in implementing the present methods.

Thus, provided herein is a kit for detecting the presence of a sequence variant in a two-strand nucleic acid target sequence comprising a nucleotide(s) of interest (NOI), wherein said target nucleic acid sequence consists of a sequence variant or a reference sequence, this set includes:

a) a reporter oligonucleotide comprising a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from 3' -end, wherein the reporter oligonucleotide is a sequence 10 to 50 in length into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence H,

wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And

in this case, the hydrophobic nucleotide has the structure

X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence including the control sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence; and b) a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid.

In addition to the specified reporter oligonucleotide and the specified set of primers, the kit may also include additional components. For example, the kit may further include PCR reagents. The kit may also include a detection probe, for example a probe capable of detecting the production of a PCR product in real time.

The above kits are particularly useful for implementing the methods described herein, in particular for detecting sequence variants of a target nucleic acid sequence, preferably including repeats, such as microsatellites. In particular, microsatellites where the wild type or control sequence is 15 nucleotides or more in length, as described in more detail above.

Reporter oligonucleotide

As described in detail above, the present methods require a reporter oligonucleotide to perform a melting assay, such as to perform an HRM assay. While not wishing to be bound by theory, we have found that reporter oligonucleotides comprising hydrophobic nucleotides are particularly useful in the present methods as they can increase assay sensitivity. Thus, similar reporter oligonucleotides are also described here.

Also provided herein is a reporter oligonucleotide that can hybridize to a single strand of a target nucleic acid consisting of two strands and comprising the nucleotide(s) of interest (NOI), preferably comprising repeats, wherein said reporter oligonucleotide includes a first fluorophore, preferably at its 5 '-end or at a distance of no more than 4 nucleotides from the 5'-end, and the first quencher, preferably at its 3'-end or at a distance of no more than 4 nucleotides from the 3'-end, wherein the reporter oligonucleotide is a sequence of length from 10 to 50, preferably from 15 to 50 nucleotides, into which from 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence H,

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid, and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeating sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize to the second strand of the first and second amplicons when the hybridization sequence hybridizes thereto.

Also provided herein is a reporter oligonucleotide comprising a first fluorophore, preferably at its 5' end or no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or no more than 4 nucleotides from the 3' end. '-end, wherein the reporter oligonucleotide is a sequence 10 to 50 in length into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence H, wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And

in this case, the hydrophobic nucleotide has the structure X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid, and wherein the hybridization sequence is complementary to a sequential fragment of the second strand sequence of the target nucleic acid.

The reporter oligonucleotide may include a second quencher. Preferably, the second quencher is a non-terminal region, that is, located in an internal region of the reporter oligonucleotide. In other words, the second quencher is not located at the 5' end or 3' end or within 4 nucleotides of the 5' end or 3' end.

The framework monomer unit X may be any of the framework monomer units described herein above in the "Scaffold Monomer Unit" section.

Intercalator Q may be any of the intercalators described herein above in the Intercalator section.

Hydrophobic nucleotides that can be used in the context of the present invention are described in detail in international application WO 2017/045689, in particular in the section entitled “Hydrophobic nucleotides” on page 30, lines 2-25.

In some embodiments, the reporter oligonucleotide has the following general structure

5'-(N) _a -Z-(N) _d -Z-(N) _e -Z-(N) _b -3' in this case

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

the total number of nucleotides or nucleotide analogues is at least 10; And

a and b individually represent integers in the range from 0 to 4; And

d and e individually represent integers ranging from 1 to 19; And

a+b+d+e is at least 10;

and (N) _a -(N) _d -(N) _e -(N) _b is identical to the control sequence.

In other embodiments, the reporter oligonucleotide has the following general structure

5'-(N) _a -Z-(N) _f -Z-(N) _g -Z-(N) _h -Z-(N) _b -3' in this case

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

a and b individually represent integers in the range from 0 to 4; And

f, g and h individually represent integers ranging from 1 to 18; And

a+b+f+g+h is at least 10 and does not exceed 50;

and (N)a-(N) _f -(N) _g -(N) _h -(N) _b is identical to a fragment of the target nucleic acid sequence including the control sequence; or

has the following general structure

5'-(N) _a -Z-(N) _i -Z-(N) _j -Z-(N) _k -Z-(N) _i -Z-(N) _b -3' in this case

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

a and b individually represent integers ranging from 0 to 4; And

i, j, k and I individually represent integers ranging from 1 to 17; And

a+b+i+j+k+I is at least 10 and does not exceed 50; and (N) _a -(N) _i -(N) _j -(N) _k -(N) _I -(N) _b is identical to a fragment of the target nucleic acid sequence including the control sequence; or

has the following general structure

5'-(N) _a -Z-(N) _m -Z-(N) _n -Z-(N) _o -Z-(N) _p -Z-(N) _q -Z-(N) _b - 3'

wherein

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1; And

a and b individually represent integers in the range from 0 to 4; And

m, n, o, p and q are individually integers ranging from 1 to 16; And

a+b+m+n+o+p+q is at least 10 and does not exceed 50; and (N) _a -(N) _m -(N) _n -(N) _o -(N) _p -Z-(N) _q -(N) _b is identical to a portion of the target nucleic acid sequence including the control sequence.

The reporter oligonucleotide may include an overhang, that is, it may include nucleotides at one of its ends that do not hybridize to the target nucleic acid sequence. The overhang may be 3'-terminal or 5'-terminal. Preferably, the reporter oligonucleotide includes a 5' terminal sequence that forms a 5' overhang relative to the strand of the first and second amplicon to which the hybridization sequence can hybridize.

The reporter oligonucleotide includes at least a first fluorophore and at least a first quencher. They are useful in terms of melting analysis and/or HRM analysis. Preferably, the reporter oligonucleotide includes a first fluorophore at least at its 5' end or at least at its 3' end, or at a distance of no more than 4 nucleotides from the 5' end or 3' end. The reporter oligonucleotide preferably includes a first quencher at at least its 5' end or at least its 3' end, or at a distance of no more than 4 nucleotides from the 5' end or 3' end. Thus, the fluorophore and quencher may be located at the 5' end or 3' end, or at a distance of no more than 4 nucleotides from the 5' end or 3' end, but are not necessarily located at the last nucleotide of the reporter oligonucleotide. Preferably, if the first fluorophore is located at the 5' end or at a distance of no more than 4 nucleotides from the 5' end, the first quencher is not located at the 5' end or at a distance of no more than 4 nucleotides from the 5' end. Instead, it is located at the 3' end or at a distance of no more than 4 nucleotides from the 3' end or in the internal region of the reporter. Conversely, if the first fluorophore is located at the 3' end or at a distance of no more than 4 nucleotides from the 3' end, then the first quencher is not located at the 3' end or at a distance of no more than 4 nucleotides from the 3' end. Instead, it is located at the 5' end or at a distance of no more than 4 nucleotides from the 5' end or in the internal region of the reporter. The term "internal region" refers here to a region of a reporter oligonucleotide that does not include the 5 terminal nucleotides at each end. Preferably, the first fluorophore and the first quencher are not located close to each other.

Suitable fluorophores and quenchers are readily available to those skilled in the art to select them for use in the present methods.

A reporter oligonucleotide is used to implement a melting assay, such as an HRM assay. Thus, the reporter nucleotide sequence is subject to certain restrictions that depend on the sequence that it hybridizes and detects. The reporter oligonucleotide includes a hybridization sequence H that, for example, is identical to the NOI and that hybridizes to a strand that is complementary to the NOI. Thus, within embodiments in which the NOI includes repeats, the reporter oligonucleotide also includes repeats, that is, the H hybridization sequence also includes repeats, as described in more detail below.

The reporter oligonucleotide may, in addition to a sequence that is identical to the NOI, also include additional nucleotides in the hybridization sequence. For example, as shown in the examples below, this may be particularly relevant in cases where the target nucleic acid is a microsatellite. If the microsatellite has M tandem repeats having a total length of n nucleotides in the control sequence, then the reporter oligonucleotide hybridization sequence preferably has M" tandem repeats, with M"≥M+1, preferably M"≥M+1 or M"≥M+2. If the tandem repeat is a mononucleotide repeat, then the hybridization sequence is n" nucleotides long, with n"≥n+1, preferably n"≥n+1 or n"≥n+2. In other words, the hybridization sequence may include at least one or two additional nucleotides.

This provides more sensitive discrimination between a sequence variant and a reference sequence, especially when the sequence variant includes an insertion.

In some embodiments, particularly where the NOI is a microsatellite, the reporter oligonucleotide hybridization sequence preferably includes a sequence that consists of repeats that are identical or complementary to the NOI repeats, and may usefully also include a terminal sequence, such as one or two terminal sequences that referred to herein as the helper sequence(s) that hybridize to the first and second amplicon immediately upstream or downstream of these repeats when the hybridization sequence hybridizes to them. In other words, the reporter oligonucleotide hybridization sequence preferably includes repeats and further includes from 1 to 20 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that hybridize to the NOI or its complementary strand immediately upstream or downstream of the repeat sequence.

The helper sequence can be 3'-terminal or 5'-terminal. Preferably, the reporter oligonucleotide includes a 5'-terminal helper sequence that hybridizes to a strand of the first and second amplicons immediately upstream or immediately downstream of these repeats when the hybridization sequence hybridizes to them. The helper sequence provides discrimination of melting profiles, as shown in Example 13. A helper sequence is designated as strong if it has a high T= _m when hybridizing with the first or second amplicon. It is well known to one skilled in the art that hybridization between two sequences is stronger if the sequences comprise a higher G/C ratio compared to two hybridized sequences comprising a higher A/T ratio; thus, a helper sequence comprising a higher G/C ratio than A/T will be more potent than a helper sequence comprising a lower G/C ratio than A/T. It is also known that longer sequences provide stronger hybridization than shorter sequences, so the strength of the helper sequence can be adjusted by increasing the length that hybridizes to the first and/or second amplicon.

In some embodiments, a reporter oligonucleotide can be used to detect a sequence variant indicative of microsatellite instability, that is, the control sequence includes a microsatellite sequence. When the control sequence includes a microsatellite sequence of M tandem repeats having a total length of n nucleotides, the length of the hybridization sequence H of the reporter oligonucleotide is n", wherein n"≥n+1, preferably n"≥n+1 or n"≥n +2.

The above reporter oligonucleotides may include a second quencher. Preferably, the second quencher is a non-terminal region, that is, located in an internal region of the reporter oligonucleotide. In other words, the second quencher is not located at the 5' end or at the 3' end or within 4 nucleotides of the 5' end or 3' end.

The fluorophores and quenchers that can be used are known in the art and are readily available to one skilled in the art to select suitable fluorophores and quenchers.

Also contemplated herein is the use of similar reporter oligonucleotides comprising hydrophobic nucleotides within methods for detecting a nucleic acid variant, particularly their use within the methods described herein. Reporter oligonucleotides can also be used as part of methods for predicting the effectiveness of a treatment for a clinical condition and as part of methods for predicting the presence of a clinical condition, particularly as described herein below.

Predicting the effectiveness of treatment for a clinical condition

The present invention also provides methods for predicting the effectiveness of treating a clinical condition in a subject in need thereof with a predetermined drug, wherein the effectiveness of treating said clinical condition with said drug is associated with the presence of a sequence variant.

Thus, certain mutations may indicate whether a certain drug may be effective in treating a subject. In particular, certain mutations may indicate a certain response to treatment with a predetermined drug. For example, the presence of a sequence variant may be indicative of whether a subject will respond favorably to treatment with a specified drug, the resistance of a subject's disease to treatment with a corresponding drug, or the ability/inability of a subject to tolerate treatment with a specific drug.

Methods for predicting the effectiveness of a treatment for a clinical condition in a subject may include the following steps:

a. obtaining a sample from a specified subject

b. implementing a method for detecting the presence of a sequence variant, as described herein, to determine whether a sample includes a sequence variant

wherein the presence of said sequence variant indicates whether said drug is effective in treating said clinical condition in said subject.

The clinical condition may be, for example, cancer, or any of the clinical conditions, diseases or disorders described herein. Many mutations have been identified that indicate whether a particular cancer drug or combination of drugs is effective in treating a particular type of cancer.

In some embodiments, the sequence variant is a microsatellite variant, as described in detail above.

Predicting the presence of a clinical condition

The present methods can also be used to predict or even diagnose the presence of any clinical condition associated with a particular mutation in a subject.

Such methods may include the following steps:

a) obtaining a sample from a specified subject

b) detecting the presence of a mutation associated with a clinical condition in a sample by performing the sequence variant detection methods described herein;

wherein the presence of said sequence variant indicates that said subject has said clinical condition.

It is known that many clinical conditions are associated with specific mutations, and reporter oligonucleotides and primer sets can be designed to detect any such mutation. The present methods are particularly useful for detecting conditions associated with microsatellite instability when they are used to detect a microsatellite sequence variant, preferably where the corresponding wild-type or control sequence is at least 15 nucleotides in length, as detailed above.

In one embodiment, the clinical condition is cancer, such as hereditary nonpolyposis colon cancer. In another embodiment, the clinical condition is Lynch syndrome.

The method may further include the step of treating said clinical condition. Upon detection of a sequence variant indicative of the presence of a clinical condition, the subject is classified as having said clinical condition, wherein the method may further comprise the step of administering an effective amount of a therapeutic agent to the subject.

Examples

Example 1: Microsatellite instability does not necessarily lead to changes in melting point.

BAT25 analysis using asymmetric PCR with primers that hybridize upstream and downstream of the region containing mononucleotide repeats. This assay is designed to detect sequence variants in the BAT25 microsatellite. The reporter oligonucleotide used in this example included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal quencher. The reporter oligonucleotide hybridizes to positions 142-171 of SEQ ID NO: 1 and has an overhang at its 3' end and a helper sequence at its 5' end that hybridizes upstream of the mononucleotide repeats. In addition, it includes a hydrophobic nucleotide no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide no more than 10 nucleotides from the 3' end. 1-2 ng/μl of normal tissue DNA and tumor tissue DNA from CRC or intrauterine samples from a patient with microsatellite instability were tested. The initial normalization interval was 39-40°C, and the final normalization interval was 63-64°C. Two melting curves were obtained (Figure 2A), which were transformed into first negative derivative curves (Figure 2B). These two curves were not identical (the maximum difference between the amplitudes of the two curves was -0.07 RFU (absolute value), not shown) when applying bilinear normalization and adjusting for temperature by applying a temperature shift intensity threshold of 0.1 RFU, which exceeds the threshold value 0.05 RFU. A difference of 0.07 exceeds the 0.05 RFU threshold established for this particular assay and thus classifies the sample as BAT25 unstable. The first negative derivative curves are shown in Fig. 2 B. As can be seen, normal tissue had a T _{m of} 57.31°C, and the tumor tissue had a T _m of 57.41°C. Such small differences are usually insignificant and may be due to different salt concentrations in the samples or instrument variability.

Based on this example, it can be concluded that the melting temperature does not necessarily change significantly from a normal tissue sample to a mutation-associated tumor tissue sample, but the shape of the melting curve does change, which is even more easily visually determined when applying bilinear normalization and temperature shift. Thus, normal and tumor tissue samples can be distinguished based on the different shapes of the HRM curves even with very small differences in plan

_Tm .

Example 2: Bilinear Normalization

NR22 analysis using asymmetric PCR with primers that hybridize upstream and downstream of the mononucleotide repeat region. This assay is designed to detect a sequence variant in the NR22 microsatellite. The reporter oligonucleotide used in this example included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The reporter oligonucleotide hybridizes to positions 143-172 of SEQ ID NO: 4 and has an overhang (2 nucleotides) at its 3' end and a helper sequence (9 nucleotides; resulting in an increase in T _m by 14.2°C) at its 5' end. end, which hybridizes above the mononucleotide repeats. In addition, it includes a hydrophobic nucleotide no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide no more than 10 nucleotides from the 3' end. 1-2 ng/μl DNA of FFPE purified normal and tumor tissue from a patient with microsatellite instability was tested. Two melting curves were obtained (Figure 3). In FIG. 3A shows the melting curve without applying bilinear normalization or temperature shift. In FIG. Figure 3B shows the melting curve after applying bilinear normalization (without temperature shift).

In FIG. 4. shows the difference in D _t in fluorescence between the control (fluorescence equal to zero at any temperature; a sample from healthy cells of the patient) and a sample of tumor tissue depending on temperature.

This is also called a difference plot or difference curve. The maximum difference, maxD _t , is measured as the absolute value of the maximum amplitude of the difference curve. Data are shown after applying standard normalization (Figure 4A) or after applying bilinear normalization (without temperature shift) (Figure 4B).

This example shows that bilinear normalization can compensate for the different decreases in fluorescence intensity observed before and after melting of different samples, and allows the HRM profiles to be differentiated between a normal (healthy) tissue sample and a tumor tissue sample.

Example 3: Bilinear normalization and temperature shift can reduce the difference between melting curves

NR24 analysis using asymmetric PCR. This assay is designed to detect a sequence variant in the NR24 microsatellite. The reporter oligonucleotide used in this example included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal quencher. The reporter oligonucleotide hybridizes to positions 158-187 of SEQ ID NO: 5 and has an overhang at its 3' end and a helper sequence (resulting in an increase in T _m by 14.8°C) at its 5' end, which hybridizes upstream of the mononucleotide repeats . In addition, it includes a hydrophobic nucleotide no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide no more than 10 nucleotides from the 3' end. 1-2 ng/μl of normal tissue DNA and tumor tissue DNA from one microsatellite stable patient were tested. Standard normalization was used with a starting interval of 44–45°C (set as a value of 1) and a final interval of 66–67°C (set as a value of 0). The results are shown in Fig. 5.

When bilinear normalization and temperature shift intensity thresholding were not applied, the difference between the curves was ∼0.04 RFU ( Figure 5A ). When applying bilinear normalization, but not temperature shift intensity thresholding, the difference between the curves was ∼0.02 RFU (Figure 5B). When applying bilinear normalization and a temperature shift intensity threshold of 0.1 RFU, the difference between the curves was ∼0.01 RFU (Figure 5C).

Based on this example, it can be concluded that bilinear normalization and temperature shift can reduce the differences between the melting curves of healthy and normal tissue samples from microsatellite stable patients. This reduces the risk of false positive results.

Example 4: Temperature Shift Intensity Threshold Can Provide Neutralization of Melting Temperature Changes Caused by Different Salt Concentrations in DNA Buffers

NR24 analysis using asymmetric PCR. This assay is designed to detect a sequence variant in the NR24 microsatellite. The reporter oligonucleotide used in this example included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The reporter oligonucleotide hybridizes to positions 158-187 of SEQ ID NO: 5 and has an overhang at its 3' end (2 nucleotides) and a helper sequence (resulting in an increase in T _m by 14.8°C) at its 5' end, which hybridizes above mononucleotide repeats. In addition, it includes a hydrophobic nucleotide no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide no more than 10 nucleotides from the 3' end. Normal tissue DNA was diluted from 40 ng/μl to 2 ng/μl in water and TE buffer, respectively. The initial normalization was carried out in the region of 44-45°C, and the final normalization was carried out in the region of 66-67°C. The results are shown in Fig. 6.

When applying bilinear normalization but not temperature shift, the maximum difference, maxD _t , between the curves was ∼0.07 RFU (Figure 6A). When applying bilinear normalization and temperature shift with an intensity threshold of 0.1 RFU, maxD _t was ~0.01 RFU (Fig. 6B).

Based on this example, it can be concluded that the use of a temperature shift can reduce the difference between two samples when the difference is caused by different salt concentrations in the DNA buffers. Thus, after applying bilinear normalization and temperature shift, aliquots of the same sample can provide identical HRM profiles, even if the aliquots were obtained using different buffers. This significantly reduces the risk of false positives because the assay is less vulnerable to varying salt and buffer concentrations between samples being compared.

Example 5: Temperature shift creates the ability to use one universal control sample

MON027 analysis using asymmetric PCR with primers that hybridize upstream and downstream of the mononucleotide repeat region. This assay is designed to detect sequence variants in the MON27 microsatellite. The reporter oligonucleotide used in this example included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The reporter oligonucleotide hybridizes to positions 300-333 of SEQ ID NO: 6 and has an overhang at its 3' end and a helper sequence (resulting in an increase in T _m by 10.8°C) at its 5' end, which hybridizes upstream of the mononucleotide repeats . In addition, it includes a hydrophobic nucleotide no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide no more than 10 nucleotides from the 3' end. 1-2 ng/μl DNA of normal FFPE purified tissue from 16 different patients was tested. The initial normalization interval was 44-45°C, and the final normalization interval was 66-67°C.

In FIG. Figure 7A shows HRM curves where bilinear normalization was applied, but no temperature shift was applied. The maximum difference maxD _T between the curves was ~0.25 RFU. In FIG. Figure 7B shows HRM curves where bilinear normalization and temperature shift were applied with an intensity threshold of 0.1 RFU, while maxDT was reduced to ~0.06 RFU.

In FIG. 8A and FIG. 8B shows the difference graphs of the HRM curves of FIG. 7A and Fig. 7V, respectively.

Based on this experiment, it can be concluded that the application of a temperature shift reduces the variability between healthy tissue samples from different subjects. This creates the opportunity to use one universal control sample instead of pairs of normal and tumor tissue samples for each patient.

Example 6: Asymmetric PCR produces more single-stranded amplicon NR22 analysis using asymmetric PCR with primers that hybridize upstream and downstream of the mononucleotide repeat region. This assay is designed to detect a sequence variant in the NR22 microsatellite. The reporter oligonucleotide used in this example included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The reporter oligonucleotide hybridizes to positions 142-172 of SEQ ID NO: 4 and has an overhang at its 3' end and a helper sequence (resulting in an increase in T _m by 14.2°C) at its 5' end, which hybridizes upstream of the mononucleotide repeats . In addition, it includes a hydrophobic nucleotide no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide no more than 10 nucleotides from the 3' end.

1-2 ng/μl of blood DNA from normal purified tissue was tested. The figure shows real-time PCR curves. The symmetric PCR curve stops increasing in terms of RFU after cycle 45, and for asymmetric PCR amplification continues after the exponential phase in the form of linear amplification.

Based on this experiment, it is clear that asymmetric PCR produces more of a single strand of DNA that includes a sequence to which the reporter oligonucleotide hybridization sequence can bind.

Example 7: Asymmetric PCR produces higher signal-to-noise ratio and sharper melting curves

This analysis was performed as described in Example 6. Normalized HRM curves were obtained and are shown in FIG. 10.

Based on this experiment, it can be concluded that asymmetric PCR provides a higher signal-to-noise ratio and a sharper melting profile.

Example 8: Asymmetric PCR facilitates discrimination between MSS and MSI patients.

The NR22 assay was performed as described in Example 6. 1-2 ng/μl DNA of FFPE purified normal and tumor tissue from a patient with microsatellite instability was tested.

In FIG. 11 shows HRM curves obtained after applying standard normalization with asymmetric PCR (Fig. 11A) or symmetric PCR (Fig. 11B). The corresponding difference curves are shown in Fig. 12A and FIG. 12V, respectively.

Based on this example, it can be concluded that amplification using asymmetric PCR creates a greater difference between the normal and tumor tissue curve for a patient with microsatellite instability compared to symmetric PCR. Thus, asymmetric PCR facilitates the discrimination between patients with MSS and MSI.

Example 9: One-way Overhang in a Reporter Oligonucleotide Can Lead to an Increase in Melting Temperature

NR22 analysis using asymmetric PCR with primers that hybridize upstream and downstream of the mononucleotide repeat region. This assay is designed to detect a sequence variant in the NR22 microsatellite.

Two different reporter oligopeptides were used in this experiment. The first reporter oligonucleotide used in this example included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end. The first reporter oligonucleotide hybridizes to positions 148-177 of SEQ ID NO: 4. The hybridization sequence also includes a 4 nucleotide long helper sequence (resulting in a 7.5°C increase in _Tm ) at the 5' end and a 5 nucleotide long helper sequence (resulting in to an increase in T _m by 3.6°C) at the 3' end, which are complementary to the NR22 regions immediately upstream and downstream of these repeats. The second reporter oligonucleotide included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal quencher. The second reporter oligonucleotide hybridizes to positions 143-172 of SEQ ID NO: 4 and has an overhang at its 3' end and a 9 nucleotide long helper sequence only at its 5' end (resulting in an increase in T _m by 14.2°C), which hybridizes above mononucleotide repeats. In addition, both reporter oligonucleotides include a hydrophobic nucleotide no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide no more than 10 nucleotides from the 3' end. 1-2 ng/μl of blood DNA from normal purified tissue was tested.

The results of the assay using the first reporter oligonucleotide are shown as dashed lines in FIG. 13; results using the second reporter oligonucleotide are shown as solid lines. The melting curves are shown in Fig. 13A, and the derivative curves are shown in FIG. 13V. Bilinear normalization and temperature shift were not applied.

It can be seen that the melting temperature for the second reporter oligonucleotide is higher than for the first reporter oligonucleotide. The lower background fluorescence for the second reporter oligonucleotide is likely due to the presence of an additional internal quencher.

Based on this experiment, it can be seen that having one longer helper sequence at one end instead of two shorter helper sequences at each end can increase the melting temperature of the probe and may be an advantage when analyzing mononucleotide repeat microsatellites.

Example 10: Double Quenching of Reporter Oligonucleotide Increases Signal-to-Noise Ratio

BAT26 analysis using asymmetric PCR with primers that hybridize upstream and downstream of the region containing mononucleotide repeats. This assay is designed to detect sequence variants in the BAT26 microsatellite. 1-2 ng/μl of blood DNA from normal purified tissue was tested. A: HRM curves using standard normalization.

In this example, two reporter oligonucleotides were used, both of which included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end. As an example of a single quench reporter oligonucleotide other than DQ, BAT26 reporter oligonucleotide FAM 5'-CZCCTTTTTTTTTTTTTTTTTTTTTTTTTTAZC-3' BHQ-1 (SEQ ID NO: 7) was used. BAT26 reporter oligonucleotide FAM 5'-CZCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTAZC-3' BHQ-1 (SEQ ID NO: 8) was used as an example of a double quenched reporter oligonucleotide, DQ; X denotes internal damper.

The reporter oligonucleotide hybridizes to positions 220-251 of SEQ ID NO: 2 and has an overhang at its 3' end and a helper sequence at both its 5' end (resulting in an increase in _Tm by 5.2°C) and at its 3'-end (leading to an increase in _Tt by 1.9°C), which hybridizes above and below the mononucleotide repeats. In addition, it includes a hydrophobic nucleotide no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide no more than 10 nucleotides from the 3' end.

HRM curves and first negative derivative curves are shown in Fig. 14A and B, respectively. It can be seen that the background fluorescence of the single-quenched reporter oligonucleotide (non-DQ probe) is higher compared to the double-quenched reporter oligonucleotide (DQ probe). Based on this experiment, it can be concluded that DQ reporter oligonucleotides reduce fluorescence noise and sharpen melting curves compared to single quenched reporter oligonucleotides. Thus, the use of double-quenched reporter oligonucleotides can discriminate between normal and tumor tissue samples.

Example 11: Additional nucleotide repeats in the hybridization sequence of the reporter oligonucleotide provide the ability to detect longer microsatellites NR21 analysis was performed using asymmetric PCR. This assay is designed to detect a sequence variant in the NR21 microsatellite. The reporter oligonucleotide used in this example included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal quencher. The reporter oligonucleotide hybridizes to positions 189-214 of SEQ ID NO: 2 and has an overhang at its 3' end and a helper sequence at its 5' end (resulting in an increase in T _m by 15.2°C) that hybridizes downstream of the mononucleotide repeats . In addition, the reporter oligonucleotide includes 2 hydrophobic nucleotides at a distance of no more than 5 nucleotides from the 5' end and one hydrophobic nucleotide at a distance of no more than 5 nucleotides from the 3' end. The reporter oligonucleotide hybridization sequence includes 22 T nucleotide repeats; The NR21 microsatellite consists of 21 repeats in most healthy cells. Thus, the reporter oligonucleotide has one extra T compared to the control sequence.

The reporter oligonucleotide was tested using artificial targets of 21, 22, or 23 adenine repeats.

In FIG. Figure 15 shows the HRM curves after applying bilinear normalization, but without temperature shift. Based on this experiment, it can be concluded that the addition of one additional repeat to the hybridization sequence in the reporter oligonucleotide provides detection of microsatellites having a length exceeding 21 control repeats.

Example 12: A point mutation results in a change in melting temperature of several degrees

KIT gene exon 13-based assay to detect a sequence variant in exon 13 of the KIT gene. PCR was performed as asymmetric PCR. The reporter oligonucleotide included a FAM fluorophore at its 5' end and a BHQ1 quencher at its 3' end. 1 ng/μl DNA of FFPE-purified tissue from a wild-type sample and FFPE-purified tumor tissue from a patient sample were tested. The initial normalization interval was 70-71°C, and the final normalization interval was 80-81°C. The results are shown in Fig. 16. Wild-type tissue provides T _m 79.1°C, and tumor tissue provides two T _m values: 75.7°C and 79.1°C.

Based on this experiment, it can be concluded that the melting temperature of the probe changes by up to 3.4°C, and that the shape of the melting curve changes significantly in the case of a single point mutation. In this way, samples of healthy and tumor tissue can be distinguished.

Example 13: A stronger single-terminal helper sequence increases the discrimination and allows discrimination between mutant and wild type NR24 analysis using asymmetric PCR with primers hybridizing upstream and downstream of the mononucleotide repeat region. This assay is designed to detect a sequence variant in the NR24 microsatellite.

Two different reporter oligopeptides were used in this experiment. The first reporter oligonucleotide included a FAM fluorophore at the 5' end and a BHQ-1 quencher at the 3' end, as well as an internal BHQ-1 quencher. The first reporter oligonucleotide hybridizes to positions 159-187 of SEQ ID NO: 5 and has an overhang at its 3' end (2 nucleotides) and a 5 nucleotide long helper sequence (resulting in an increase in T _m by 10.5°C) at its 5' -end, which hybridizes above the mononucleotide repeats. In addition, it includes two hydrophobic nucleotides at a distance of no more than 10 nucleotides from the 5' end and a hydrophobic nucleotide at a distance of no more than 10 nucleotides from the 3' end. The second reporter is described in Example 4.

1-2 ng/μl DNA of FFPE purified normal and tumor tissue from a patient with microsatellite instability was tested.

The initial normalization was carried out in the region of 40.5-41.5°C, and the final normalization was carried out in the region of 66-67°C. Bilinear normalization and temperature shift at 0.05 RFU were used. The results are shown in Fig. 17 and 18; see Fig. 17A and 18A (results using the first reporter oligonucleotide) and FIG. 17B and 18B (results using the second reporter oligonucleotide).

The second helper sequence, when hybridized, results in a higher increase in _Tm (14.8°C, ie 4.3°C higher) than the first helper sequence, which helps distinguish between the two melting profiles.

It can be seen that a stronger helper sequence increases the maxD _t of the mutant. The first reporter oligonucleotide with a weak helper sequence provided a maxD _t value of ~0.03 RFU between normal and tumor tissue. This value is below the threshold for the NR24 marker and the mutant will therefore be incorrectly classified as wild type (FIG. 18A). A second reporter oligonucleotide with a stronger helper sequence provided a maxD _T value of ~0.17, and thus the sample was correctly classified as a mutant (Figure 18B).

Based on this experiment, it can be concluded that a stronger helper sequence will increase the discrimination between wild type and mutants, which may be useful in the analysis of mononucleotide repeat microsatellites.

Links

Boland et al. (1998) "A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer," Cancer Res 58:5248-5257 Rodriguez-Bigas et al. (1997) "A National Cancer Institute workshop on hereditary nonpolyposis colorectal cancer syndrome: meeting highlights and Bethesda guidelines," J Natl Cancer Inst 89:1758-1762 Hunter and Sanders (1990) J, Am Chem. Soc. 112: 5525-5534 WO 2017/045689 WO 03/052132

Items

1. A method for detecting the presence of a sequence variant in a target nucleic acid sequence consisting of two strands and comprising a nucleotide(s) of interest (NOI), wherein said target nucleic acid sequence consists of a sequence variant or a control sequence, wherein said method includes the following steps:

a) Obtaining a first sample comprising nucleic acids believed to include the specified sequence variant;

b) Obtaining a second sample comprising nucleic acids comprising said control sequence, wherein the second sample is a control sample;

c) Preparation of a reporter oligonucleotide;

d) Providing a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid sequence;

e) Amplifying a target nucleic acid sequence in the presence of said first sample, said first primer and said second primer, thereby producing a first amplicon comprising nucleic acids believed to comprise the sequence variant; and amplifying a target nucleic acid sequence in the presence of said second sample, said first primer and said second primer, thereby providing a second amplicon including a control sequence, wherein the second amplicon is a control amplicon;

f) Performing a high-sensitivity melting curve analysis (HRM) of the first amplicon, thereby obtaining a first HRM profile characterized by a first melting curve, and performing an HRM analysis of a second amplicon, thereby obtaining a second HRM profile characterized by a second melting curve, wherein the second HRM the profile is a control profile characterized by a control melting curve; wherein each amplicon includes a first strand and a second strand, wherein the HRM analysis includes hybridizing a reporter oligonucleotide to one strand of each amplicon, detecting a signal emitted by a fluorophore, and obtaining a first and second melting curve;

wherein the reporter oligonucleotide is a sequence from 10 to 50 in length, into which from 2 to 10 hydrophobic nucleotides were inserted,

wherein the reporter oligonucleotide includes a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' end end, and wherein the reporter oligonucleotide includes a hybridization sequence H, wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And

in this case, the hydrophobic nucleotide has the structure X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence; And

g) Comparing the first HRM profile with a control HRM profile, with the difference between the first HRM profile and the control HRM profile indicating that the first sample contains a sequence variant.

2. The method according to claim 1, wherein the NOI includes repeats, and wherein the hybridization sequence of the reporter oligonucleotide consists of a repeat sequence and a helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize to the first or second strand of the first and second amplicon when the hybridization sequence hybridizes thereto, preferably the reporter oligonucleotide consisting of a repeat sequence and only one helper sequence at its 5' end or at its 3' end .

3. The method according to claim 2, wherein the helper sequence includes or consists of from 1 to 20 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 or 20 nucleotides, preferably the helper sequence includes at least one hydrophobic oligonucleotide as specified in paragraph 1.

4. The method according to any one of the previous paragraphs, wherein at least one hydrophobic nucleotide as specified in paragraph 1, for example 1, 2, or 3 hydrophobic nucleotides, is inserted at a distance of no more than 1 to 10 nucleotides from the 3'- end of the reporter oligonucleotide, for example at a distance of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides from the 3' end.

5. The method according to any one of the previous paragraphs, wherein at least one hydrophobic nucleotide as specified in paragraph 1, for example 1, 2, or 3 hydrophobic nucleotides, is inserted at a distance of no more than 1 to 10 nucleotides from the 5'- end of the reporter oligonucleotide, for example at a distance of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides from the 5' end.

6. The method according to any one of the previous claims, wherein the plurality of target nucleic acid sequences consists of BAT25 and BAT26, preferably the plurality consists of BAT25 according to SEQ ID NO: 1 and BAT26 according to SEQ ID NO: 2.

7. The method according to any one of the preceding claims, wherein the target nucleic acid sequence is one or more nucleic acid sequences including one or more of BAT25, BAT26, NR21, NR22, NR24 and MON027, preferably BAT25 according to SEQ ID NO: 1, BAT26 as per SEQ ID NO: 2, NR21 as per SEQ ID NO: 3, NR22 as per SEQ ID NO: 4, NR24 as per SEQ ID NO: 5 and MON027 as per SEQ ID NO: 6.

8. The method according to any one of the previous claims, wherein the target nucleic acids are a plurality of target nucleic acid sequences consisting of BAT25, BAT26, NR21, NR22 and NR24, preferably the plurality consists of BAT25 according to SEQ ID NO:1, BAT26 as per SEQ ID NO: 2, NR21 as per SEQ ID NO: 3, NR22 as per SEQ ID NO: 4, and NR24 as per SEQ ID NO: 5.

9. The method according to any one of the previous claims, wherein the target nucleic acids are a plurality of target nucleic acid sequences consisting of BAT25, BAT26, NR22, NR24 and MON027, preferably the plurality consists of BAT25 according to SEQ ID NO:1, BAT26 as per SEQ ID NO: 2, NR22 as per SEQ ID NO: 4, NR24 as per SEQ ID NO: 5 and MON027 as per SEQ ID NO: 6.

10. The method according to any one of the previous claims, wherein the target nucleic acid sequences are a plurality of target nucleic acid sequences consisting of BAT25, BAT26, NR21, NR22, NR24 and MON027, preferably the plurality of target nucleic acid sequences consisting of BAT25 according to SEQ ID NO:1, BAT26 as per SEQ ID NO: 2, NR21 as per SEQ ID NO: 3, NR22 as per SEQ ID NO: 4, NR24 as per SEQ ID NO: 5 and MON027 as per SEQ ID NO: 6.

11. The method according to any one of the previous claims, wherein the amplification in step e) is carried out using a polymerase chain reaction (PCR), preferably using an asymmetric PCR in which the first and second primers are present in different amounts, thereby directing the PCR to amplification in more of the second strand of each amplicon than the first strand of each amplicon.

12. The method of any one of the preceding claims, wherein the first sample is obtained from a subject suffering or suspected of suffering from a disease, such as cancer, wherein the cancer is preferably hereditary non-polyposis colon cancer.

13. The method according to any one of the previous claims, wherein the NOI is a microsatellite.

14. The method according to any one of the previous claims, wherein the control sequence includes a microsatellite sequence of M tandem repeats having a total length of n nucleotides, and wherein the sequence variant has M' tandem repeats having a total length of n' nucleotides, wherein M and M' represent distinct integers.

15. The method according to any one of the previous paragraphs, wherein the length of the hybridization sequence H of the reporter oligonucleotide is n", wherein n"≥n+1, preferably n"≥n+1 or n"≥n+2.

16. The method of any one of the preceding claims, wherein the reporter oligonucleotide includes a second quencher located at a non-terminal position of the reporter oligonucleotide.

17. The method according to any one of the previous claims, wherein the first sample is a tissue sample comprising or suspected of containing cells with mutations characteristic of the specified disease.

18. The method of any one of the preceding claims, wherein the control sample is obtained from the same subject as the first sample, optionally from non-diseased tissue, or wherein the control sample is obtained from a healthy subject.

19. The method according to any one of the previous claims, wherein step f) includes the step of transforming the first and second HRM melting curves to obtain a first negative derivative of the first and second melting curves, wherein the difference between the first HRM profile and the reference HRM profile is the difference between the first negative derivatives of the first and second melting curves.

20. The method of any one of the preceding claims, wherein the fluorescence of the first melting curve and the fluorescence of the second melting curve are normalized, and wherein the difference between the HRM profiles is the difference between the fluorescence of said first and second melting curve as a function of temperature.

21. The method of any one of the preceding claims, wherein the difference between the first HRM profile and the control HRM profile is measured as the absolute maximum difference in relative fluorescence units within the upper and lower normalization region between the first and second HRM curve.

22. The method according to any one of the previous paragraphs, wherein the HRM analysis includes the step of bilinear normalization of the first and second melting curves.

23. The method according to any one of the preceding claims, wherein the HRM analysis includes the step of applying a temperature correction at a certain fluorescence intensity to the first and second melting curves and/or the first negative derivatives of the first and second melting curves.

24. The method of any one of the preceding claims, wherein the second primer includes a sequence of at least 15 nucleotides in length that is complementary to a contiguous sequence of the target nucleic acid sequence.

25. The method of any one of the preceding claims, wherein the sequence variant includes or consists of an insertion of one or more nucleotides relative to the reference sequence.

26. The method of any one of the preceding claims, wherein the sequence variant includes or consists of a deletion of one or more nucleotides compared to the reference sequence.

27. The method according to any one of the previous paragraphs, wherein the reporter oligonucleotide has the following general structure

5'-(N) _a -Z-(N) _d -Z-(N) _e -Z-(N) _b -3' in this case

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

the total number of nucleotides or nucleotide analogues is at least 10; And

a and b individually represent integers in the range from 0 to 4; And

d and e individually represent integers ranging from 1 to 19; And

a+b+d+e is at least 10; And

(N) _a -(N) _d -(N) _e -(N) _b is identical to the control sequence.

28. The method according to any one of the previous paragraphs, wherein the reporter oligonucleotide has the following general structure:

5'-(N) _a -Z-(N) _f -Z-(N) _g -Z-(N) _h -Z-(N) _b -3' in this case

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

a and b individually represent integers in the range from 0 to 4; And

f, g and h individually represent integers ranging from 1 to 18; And

a+b+f+g+h is at least 10 and does not exceed 50; and (N) _a -(N) _f -(N) _g -(N) _h -(N) _b is identical to a fragment of a target nucleic acid sequence including a control sequence; or

has the following general structure

5'-(N) _a -Z-(N) _i -Z-(N) _j -Z-(N) _k -Z-(N) _I -Z-(N) _b -3' in this case

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

a and b individually represent integers in the range from 0 to 4; And

i, j, k and I individually represent integers ranging from 1 to 17; And

a+b+i+j+k+I is at least 10 and does not exceed 50; and (N) _a -(N) _i -(N) _j -(N) _k -(N)r(N)b is identical to a fragment of a target nucleic acid sequence including a control sequence; or

has the following general structure

5'-(N) _a -Z-(N) _m -Z-(N) _n -Z-(N) ₀ -Z-(N) _p -Z-(N) _q -Z-(N) _b - 3'

wherein

N is any nucleotide or nucleotide analogue; And

Z is a hydrophobic nucleotide as defined in paragraph 1;

And

a and b individually represent integers in the range from 0 to 4; And

m, n, o, p and q are individually integers ranging from 1 to 16; And

a+b+m+n+o+p+q is at least 10 and does not exceed 50; and (N) _a -(N) _m -(N) _n -(N) _o -(N) _p -Z-(N) _q -(N) _b is identical to a portion of the target nucleic acid sequence including the control sequence.

29. The method according to any one of the previous claims, wherein at least one intercalator, Q, is selected from the group consisting of polyaromatic and heteropolyaromatic compounds, optionally substituted with one or more substituents selected from the group consisting of hydroxyl, bromo- , fluoro-, chloro-, iodo-, mercapto-, thio-, cyano-, alkylthio-, heterocycle, aryl, heteroaryl, carboxyl, carboalkyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl, carbonyl and amido.

30. The method according to any one of the previous paragraphs, wherein the intercalator is selected from the group consisting of benzene, pentalene, indene, naphthalene, azulene, as-indacene, s-indacene, biphenylene, acenaphthylene, phenalene, heptalene, phenanthrane, fluoranthene, phenanthroline , phenazine, phenanthridine, anthraquinone, pyrene, anthracene, naphthene, phenanthrene, flurene, pickene, chrysene, naphthacene, acridones, benzanthracenes, stilbenes, oxalo-pyridocarbazenes, azidobenzenes, porphyrins and psoralenes and their derivatives.

31. The method according to any one of the previous claims, wherein at least one, for example all of the X framework monomer units are phosphoramidite.

32. The method according to any one of the previous claims, wherein at least one linker, Y, includes a chain of x atoms selected from the group consisting of C, O, S, N and P, optionally wherein said chain is replaced by one or several substituents selected from the group consisting of C, H, O, S, N and P.

33. The method of any one of the preceding claims, wherein the target nucleic acid sequence is a plurality of target nucleic acid sequences.

34. A kit for detecting the presence of a sequence variant in a two-strand nucleic acid target sequence comprising a nucleotide(s) of interest (NOI), wherein said target nucleic acid sequence consists of a sequence variant or a control sequence, wherein said set includes:

a) a reporter oligonucleotide comprising a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' -end, wherein the reporter oligonucleotide is a sequence 10 to 50 in length into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes a hybridization sequence H,

wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And

in this case, the hydrophobic nucleotide has the structure

X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid, and wherein the hybridization sequence is complementary to a sequential fragment of the second strand sequence of the target nucleic acid;

and b) a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid.

35. The set according to paragraph 34, wherein the reporter oligonucleotide and the first primer and second primer are defined in any one of the previous paragraphs.

36. A reporter oligonucleotide that can hybridize to one strand of a target nucleic acid, wherein said reporter oligonucleotide includes a first fluorophore, preferably at its 5' end or at a distance of no more than 4 nucleotides from the 5' end, and a first quencher, preferably at its 3' end or at a distance of no more than 4 nucleotides from the 3' end, wherein the reporter oligonucleotide is a sequence 10 to 50 in length into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes the sequence hybridization H,

wherein

at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or

at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And

in this case, the hydrophobic nucleotide has the structure X-Y-Q

wherein

X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue,

Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And

Y is a linker moiety linking said nucleotide or nucleotide analogue or framework monomer unit and said intercalator; And

wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid, and wherein the hybridization sequence is complementary to a sequential fragment of the second strand sequence of the target nucleic acid.

37. The reporter oligonucleotide of claim 36, wherein the NOI includes repeats, and wherein the hybridization sequence of the reporter oligonucleotide consists of a repeat sequence and a helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence is not includes repeats and can hybridize to the second strand of the target nucleic acid when the hybridization sequence hybridizes thereto, preferably the reporter oligonucleotide consists of a repeat sequence and only one helper sequence at its 5' end or at its 3' end.

38. The reporter oligonucleotide according to any one of paragraphs 36-37, wherein the helper sequence includes or consists of from 1 to 20 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides, preferably the helper sequence includes at least one hydrophobic oligonucleotide as specified in paragraph 1.

39. The reporter oligonucleotide according to any one of paragraphs 36-38, wherein the control sequence includes a microsatellite sequence of M tandem repeats having a total length of n nucleotides, and wherein the length of the hybridization sequence H of the reporter oligonucleotide is n", wherein n"≥ n+1, preferably n"≥n+1 or n"≥n+2.

40. The reporter oligonucleotide according to any one of paragraphs 36-39, wherein the reporter oligonucleotide includes a second quencher located at a non-terminal position of the reporter oligonucleotide.

41. A method of predicting the effectiveness of treating a clinical condition in a subject in need thereof with a predetermined drug, wherein the effectiveness of treating said clinical condition with said drug is associated with the presence of a sequence variant, wherein the method includes the following steps:

a. obtaining a sample from a specified subject

b. implementing the method of any one of paragraphs 1-33 for determining the presence of the specified sequence variant;

wherein the presence of said sequence variant indicates whether said drug is effective in treating said clinical condition in said subject.

42. A method for predicting the presence of a clinical condition in a subject in need thereof, wherein said clinical condition is associated with the presence of a target nucleic acid sequence including a sequence variant, wherein said method includes the following steps:

a. obtaining a sample from a specified subject

b. implementing the method of any one of paragraphs 1-33 for determining the presence of a sequence variant;

wherein the presence of the sequence variant indicates that the specified subject has the specified clinical condition.

43. The method according to any one of claims 41-42, wherein the clinical condition is cancer, preferably hereditary non-polyposis colon cancer.

44. The method of any one of claims 41-43, further comprising the step of administering an effective amount of the therapeutic agent to said subject.

Claims (80)

1. A method for detecting the presence of a sequence variant in a target nucleic acid sequence consisting of two strands and including a nucleotide(s) of interest (NOI), where the NOI is a microsatellite having a microsatellite sequence of M tandem repeats having a total length of n nucleotides, and wherein the sequence variant has M' tandem repeats having a total length of n' nucleotides, wherein M and M' are distinct integers, wherein n is 15 or more, and wherein said target nucleic acid sequence consists of the variant sequence or control sequence, wherein said method includes the following steps: a) Obtaining a first sample comprising nucleic acids believed to include the specified sequence variant; b) Obtaining a second sample comprising nucleic acids comprising said control sequence, wherein the second sample is a control sample; c) Preparation of a reporter oligonucleotide; d) Providing a primer set consisting of a first primer and a second primer, wherein the primers of the set together are capable of amplifying a target nucleic acid sequence including an NOI; e) Amplifying a target nucleic acid sequence in the presence of said first sample, said first primer and said second primer, thereby producing a first amplicon comprising nucleic acids believed to comprise the sequence variant; and amplifying a target nucleic acid sequence in the presence of said second sample, said first primer and said second primer, thereby providing a second amplicon including a control sequence, wherein the second amplicon is a control amplicon; f) Performing a melting analysis, such as high sensitivity melting curve analysis (HRM), on the first amplicon, thereby obtaining a first profile characterized by the first melting curve, and performing a melting analysis, such as HRM analysis, on the second amplicon, thereby obtaining a second profile characterized by a second melting curve, wherein the second profile is a control profile characterized by a control melting curve; wherein each amplicon includes a first strand and a second strand, wherein the melting assay includes hybridizing a reporter oligonucleotide to one strand of each amplicon, detecting a signal emitted by a fluorophore, and obtaining a first and second melting curve; wherein the reporter oligonucleotide is a sequence from 16 to 50 nucleotides in length, into which from 2 to 10 hydrophobic nucleotides were inserted, wherein the reporter oligonucleotide includes a first fluorophore and a first quencher, and wherein the reporter oligonucleotide includes the hybridization sequence H, wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence, and where the length of the hybridization sequence H of the reporter oligonucleotide is n'', wherein n'' ≥ n + 1 or n'' ≥ n + 2; and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeat sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize with the first and second amplicons when the hybridization sequence hybridizes with them; And g) Comparing the first profile with a control profile, with the difference between the first profile and the control profile indicating that the first sample contains a sequence variant. 2. The method according to claim 1, characterized in that step g) includes or consists of the following steps: i) aligning the first and second melting curves at a certain fluorescence intensity along the temperature axis, thereby eliminating differences in terms of melting temperature between the first and second melting curves; ii) determining the difference in the signal emitted by the fluorophore between the first and second melting curve, preferably the difference being a quantitative difference; And iii) comparing the difference determined in step ii) with a threshold value, wherein a difference exceeding the threshold value indicates that the first sample includes a sequence variant, and a difference not exceeding the threshold value indicates that the first sample includes a reference sequence. 3. The method according to any one of the previous claims, characterized in that the control sequence is 15 nucleotides or more in length, for example 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides or more. 4. A method according to any one of the preceding claims, characterized in that the reporter oligonucleotide consists of a repeat sequence and only one helper sequence at its 5' end or at its 3' end, or wherein the reporter oligonucleotide consists of a repeat sequence and two helper sequences, for example one helper sequence at both the 5' end and the 3' end of the reporter oligonucleotide. 5. The method according to any one of the previous paragraphs, characterized in that at least one of said hydrophobic nucleotides is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or at least one hydrophobic nucleotide is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And in this case, the hydrophobic nucleotide has the structure X-Y-Q, wherein X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue, Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And Y is a linker moiety linking said nucleotide or nucleotide analogue or scaffold monomer unit and said intercalator. 6. The method according to any one of the previous claims, characterized in that the helper sequence includes or consists of from 1 to 20 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides. 7. The method according to any one of the previous claims, characterized in that the helper sequence includes at least one hydrophobic nucleotide as specified in claim 5. 8. The method according to any one of the previous claims, characterized in that at least one hydrophobic nucleotide as specified in claim 5, for example 1, 2, or 3 hydrophobic nucleotides, is inserted at a distance of no more than 1 to 10 nucleotides from the 3' end of the reporter oligonucleotide, for example at a distance of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides from the 3' end. 9. The method according to any one of the previous claims, characterized in that at least one hydrophobic nucleotide as specified in claim 5, for example 1, 2, or 3 hydrophobic nucleotides, is inserted at a distance of no more than 1 to 10 nucleotides from the 5' end of the reporter oligonucleotide, for example at a distance of no more than 1, 2, 5, 4, 5, 6, 7, 8, 9 or 10 nucleotides from the 5' end. 10. Method according to any one of the previous claims, characterized in that the amplification in step e) is carried out using polymerase chain reaction (PCR), preferably using asymmetric PCR, in which the first and second primers are present in different amounts, thereby directing the PCR to amplify more of one strand of each amplicon than the other strand of each amplicon. 11. The method of any one of the preceding claims, wherein the first sample is obtained from a subject suffering or suspected of suffering from a disease, such as cancer, the cancer preferably being hereditary non-polyposis colon cancer, or a disorder, preferably a disorder associated with microsatellite instability. 12. The method of any one of the preceding claims, wherein the first sample is a tissue sample comprising or suspected of containing cells with mutations characteristic of the disease or disorder. 13. A kit for detecting the presence of a sequence variant in a two-stranded nucleic acid target sequence comprising a nucleotide(s) of interest (NOI), wherein the NOI is a microsatellite having a microsatellite sequence of M tandem repeats having a total length of n nucleotides wherein the sequence variant has M' tandem repeats having a total length of n' nucleotides, wherein M and M' are distinct integers, wherein n is 15 or more, wherein said target nucleic acid sequence consists of the variant sequence or control sequence, wherein said set includes: a) a reporter oligonucleotide including a first fluorophore and a first quencher, wherein the reporter oligonucleotide is a sequence of 16 to 50 nucleotides in length into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes an H hybridization sequence, And wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid sequence, and wherein the hybridization sequence is complementary to the sequential fragment of the second strand sequence of the target nucleic acid sequence; And where the length of the hybridization sequence H of the reporter oligonucleotide is n'', wherein n'' ≥ n + 1 or n'' ≥ n + 2; and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeat sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize with the first and second amplicons when the hybridization sequence hybridizes with them; And b) a primer set consisting of a first primer and a second primer, wherein the primers of the set are collectively capable of amplifying a target nucleic acid sequence. 14. The kit according to claim 13, preferably characterized in that the reporter oligonucleotide and the first primer and the second primer correspond to the definition in any one of claims. 1-12. 15. Set according to any one of paragraphs. 13, 14, characterized in that the reporter oligonucleotide consists of a repeat sequence and only one helper sequence at its 5' end or at its 3' end, or characterized in that the reporter oligonucleotide consists of a repeat sequence and two helper sequences, for example one helper sequence at both the 5' and 3' ends of the reporter oligonucleotide. 16. Set according to any one of paragraphs. 13-15, characterized in that at least one hydrophobic nucleotide is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or at least one of said hydrophobic nucleotides is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And in this case, the hydrophobic nucleotide has the structure X-Y-Q, wherein X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue, Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And Y is a linker moiety linking said nucleotide or nucleotide analogue or scaffold monomer unit and said intercalator. 17. A reporter oligonucleotide that can hybridize to a single strand of a target nucleic acid consisting of two strands and comprising a nucleotide of interest (NOI), where the NOI is a microsatellite, for example a microsatellite sequence of M tandem repeats having a total length of n nucleotides, wherein the sequence variant has M' tandem repeats having a total length of n' nucleotides, wherein M and M' are distinct integers, wherein n is 15 or more, wherein said reporter oligonucleotide includes a first fluorophore, and a first quencher, wherein the reporter oligonucleotide is a sequence of 16 to 50 nucleotides in length into which 2 to 10 hydrophobic nucleotides have been inserted, and wherein the reporter oligonucleotide includes an H hybridization sequence, wherein the hybridization sequence is identical to a sequential fragment of the first strand sequence of the target nucleic acid, and wherein the hybridization sequence is complementary to a sequential fragment of the second strand sequence of the target nucleic acid, and where the length of the hybridization sequence H of the reporter oligonucleotide is n'', wherein n'' ≥ n + 1 or n'' ≥ n + 2; and wherein the hybridization sequence of the reporter oligonucleotide includes or consists of a repeat sequence and at least one helper sequence at its 5' end and/or at its 3' end, wherein said helper sequence does not include repeats and can hybridize with the second strand of the first and second amplicons when the hybridization sequence hybridizes with them. 18. The reporter oligonucleotide of claim 17, wherein the reporter oligonucleotide includes a first fluorophore at its 5' end or at a distance of no more than 4 nucleotides from the 5' end. 19. The reporter oligonucleotide of claim 17, wherein the reporter oligonucleotide includes a first quencher at its 3' end or at a distance of no more than 4 nucleotides from the 3' end. 20. Reporter oligonucleotide according to any one of paragraphs. 17-19, characterized in that the reporter oligonucleotide consists of a repeat sequence and only one helper sequence at its 5' end or at its 3' end, or characterized in that the reporter oligonucleotide consists of a repeat sequence and two helper sequences, for example one helper sequence at both the 5' and 3' ends of the reporter oligonucleotide. 21. Reporter oligonucleotide according to any one of paragraphs. 17-20, characterized in that at least one of said hydrophobic nucleotides is located at the 5' end or at a distance of no more than 10 nucleotides from the 5' end of the reporter oligonucleotide; and/or at least one of said hydrophobic nucleotides is located at the 3' end or at a distance of no more than 10 nucleotides from the 3' end of the reporter oligonucleotide; And in this case, the hydrophobic nucleotide has the structure X-Y-Q, wherein X is a nucleotide or nucleotide analogue or a framework monomer unit capable of being incorporated into the framework of a nucleic acid or nucleic acid analogue, Q is an intercalator that does not participate in the Watson-Crick hydrogen bond; And Y is a linker moiety linking said nucleotide or nucleotide analogue or scaffold monomer unit and said intercalator. 22. Reporter oligonucleotide according to any one of paragraphs. 17-21, characterized in that the helper sequence includes or consists of from 1 to 20 nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20 nucleotides, preferably the helper sequence includes at least one hydrophobic nucleotide, as specified in paragraph 5. 23. A method of predicting the effectiveness of treating a clinical condition in a subject in need thereof with a predetermined drug, wherein the effectiveness of treating said clinical condition with said drug is associated with the presence of a sequence variant, wherein the method includes the following steps: a) obtaining a sample from a specified subject b) implementation of the method according to any one of paragraphs. 1-12 to determine the presence of the specified sequence variant; wherein the presence of said sequence variant indicates whether said drug is effective in treating said clinical condition in said subject. 24. A method of predicting the presence of a clinical condition in a subject in need thereof, wherein said clinical condition is associated with the presence of a target nucleic acid sequence including a sequence variant, wherein said method includes the following steps: a) obtaining a sample from a specified subject; b) implementation of the method according to any one of paragraphs. 1-12 to determine the presence of a sequence variant; wherein the presence of the sequence variant indicates that the specified subject has the specified clinical condition.