[go: up one dir, main page]

WO2025027080A1 - Utilisation d'amorces longues à queue en 5' pour améliorer les performances d'amplification lors du ciblage de sites de liaison d'amorce courts - Google Patents

Utilisation d'amorces longues à queue en 5' pour améliorer les performances d'amplification lors du ciblage de sites de liaison d'amorce courts Download PDF

Info

Publication number
WO2025027080A1
WO2025027080A1 PCT/EP2024/071686 EP2024071686W WO2025027080A1 WO 2025027080 A1 WO2025027080 A1 WO 2025027080A1 EP 2024071686 W EP2024071686 W EP 2024071686W WO 2025027080 A1 WO2025027080 A1 WO 2025027080A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
primer
long
target nucleic
primers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/071686
Other languages
English (en)
Inventor
Brian Christopher Godwin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Roche Sequencing Solutions Inc
Original Assignee
F Hoffmann La Roche AG
Roche Sequencing Solutions Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG, Roche Sequencing Solutions Inc filed Critical F Hoffmann La Roche AG
Publication of WO2025027080A1 publication Critical patent/WO2025027080A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/155—Modifications characterised by incorporating/generating a new priming site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/161—Modifications characterised by incorporating target specific and non-target specific sites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/204—Modifications characterised by specific length of the oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/107—Temperature of melting, i.e. Tm

Definitions

  • the present disclosure relates to the field of in vitro diagnostics and nucleic acid analysis.
  • the present invention concerns: (a) the amplification of a target nucleic acid and/or (b) the amplification and detection of a target nucleic acid that may be present in a sample.
  • the present invention is directed to overcoming the problem of poor amplification performance of a target nucleic acid, where the target nucleic acid has short primer-binding sites. This problem is overcome by employing longer primers, where the longer primers have one region that anneals/hybridizes to the target nucleic acid, and one region that does not anneal/hybridize to the target nucleic acid.
  • Short target primer binding sites e.g. 15 bases
  • PCR polymerase chain reactions
  • ⁇ 55°C annealing temperature
  • this poor annealing per cycle impacts final yield of amplification product in an exponential manner, due to the doubling nature per cycle of PCR and multiple rounds of cycling.
  • the initial cycles of PCR will be different than the subsequent cycles.
  • the initial cycle of PCR using longer forward primers will occur while using only the shortened matching primer binding sites of the longer forward primers.
  • the 3’ end of the long forward primer will anneal to the corresponding portion of the template (z.e., matching), whereas the 5’ end will not anneal to anything (z.e., nonmatching), and the first extension product generated will contain the non-matching 5’ tail of the forward primer, the 3’ matching end of the forward primer, the reverse complement of the initial template, and the short reverse primer-binding site.
  • the template is the primer extension product generated from the first PCR cycle. That is, the template for the second cycle of PCR will contain the non-matching 5’ tail of the forward primer, the 3’ matching end of the forward primer, the reverse complement of the initial template, and the short reverse primer-binding site.
  • the product of the second PCR cycle is the template nucleic acid flanked by, now, primer-binding sites for: (a) the full-length longer forward primer, and (b) the full-length longer reverse primer.
  • the template is the primer extension product generated from the 2nd PCR cycle. That is, the template for the third cycle of PCR will contain the template nucleic acids flanked by the primer binding sites for: (a) the full-length longer forward primer, and (b) the full-length longer reverse primer. That is, the template for the third PCR cycle will contain a full-length primer binding site (for example > 18 bases long), with a typical PCR annealing temperature (> 55°C).
  • a full-length primer binding site for example > 18 bases long
  • a typical PCR annealing temperature > 55°C
  • the long forward primer anneals/matches completely to the long forward primer-binding site, and generates a primer extension product that is the template nucleic acid flanked by primer-binding sites for: (a) the full-length longer forward primer, and (b) the full-length longer reverse primer.
  • subsequent PCR cycles should also employ templates having primer-binding sites that accommodate/anneal to the longer forward primers and the longer reverse primers, thereby eliminating the problems of short primer-binding sites stemming from the original template.
  • extension can occur in the two directions (z.e., (a) extension of the longer forward primer, and (b) extension of the template).
  • PCR Polymerase Chain Reaction
  • Other amplification techniques include Ligase Chain Reaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3 SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and QP-amplification.
  • SDA Strand Displacement Amplification
  • TMA Transcription Mediated Amplification
  • QP-amplification QP-amplification
  • Short target primer binding sites e.g. 15 bases
  • PCR polymerase chain reactions
  • ⁇ 55°C annealing temperature
  • This is due to the low potential of primer annealing events to occur during typical thermal-cycling temperatures (> 55°C).
  • this poor annealing per cycle impacts final yield of amplification product in an exponential manner, due to the doubling nature per cycle of PCR and multiple rounds of cycling.
  • the methods disclosed, herein, describe a method for overcoming the problems of short target primer-binding sites, by employing longer forward primers and longer reverse primers.
  • Certain embodiments in the present disclosure relate to methods for the amplification and/or detection of the presence or absence of a template/target nucleic acid, by a polymerase chain reaction (PCR), using longer forward primers and longer reverse primers.
  • Embodiments include methods of amplification and/or detection of template/target nucleic acids, comprising performing at least one cycling step, which may include an amplifying step and a hybridizing step, using longer forward primers and longer reverse primers.
  • embodiments include primers, probes, and kits that are designed for the amplification and/or detection template/target nucleic acids.
  • One embodiment is directed to a method for amplifying a target nucleic acid, wherein the method comprises the following steps: (a) obtaining one or more forward primers and one or more reverse primers, wherein the forward primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid, wherein the reverse primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid; and (b) performing one or more amplification steps, wherein the amplification step comprises contacting the target nucleic acid with the one or more forward primers and one or more reverse primers, in the presence of a polymerase, to produce one or more amplification products.
  • the region of the forward primer that is complementary to the target nucleic acid is ⁇ 20 base pairs long. In another embodiment, the region of the forward primer that is complementary to the target nucleic acid is ⁇ 15 base pairs long. In another embodiment, the region of the forward primer that is complementary to the target nucleic acid is ⁇ 10 base pairs long. In another embodiment, the region of the reverse primer that is complementary to the target nucleic acid is ⁇ 20 base pairs long. In another embodiment, the region of the reverse primer that is complementary to the target nucleic acid is ⁇ 15 base pairs long. In one embodiment, the region of the reverse primer that is complementary to the target nucleic acid is ⁇ 10 base pairs long. In one embodiment, the forward primer is > 17 base pairs long.
  • the forward primer is > 20 base pairs long. In one embodiment, the forward primer is > 25 base pairs long. In another embedment, the reverse primer is > 17 base pairs long. In another embodiment, the reverse primer is > 20 base pairs long. In another embodiment, the reverse primer is > 25 base pairs long. In another embodiment, the target nucleic acid is between 110-130 base pairs long. In one embodiment, the target nucleic acid about 120 base pairs long.
  • Another embodiment is directed to a method for detecting a target nucleic acid in a sample, wherein the method comprises the following steps: (a) obtaining one or more forward primers and one or more reverse primers, wherein the forward primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid, wherein the reverse primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid; and (b) performing one or more amplification steps, wherein the amplification step comprises contacting the sample with the one or more forward primers and one or more reverse primers, in the presence of a polymerase, to produce one or more amplification products, if the target nucleic acid is present in the sample; (c) performing a hybridization step, wherein the hybridization step comprises contacting one or more probes with the one or more amplification products from step (b); and (
  • the region of the forward primer that is complementary to the target nucleic acid is ⁇ 20 base pairs long. In another embodiment, the region of the forward primer that is complementary to the target nucleic acid is ⁇ 15 base pairs long. In another embodiment, the region of the forward primer that is complementary to the target nucleic acid is ⁇ 10 base pairs long. In another embodiment, the region of the reverse primer that is complementary to the target nucleic acid is ⁇ 20 base pairs long. In another embodiment, the region of the reverse primer that is complementary to the target nucleic acid is ⁇ 15 base pairs long. In one embodiment, the region of the reverse primer that is complementary to the target nucleic acid is ⁇ 10 base pairs long. In one embodiment, the forward primer is > 17 base pairs long.
  • the forward primer is > 20 base pairs long. In one embodiment, the forward primer is > 25 base pairs long. In another embedment, the reverse primer is > 17 base pairs long. In another embodiment, the reverse primer is > 20 base pairs long. In another embodiment, the reverse primer is > 25 base pairs long. In another embodiment, the target nucleic acid is between 110-130 base pairs long. In one embodiment, the target nucleic acid about 120 base pairs long.
  • an oligonucleotide comprising or consisting of a sequence of nucleotides selected from SEQ ID NOs:l-4, or complements thereof, which oligonucleotide has 100 or fewer nucleotides.
  • the present disclosure provides an oligonucleotide that includes a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) to one of SEQ ID NOs:l-4, or a complement thereof, which oligonucleotide has 100 or fewer nucleotides.
  • these oligonucleotides may be primer nucleic acids, probe nucleic acids, or the like in these embodiments.
  • the oligonucleotides have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides, 20 or fewer nucleotides, 15 or fewer nucleotides, etc.).
  • the oligonucleotides comprise at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability relative to unmodified nucleotides.
  • the oligonucleotides comprise at least one label and optionally at least one quencher moiety.
  • the oligonucleotides include at least one conservatively modified variation.
  • “Conservatively modified variations” or, simply, “conservative variations” of a particular nucleic acid sequence refers to those nucleic acids, which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • One of skill in the art will recognize that individual substitutions, deletions or additions which alter, add or delete a single nucleotide or a small percentage of nucleotides (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are “conservatively modified variations” where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.
  • amplification can employ a polymerase enzyme having 5’ to 3’ nuclease activity.
  • the donor fluorescent moiety and the acceptor moiety e.g., a quencher
  • the probe includes a nucleic acid sequence that permits secondary structure formation. Such secondary structure formation may result in spatial proximity between the first and second fluorescent moiety.
  • the second fluorescent moiety on the probe can be a quencher.
  • the present disclosure also provides for methods of detecting the presence or absence of template/target nucleic acids.
  • Such methods generally include performing at least one cycling step, which includes an amplifying step and a dyebinding step.
  • the amplifying step includes contacting the sample with a plurality of pairs of oligonucleotide primers to produce one or more amplification products if a nucleic acid molecule is present in the sample
  • the dye-binding step includes contacting the amplification product with a double-stranded DNA binding dye.
  • Such methods also include detecting the presence or absence of binding of the double-stranded DNA binding dye into the amplification product, wherein the presence of binding is indicative of the presence of template/target nucleic acids in the sample, and wherein the absence of binding is indicative of the absence of template/target nucleic acids in the sample.
  • a representative double-stranded DNA binding dye is ethidium bromide.
  • Other nucleic acid-binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YOYO® and SYBR® Green.
  • such methods also can include determining the melting temperature between the amplification product and the double-stranded DNA binding dye, wherein the melting temperature confirms the presence or absence of the template/target nucleic acid.
  • kits for detecting and/or quantitating one or more nucleic acids of template/target nucleic acids can include one or more sets of primers specific for amplification of the gene target; and one or more detectable oligonucleotide probes specific for detection of the amplification products.
  • the kit can include probes already labeled with donor and corresponding acceptor moieties, e.g., another fluorescent moiety or a dark quencher, or can include fluorophoric moieties for labeling the probes.
  • the kit can also include nucleoside triphosphates, nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase.
  • the kit can also include a package insert and instructions for using the primers, probes, and fluorophoric moieties to detect the presence or absence of the template/target nucleic acid.
  • FIG. 1A shows alignment of an exemplary long forward primer (SEQ ID NO: 1), and the short version of the primer-binding site for the long forward primer (SEQ ID NO:2).
  • FIG. IB shows alignment of an exemplary long forward primer (SEQ ID NO: 1), and the long version of the primer-binding site for the long forward primer (SEQ ID NO:3).
  • FIG. 2A shows alignment of an exemplary long reverse primer (SEQ ID NO:4), and the short version of the primer-binding site for the long reverse primer (SEQ ID NO:5).
  • FIG. 2B shows alignment of an exemplary long forward primer (SEQ ID NO:4), and the long version of the primer-binding site for the long reverse primer (SEQ ID NO:6).
  • FIG. 3A shows a first PCR cycle, using a long forward primer (LFP) annealing to a short forward primer-binding site (SFPBS) of a template nucleic acid/insert that also contains a template reverse primer-binding site (TRPBS).
  • LFP long forward primer
  • SFPBS short forward primer-binding site
  • TRPBS template reverse primer-binding site
  • FIG. 3B shows a second PCR cycle, using a long reverse primer (LRP) annealing to a short reverse primer-binding site (SRPBS) of the primer extension product from the first PCR cycle, which also contains a long forward primer (LFP).
  • LRP long reverse primer
  • SRPBS short reverse primer-binding site
  • LFP long forward primer
  • FIG. 3C shows a third PCR cycle, using a long forward primer (LFP) annealing to a long forward primer-binding site (LFPBS) of the primer extension product from the second PCR cycle, which also contains a long reverse primer (LRP).
  • LFP long forward primer
  • LFPBS long forward primer-binding site
  • LRPBS long reverse primer
  • FIG. 4 shows additional priming that may occur when using long forward primers (LFP) and/or long reverse primers (LRP).
  • LFP long forward primers
  • LRP long reverse primers
  • the non- hybridizing/unmatched region of the long forward primer can act as a template, and the template/PCR insert can act as a primer.
  • the long forward primer (LFP) also acts as a primer and extends (as shown in FIG. 3A).
  • FIG. 6 shows simultaneous priming and extension reactions in two directions.
  • Described herein is a method for amplifying a template/target nucleic acid, by using long forward primers and long reverse primers.
  • Use of these long forward primers and long reverse primers overcome the situation or problem of when the primer-binding region is short (e.g., around 15 bases). In such cases, short primerbinding sites then result in lower inherent annealing temperatures ( ⁇ 55°C), which make it challenging for conventional primers to prime.
  • the primerbinding site is increased and is no longer short, which results in higher annealing temperatures (> 55°C), which improves PCR yields.
  • the long forward primers and the long reverse primers have a region that is complementary to the target nucleic acid (z.e., matched), and a region that is not complementary to the target nucleic acid (ie., unmatched). Therefore, the long forward primers and the long reverse primers are considered to be “long” or “longer,” by virtue of having regions that do not hybridize/anneal to the target nucleic acid, which appear as single-stranded overhang regions.
  • the melting temperature (T m ) is lower.
  • the template/target nucleic acid will lengthen, due to addition of the unmatched regions of the long forward primer and the long reverse primer. Accordingly, during later cycles of PCR, the length of the primer-binding region matches the length of the long forward/reverse primers, and the T m is higher, allowing for more efficient cycling and PCR.
  • this method is useful for amplification of template/target nucleic acid and/or for detecting a template/target nucleic acid in a sample by nucleic acid amplification, thereby providing a method for rapidly, accurately, reliably, specifically, and sensitively detecting and/or quantitating a template/target nucleic acid.
  • Primers where the oligonucleotide primers are long forward primers and long reverse primers
  • probes for amplification and/or detecting of a template/target nucleic acid are provided, as are articles of manufacture or kits containing such primers (where the oligonucleotide primers are long forward primers and long reverse primers) and/or probes.
  • the present disclosure includes oligonucleotide primers (where the oligonucleotide primers are long forward primers and long reverse primers) and/or fluorescent labeled hydrolysis probes that hybridize to template/target nucleic acid.
  • the disclosed methods may include performing at least one cycling step that includes amplifying one or more portions of the nucleic acid molecule gene target/template using one or more pairs of long forward primers and long reverse primers (where the oligonucleotide primers are long forward primers and long reverse primers).
  • Each of the discussed long primers anneals to a template/target nucleic acid, such that at least a portion of each amplification product contains nucleic acid sequence corresponding to the template/target nucleic acid.
  • Each cycling step includes an amplification step, and a hybridization step.
  • amplifying refers to the process of synthesizing nucleic acid molecules that are complementary to one or both strands of a template nucleic acid molecule (e.g., nucleic acid molecules from the Plasmodium genome). Amplifying a nucleic acid molecule typically includes denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature that is below the melting temperatures of the primers, and enzymatically elongating from the primers to generate an amplification product.
  • Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme (e.g., MgCh and/or KC1).
  • a DNA polymerase enzyme e.g., Platinum® Taq
  • an appropriate buffer and/or co-factors for optimal activity of the polymerase enzyme e.g., MgCh and/or KC1.
  • oligonucleotide refers to oligomeric compounds, primarily to oligonucleotides but also to modified oligonucleotides that are able to “prime” DNA synthesis by a template-dependent DNA polymerase, z.e., the 3’-end of the, e.g., oligonucleotide provides a free 3’-OH group where further "nucleotides” may be attached by a template-dependent DNA polymerase establishing 3’ to 5’ phosphodiester linkage whereby deoxynucleoside triphosphates are used and whereby pyrophosphate is released.
  • hybridizing refers to the annealing of one or more probes to an amplification product.
  • Hybridization conditions typically include a temperature that is below the melting temperature of the probes but that avoids non-specific hybridization of the probes.
  • nuclease activity refers to an activity of a nucleic acid polymerase, typically associated with the nucleic acid strand synthesis, whereby nucleotides are removed from the 5’ end of nucleic acid strand.
  • thermostable polymerase refers to a polymerase enzyme that is heat stable, z.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of doublestranded template nucleic acids. Generally, the synthesis is initiated at the 3’ end of each primer and proceeds in the 5’ to 3’ direction along the template strand.
  • Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR assays provided the enzyme is replenished, if necessary.
  • nucleic acid that is both the same length as, and exactly complementary to, a given nucleic acid.
  • nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase that typically adds nucleotides at the 3’ terminal end of a nucleic acid.
  • a nucleotide incorporating biocatalyst such as a polymerase that typically adds nucleotides at the 3’ terminal end of a nucleic acid.
  • nucleic acid sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection.
  • sequence comparison algorithms available to persons of skill or by visual inspection.
  • Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) “Basic local alignment search tool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification of protein coding regions by database similarity search” Nature Genet.
  • modified nucleotide in the context of an oligonucleotide refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced by a different nucleotide that provides a desired property to the oligonucleotide.
  • Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, e.g., a t-butyl benzyl, a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5- propynyl-dU, a C7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, a C7-propargylamino-dA, a C7-propargylamino-dG, a 7- deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU
  • modified nucleotide substitutions modify melting temperatures (Tm) of the oligonucleotides relative to the melting temperatures of corresponding unmodified oligonucleotides.
  • Tm melting temperatures
  • certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation or the like), increase the yield of an intended target amplicon, and/or the like in some embodiments. Examples of these types of nucleic acid modifications are described in, e.g., U.S. Patent No. 6,001,611, which is incorporated herein by reference.
  • Other modified nucleotide substitutions may alter the stability of the oligonucleotide, or provide other desirable features. Amplification and/or Detection of Template/Tar et Nucleic Acid
  • the present disclosure provides methods to amplify a template/target nucleic acid. Specifically, long primers and/or long primers and probes to amplify and detect and/or quantitate template and/or target nucleic acids are provided by the embodiments in the present disclosure.
  • long primers and/or long primers and probes to amplify and detect/quantitate the template/target nucleic acids are provided.
  • Long primer nucleic acids other than those exemplified herein can also be used to amplify template/target nucleic acid.
  • functional variants can be evaluated for specificity and/or sensitivity by those of skill in the art using routine methods.
  • Representative functional variants can include, e.g., one or more deletions, insertions, and/or substitutions in the long primer nucleic acids disclosed herein.
  • oligonucleotides a substantially identical variant thereof in which the variant has at least, e.g., 80%, 90%, or 95% sequence identity to one of the sequences of long primers.
  • the above-described sets of long primers and/or long primers and probes are used in order to provide for amplification and/or detection of a template/target nucleic acid. In one embodiment, the above described sets of long primers and/or long primers and probes are used in order to provide for amplification and/or detection of a template/target nucleic acid.
  • variants of the long primers and/or the long primers and probe may be employed.
  • the variants may vary from sequences by one or more nucleotide additions, deletions or substitutions such as one or more nucleotide additions, deletions or substitutions at the 5’ end and/or the 3’ end of a sequence.
  • a long primer and/or probe may be chemically modified, z.e., a long primer and/or probe may comprise a modified nucleotide or a non-nucleotide compound.
  • a probe (or a long primer) is then a modified oligonucleotide.
  • Modified nucleotides differ from a natural “nucleotide” by some modification but still consist of a base or base-like compound, a pentofuranosyl sugar or a pentofuranosyl sugar-like compound, a phosphate portion or phosphate-like portion, or combinations thereof.
  • a “label” may be attached to the base portion of a “nucleotide” whereby a “modified nucleotide” is obtained.
  • a natural base in a “nucleotide” may also be replaced by, e.g., a 7- desazapurine whereby a “modified nucleotide” is obtained as well.
  • modified nucleotide or “nucleotide analog” are used interchangeably in the present application.
  • a “modified nucleoside” (or “nucleoside analog”) differs from a natural nucleoside by some modification in the manner as outlined above for a “modified nucleotide” (or a “nucleotide analog”).
  • Oligonucleotides including modified oligonucleotides and oligonucleotide analogs that detect and amplify a given template/target nucleic acid can be designed using, for example, a computer program such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.).
  • oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of a pair of primers, and the length of each primer (z.e., the primers need to be long enough to anneal with sequence-specificity and to initiate synthesis but not so long that fidelity is reduced during oligonucleotide synthesis).
  • the methods may use one or more probes in order to detect and amplify a template/target nucleic acid.
  • probe refers to synthetically or biologically produced nucleic acids (DNA or RNA), which by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies specifically (z.e., preferentially) to “target nucleic acids”, in the present case to a Plasmodium (target) nucleic acid.
  • a “probe” can be referred to as a “detection probe” meaning that it detects the target nucleic acid.
  • the described probes can be labeled with at least one fluorescent label.
  • the probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher.
  • oligonucleotides to be used as probes can be performed in a manner similar to the design of primers.
  • Embodiments may use a single probe or a pair of probes for detection of the amplification product.
  • the probe(s) use may comprise at least one label and/or at least one quencher moiety.
  • the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur but not so long that fidelity is reduced during synthesis.
  • Oligonucleotide probes are generally 15 to 40 (e.g., 15, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, or 40) nucleotides in length.
  • Constructs can include vectors each containing one of long primers and/or probes nucleic acid molecules. Constructs can be used, for example, as control template nucleic acid molecules. Vectors suitable for use are commercially available and/or produced by recombinant nucleic acid technology methods routine in the art.
  • Constructs suitable for use in the methods typically include long primers and/or probes for amplification and/or detection of a template/target nucleic acid, sequences encoding a selectable marker (e.g., an antibiotic resistance gene) for selecting desired constructs and/or transformants, and an origin of replication.
  • a selectable marker e.g., an antibiotic resistance gene
  • the choice of vector systems usually depends upon several factors, including, but not limited to, the choice of host cells, replication efficiency, selectability, inducibility, and the ease of recovery.
  • Constructs containing long primers and/or probes for amplification and/or detection of a template/target nucleic acid can be propagated in a host cell.
  • the term host cell is meant to include prokaryotes and eukaryotes such as yeast, plant and animal cells.
  • Prokaryotic hosts may include E. coh. Salmonella typhimurium, Serratia marcescens. and Bacillus subtilis.
  • Eukaryotic hosts include yeasts such as S. cerevisiae. S. pombe. Pichia pasloris. mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum.
  • a construct can be introduced into a host cell using any of the techniques commonly known to those of ordinary skill in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral -mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells.
  • naked DNA can be delivered directly to cells (see, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466).
  • PCR Polymerase Chain Reaction
  • PCR typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA).
  • Primers useful in some embodiments include oligonucleotides capable of acting as points of initiation of nucleic acid synthesis.
  • a primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically.
  • the primer is preferably single-stranded for maximum efficiency in amplification, but the primer can be double-stranded.
  • Double-stranded primers are first denatured, i.e., treated to separate the strands.
  • One method of denaturing double stranded nucleic acids is by heating.
  • Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means.
  • One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured).
  • the heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90°C to about 105°C for a time depending on features of the reaction such as temperature and the nucleic acid length. Denaturation is typically performed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5 min).
  • the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence.
  • the temperature for annealing is usually from about 35°C to about 65°C (e.g., about 40°C to about 60°C; about 45°C to about 50°C).
  • Annealing times can be from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec; about 30 sec to about 40 sec).
  • the reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for extension to occur from the annealed primer to generate products complementary to the template nucleic acid.
  • the temperature should be sufficient to synthesize an extension product from each primer that is annealed to a nucleic acid template, but should not be so high as to denature an extension product from its complementary template (e.g., the temperature for extension generally ranges from about 40°C to about 80°C (e.g., about 50°C to about 70°C; about 60°C). Extension times can be from about 10 sec to about 5 min (e.g., about 30 sec to about 4 min; about 1 min to about 3 min; about 1 min 30 sec to about 2 min).
  • RNA Ribonucleic acid
  • cDNA complementary DNA
  • Reverse transcriptases use an RNA template and a short primer complementary to the 3’ end of the RNA to direct synthesis of the first strand cDNA, which can then be used directly as a template for polymerase chain reaction.
  • PCR assays can employ Plasmodium nucleic acid, and/or primers/probes that amplify and/or detect Plasmodium, such as RNA or DNA (cDNA).
  • the template nucleic acid need not be purified; it may be a minor fraction of a complex mixture, such as Plasmodium nucleic acid contained in human cells.
  • Plasmodium nucleic acid molecules, and/or primers/probes that amplify and/or detect Plasmodium may be extracted from a biological sample by routine techniques such as those described in Diagnostic Molecular Microbiology. Principles and Applications (Persing, el al. (eds), 1993, American Society for Microbiology, Washington D.C.).
  • Nucleic acids can be obtained from any number of sources, such as plasmids, or natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.
  • the oligonucleotide long primers are combined with PCR reagents under reaction conditions that induce primer extension.
  • chain extension reactions generally include 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 15 mM MgCh, 0.001% (w/v) gelatin, 0.5-1.0 pg denatured template DNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO).
  • the reactions usually contain 150 to 320 pM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.
  • the newly-synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction.
  • the steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity of amplification products corresponding to the template/target nucleic acid molecules.
  • the limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction.
  • the cycling steps (z.e., denaturation, annealing, and extension) are preferably repeated at least once. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficient for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.
  • FRET Fluorescence Resonance Energy Transfer
  • FRET technology is based on a concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer takes place between the two fluorescent moieties that can be visualized or otherwise detected and/or quantitated.
  • the donor typically transfers the energy to the acceptor when the donor is excited by light radiation with a suitable wavelength.
  • the acceptor typically re-emits the transferred energy in the form of light radiation with a different wavelength.
  • non-fluorescent energy can be transferred between donor and acceptor moieties, by way of biomolecules that include substantially non-fluorescent donor moieties (see, for example, US Patent. No. 7,741,467).
  • an oligonucleotide probe can contain a donor fluorescent moiety or dye (e.g., HEX or FAM) and a corresponding quencher (e.g, BlackHole QuencherTM (BHQ) (such as BHQ-2)), which may or not be fluorescent, and which dissipates the transferred energy in a form other than light.
  • a donor fluorescent moiety or dye e.g., HEX or FAM
  • BHQ BlackHole QuencherTM
  • BHQ BlackHole Quencher
  • a probe bound to an amplification product is cleaved by the 5’ to 3’ nuclease activity of, e.g., a Taq Polymerase such that the fluorescent emission of the donor fluorescent moiety is no longer quenched.
  • a Taq Polymerase e.g., a Taq Polymerase
  • Exemplary probes for this purpose are described in, e.g., U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785.
  • Commonly used donor-acceptor pairs include the FAM-TAMRA pair.
  • Commonly used quenchers are DABCYL and TAMRA.
  • BlackHole QuencherTM (BHQ) (such as BHQ2), (Biosearch Technologies, Inc., Novato, Cal.), Iowa BlackTM, (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerryTM Quencher 650 (BBQ- 650), (Berry & Assoc., Dexter, Mich.).
  • two oligonucleotide probes each containing a fluorescent moiety, can hybridize to an amplification product at particular positions determined by the complementarity of the oligonucleotide probes to the Plasmodium target nucleic acid sequence.
  • a FRET signal is generated.
  • Hybridization temperatures can range from about 35°C. to about 65°C. for about 10 sec to about 1 min.
  • Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system, or a fluorimeter.
  • Excitation to initiate energy transfer, or to allow direct detection of a fluorophore can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a xenon lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range.
  • Hg high intensity mercury
  • corresponding refers to an acceptor fluorescent moiety or a dark quencher having an absorbance spectrum that overlaps the emission spectrum of the donor fluorescent moiety.
  • the wavelength maximum of the emission spectrum of the acceptor fluorescent moiety should be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Accordingly, efficient non-radiative energy transfer can be produced therebetween.
  • Fluorescent donor and corresponding acceptor moieties are generally chosen for (a) high efficiency Foerster energy transfer; (b) a large final Stokes shift (>100 nm); (c) shift of the emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) shift of the emission to a higher wavelength than the Raman water fluorescent emission produced by excitation at the donor excitation wavelength.
  • a donor fluorescent moiety can be chosen that has its excitation maximum near a laser line (for example, helium-cadmium 442 nm or Argon 488 nm), a high extinction coefficient, a high quantum yield, and a good overlap of its fluorescent emission with the excitation spectrum of the corresponding acceptor fluorescent moiety.
  • a corresponding acceptor fluorescent moiety can be chosen that has a high extinction coefficient, a high quantum yield, a good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red part of the visible spectrum (>600 nm).
  • Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4- acetamido-4’-isothio-cyanatostilbene-2, 2’ -disulfonic acid, 7-diethylamino-3-(4’- isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1 -pyrenebutyrate, and 4- acetamido-4’-isothiocyanatostilbene-2,2’-disulfonic acid derivatives.
  • acceptor fluorescent moieties depending upon the donor fluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of Lanthanide ions (e.g., Europium, or Terbium).
  • Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).
  • the donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm.
  • the length of each linker arm is important, as the linker arms will affect the distance between the donor and acceptor fluorescent moieties.
  • the length of a linker arm can be the distance in Angstroms (A) from the nucleotide base to the fluorescent moiety. In general, a linker arm is from about 10 A to about 25 A.
  • the linker arm may be of the kind described in International Patent Publication No. WO 84/03285.
  • WO 84/03285 also discloses methods for attaching linker arms to a particular nucleotide base, and also for attaching fluorescent moieties to a linker arm.
  • An acceptor fluorescent moiety such as an LC Red 640
  • an oligonucleotide that contains an amino linker e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, VA)
  • an amino linker e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, VA)
  • linkers to couple a donor fluorescent moiety such as fluorescein to an oligonucleotide include thiourea linkers (FITC-derived, for example, fluorescein- CPG's from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3’-amino-CPGs that require coupling of a fluorescein-NHS-ester after oligonucleotide synthesis.
  • FITC-derived for example, fluorescein- CPG's from Glen Research or ChemGene (Ashland, Mass.)
  • amide-linkers fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex (San Ramon, Calif.)
  • 3’-amino-CPGs that require coupling
  • the present disclosure provides methods for amplifying and detecting the presence or absence of template/target nucleic acid. Methods provided avoid problems of sample contamination, false negatives, and false positives.
  • the methods include performing at least one cycling step that includes amplifying a portion of target nucleic acid molecules using one or more pairs of long primers, and a FRET detecting step. Multiple cycling steps are performed, preferably in a thermocycler. Methods can be performed using the long primers and probes to amplify and/or detect a template/target nucleic acid.
  • amplification products can be detected using labeled hybridization probes that take advantage of FRET technology.
  • FRET format utilizes TaqMan® technology to detect the presence or absence of an amplification product, and hence, the presence or absence of the template/target nucleic acid.
  • TaqMan® technology utilizes one single- stranded hybridization probe labeled with, e.g., one fluorescent moiety or dye (e.g., HEX or FAM) and one quencher (e.g., BHQ-2), which may or may not be fluorescent.
  • one fluorescent moiety or dye e.g., HEX or FAM
  • quencher e.g., BHQ-2
  • the second moiety is generally a quencher molecule.
  • the labeled hybridization probe binds to the target DNA (z.e., the amplification product) and is degraded by the 5’ to 3’ nuclease activity of, e.g., the Taq Polymerase during the subsequent elongation phase.
  • the fluorescent moiety and the quencher moiety become spatially separated from one another.
  • the fluorescence emission from the first fluorescent moiety can be detected.
  • an ABI PRISM® 7700 Sequence Detection System uses TaqMan® technology, and is suitable for performing the methods described herein for amplifying and/or detecting a template/target nucleic acid.
  • Molecular beacons in conjunction with FRET can also be used to detect the presence of an amplification product using the real-time PCR methods.
  • Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety.
  • the second fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe.
  • Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g., a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution.
  • the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.
  • FRET fluorescein
  • a donor fluorescent moiety for example, fluorescein
  • fluorescein is excited at 470 nm by the light source of the LightCycler® Instrument.
  • the fluorescein transfers its energy to an acceptor fluorescent moiety such as LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705).
  • the acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument.
  • Efficient FRET can only take place when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety.
  • the intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of Plasmodium genomes). If amplification of Plasmodium target nucleic acid occurs and an amplification product is produced, the step of hybridizing results in a detectable signal based upon FRET between the members of the pair of probes.
  • the presence of FRET indicates the presence of Plasmodium in the sample
  • the absence of FRET indicates the absence of Plasmodium in the sample.
  • Inadequate specimen collection, transportation delays, inappropriate transportation conditions, or use of certain collection swabs (calcium alginate or aluminum shaft) are all conditions that can affect the success and/or accuracy of a test result, however.
  • Representative biological samples that can be used in practicing the methods include, but are not limited to whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs, wound swabs, blood cultures, skin, and soft tissue infections. Collection and storage methods of biological samples are known to those of skill in the art. Biological samples can be processed (e.g., by nucleic acid extraction methods and/or kits known in the art) to release Plasmodium nucleic acid or in some cases, the biological sample can be contacted directly with the PCR reaction components and the appropriate oligonucleotides. In some instances, the biological sample is whole blood.
  • nucleic acids within the whole blood undergo considerable amount of degradation. Therefore, it may be advantageous to collect the blood in a reagent that will lyse, denature, and stabilize whole blood components, including nucleic acids, such as a nucleic acidstabilizing solution. In such cases, the nucleic acids can be better preserved and stabilized for subsequent isolation and analysis, such as by nucleic acid test, such as PCR.
  • nucleic acid-stabilizing solution are well known in the art, including, but not limited to, cobas PCR media, which contains 4.2 M guanadinium salt (GuHCl) and 50 mM Tris, at a pH of 7.5.
  • the sample can be collected by any method or device designed to adequately hold and store the sample prior to analysis.
  • the method or device may include a blood collection vessel.
  • a blood collection vessel is well known in the art, and may include, for example, a blood collection tube.
  • a blood collection tube with an evacuated chamber, such as a vacutainer blood collection tube are well known in the art.
  • a solution that will lyse, denature, and stabilize whole blood components including nucleic acids, such as a nucleic acid-stabilizing solution, such that the whole blood being drawn immediately contacts the nucleic acid-stabilizing solution in the blood collection vessel.
  • Melting curve analysis is an additional step that can be included in a cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which half of the DNA duplexes have separated into single strands.
  • Tm melting temperature
  • the melting temperature of a DNA depends primarily upon its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than those having an abundance of A and T nucleotides.
  • the melting temperature of probes can be determined. Similarly, by detecting the temperature at which signal is generated, the annealing temperature of probes can be determined.
  • the melting temperature(s) of the Plasmodium probes from the Plasmodium amplification products can confirm the presence or absence of Plasmodium in the sample.
  • control samples can be cycled as well.
  • Positive control samples can amplify target nucleic acid control template (other than described amplification products of target genes) using, for example, control primers and control probes.
  • Positive control samples can also amplify, for example, a plasmid construct containing the target nucleic acid molecules.
  • a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run side-by-side with the patients' samples using the same primers and probe as used for detection of the intended target.
  • Such controls are indicators of the success or failure of the amplification, hybridization, and/or FRET reaction.
  • Each thermocycler run can also include a negative control that, for example, lacks target template DNA.
  • Negative control can measure contamination. This ensures that the system and reagents would not give rise to a false positive signal. Therefore, control reactions can readily determine, for example, the ability of primers to anneal with sequencespecificity and to initiate elongation, as well as the ability of probes to hybridize with sequence-specificity and for FRET to occur.
  • the methods include steps to avoid contamination.
  • an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Patent Nos. 5,035,996, 5,683,896 and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next.
  • the LightCycler® can be operated using a PC workstation and can utilize a Windows NT operating system. Signals from the samples are obtained as the machine positions the capillaries sequentially over the optical unit.
  • the software can display the fluorescence signals in real-time immediately after each measurement. Fluorescent acquisition time is 10-100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence vs. cycle number can be continually updated for all samples. The data generated can be stored for further analysis.
  • an amplification product can be detected using a double-stranded DNA binding dye such as a fluorescent DNA binding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)).
  • a double-stranded DNA binding dye such as a fluorescent DNA binding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)
  • fluorescent DNA binding dyes Upon interaction with the double-stranded nucleic acid, such fluorescent DNA binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength.
  • a doublestranded DNA binding dye such as a nucleic acid intercalating dye also can be used.
  • a melting curve analysis is usually performed for confirmation of the presence of the amplification product.
  • nucleic acid- or signalamplification methods may also be employed.
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequence-based amplification
  • SDA selfsustained sequence replication
  • SMAP 2 smart amplification process version 2
  • Embodiments of the present disclosure further provide for articles of manufacture or kits to amplifying and/or detecting template/target nucleic acid.
  • An article of manufacture can include long primers and/or long primers and probes used to amplify and/or detect a template/target nucleic acid, together with suitable packaging materials.
  • Representative long primers and probes for amplification and/or detection of template/target nucleic acid are capable of hybridizing to template/target nucleic acid.
  • the kits may also include suitably packaged reagents and materials needed for DNA immobilization, hybridization, and detection, such solid supports, buffers, enzymes, and DNA standards. Methods of designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to template/target nucleic acid.
  • Articles of manufacture can also include one or more fluorescent moieties for labeling the probes or, alternatively, the probes supplied with the kit can be labeled.
  • an article of manufacture may include a donor and/or an acceptor fluorescent moiety for labeling the Plasmodium probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.
  • Articles of manufacture can also contain a package insert or package label having instructions thereon for using the long primers and/or the long primers and probes to amplify and/or detect a template/target nucleic acid.
  • Articles of manufacture may additionally include reagents for carrying out the methods disclosed herein (e.g., buffers, polymerase enzymes, co-factors, or agents to prevent contamination). Such reagents may be specific for one of the commercially available instruments described herein.
  • Embodiments of the present disclosure also provide for a set of long primers and one or more detectable probes for the amplification and/or detection of template/target nucleic acids.
  • This disclosure provides for long forward primers and long reverse primers for amplification of a template/target nucleic acid, where the template/target nucleic acid has a short primer-binding region.
  • Long forward primers and long reverse primers were designed to have two regions: (1) a region for annealing/hybri dizing to a region of the template/target nucleic acid; and (2) a region that does not anneal/hybridize to a region of the template/target nucleic acid region.
  • the long primers are longer than the length of the primer-binding site (z.e., the site upon which the long primers are to anneal/hybridize).
  • Table 1 Exemplary long primers and the, relatively, shorter primer-binding sites.
  • Table 1 shows the sequences of exemplary long forward primers (SEQ ID NO: 1), the correspondingly (short) binding-region for the long forward primer (SEQ ID NO:2), long reverse primers (SEQ ID NO:3), and the correspondingly (short) binding-region for the long reverse primer (SEQ ID NON).
  • FIG. 1A shows alignment of an exemplary long forward primer (SEQ ID NO: 1), and the short version of the primer-binding site for the long forward primer (SEQ ID NO:2).
  • SEQ ID NO: 1 the 3 ’ region of the long forward primer hybridizes/aligns with the entirety of the short version of the primer-binding site for the long forward primer (SEQ ID NO:2), and the 5’ region of the long forward primer is not hybridized and not aligned, and therefore appears as a single-stranded tail (of 5 bases, ACTCG).
  • T m with this alignment is 51.1 °C.
  • FIG. IB shows alignment of an exemplary long forward primer (SEQ ID NO: 1), and the long version of the primer-binding site for the long forward primer (SEQ ID NON).
  • the long forward primer (SEQ ID NO: 1) hybridizes/aligns entirely with the long version of the primer-binding site for the long forward primer (SEQ ID NON) (z.e., the singled-stranded 5’ tail from FIG. 1A no longer exists).
  • the long forward primer SEQ ID NON
  • FIG. IB shows alignment of the long forward primer (SEQ ID NON) will be across the entirety of the long primer-binding region (SEQ ID NON), as depicted in FIG. IB, and the calculated T m with this alignment is 61.1°C. Therefore, over time, use of the long primers in PCR cycles results in increased T m , which results in better annealing and improved PCR yields.
  • FIG. 2A shows alignment of an exemplary long reverse primer (SEQ ID NON), and the short version of the primer-binding site for the long reverse primer (SEQ ID NON).
  • SEQ ID NON long reverse primer
  • SEQ ID NON short version of the primer-binding site for the long reverse primer
  • the 3’ region of the long reverse primer hybridizes/aligns with the entirety of the short version of the primer-binding site for the long reverse primer (SEQ ID NON)
  • the 5’ region of the long reverse primer is not hybridized and not aligned, and therefore appears as a single-stranded tail (of 5 bases, ATGCC).
  • ATGCC a single-stranded tail
  • FIG. 2B shows alignment of an exemplary long reverse primer (SEQ ID NO:4), and the long version of the primer-binding site for the long forward primer (SEQ ID NO:6).
  • the long reverse primer (SEQ ID NO: 1) hybridizes/aligns entirely with the long version of the primer-binding site for the long reverse primer (SEQ ID NO:6) (z.e., the single-stranded 5’ tail from FIG. 2A no longer exists).
  • the long reverse primer SEQ ID NO: 1
  • SEQ ID NO:3 the long primer-binding region
  • the calculated T m with this alignment is 58.4°C. Therefore, over time, use of the long primers in PCR cycles results in increased T m , which results in better annealing and improved PCR yields.
  • these exemplary sequences for long forward primers (SEQ ID NO: 1) and long reverse primers (SEQ ID NO:4) are, over time, effective at increasing T m , exhibit better annealing, and improve overall PCR yields.
  • This disclosure provides for long forward primers and long reverse primers for amplification of a tempi ate/target nucleic acid, where the template/target nucleic acid has a short primer-binding region.
  • Long forward primers and long reverse primers were designed to have two regions: (1) a region for annealing/hybri dizing to a region of the template/target nucleic acid; and (2) a region that does not anneal/hybridize to a region of the template/target nucleic acid region.
  • the long primers are longer than the length of the primer-binding site (z.e., the site upon which the long primers are to anneal/hybridize).
  • the template for primer extension is the original template/insert (shown in black), and including a template reverse primer-binding site (TRPBS) on its 5’ end (shown in purple) and a short forward primer-binding site (SFPBS) on its 3’ end (shown in light blue).
  • TRPBS template reverse primer-binding site
  • SFPBS short forward primer-binding site
  • the long forward primer (shown in light blue and dark blue) acts as the primer, upon which extension will occur, and the long forward primer (LFP) contains the following 2 regions: (1) a region for annealing/hybridizing to a primer- binding site (depicted in light blue), and (2) a region that does not anneal/hybridize to a primer-binding site (depicted in dark blue).
  • the long forward primer (LFP) anneals, in part, to the short forward primer-binding site (SFPBS) (shown in light blue), which is ⁇ 15 nucleotides, as shown in FIG. 3A.
  • a region of the 5’ end of the long forward primer (LFP) does not anneal/hybridize to any portion of the short forward primer-binding site (SFPBS), and appears as a single-stranded 5’ tail (shown in dark blue), as shown in FIG. 3A.
  • the T m of annealing is lower, but primer concentration is high, which allows for annealing to take place (albeit, with lower efficiency for this cycle).
  • Primer extension is then initiated and completed, generating a first primer extension product, depicted as a dashed line (black and purple), as shown in FIG. 3A.
  • the first primer extension product includes the long forward primer (LFP), as well as the reverse complement of the original template/insert (shown as a dashed black line), and a short reverse primer-binding site (SRPBS) (shown as a dashed purple line), as shown in FIG. 3A. That is, during the first primer extension reaction, the template/insert is copied into its reverse complement (depicted as a dashed black line), and the template reverse primerbinding site (TRPBS) is copied into the short reverse primer-binding site (SRPBS) (depicted as a dashed purple line), as shown in FIG. 3A. Due to the short forward primer-binding site (SFPBS), the T m of annealing is lower, and annealing/priming occurs at a lower efficiency for this first cycle.
  • LFP long forward primer
  • SRPBS short reverse primer-binding site
  • the template for primer extension is first primer extension product, which was generated during the first PCR cycle, which is shown in FIG. 3A. That is, the template for primer extension in the second PCR cycle includes the reverse complement of the original template/insert (shown as a dashed black line), and including a long forward primer (LFP) on its 5’ end (shown in dark blue and light blue) and a short reverse primer-binding site (SRPBS) on its 3’ end (shown as a dashed purple line).
  • LFP long forward primer
  • SRPBS short reverse primer-binding site
  • the long reverse primer (LRP) acts as the primer, upon which extension will occur, and the long reverse primer (LRP) contains the following 2 regions: (1) a region for annealing/hybridizing to a primer-binding site (depicted in purple), and (2) a region that does not anneal/hybridize to a primerbinding site (depicted in red).
  • the long reverse primer (LRP) anneals, in part, to the short reverse primer-binding site (SRPBS), which is ⁇ 15 nucleotides, and shown as a dashed purple line, as shown in FIG. 3B.
  • SRPBS short reverse primer-binding site
  • a region of the 5’ end of the long reverse primer (LRP) does not anneal/hybridize to any portion of the short reverse primer-binding site (SRPBS), and appears as a singlestranded 5’ tail (shown in red), as shown in FIG. 3B.
  • SRPBS short reverse primer-binding site
  • FIG. 3B the T m of annealing is lower, but primer concentration is high, which allows for annealing to take place (albeit, with lower efficiency for this second cycle).
  • Primer extension is then initiated and completed, generating a second primer extension product, depicted as a dotted line (black, light blue, and dark blue), as shown in FIG. 3B.
  • the second primer extension product includes the long reverse primer (LRP), as well as the reverse complement of the template/insert (shown as a dotted black line), and a long forward primer-binding site (LFPBS) (shown as a dotted light blue and dark blue line), as shown in FIG. 3B. That is, during the second primer extension reaction, the template/insert is copied into its reverse complement (depicted as a dotted black line), and the long forward primer-binding site (LFPBS) is generated from the long forward primer (LFP) in the template (depicted as a dotted light and dark blue line), as shown in FIG. 3B.
  • LRP long reverse primer
  • LFPBS long forward primer-binding site
  • the T m of annealing is lower, and annealing/priming occurs at a lower efficiency for this second cycle.
  • the primer extension product generated after the second PCR cycle yields a template having an extended version of both of the original short forward primer-binding site (SFPBS) and the original short reverse primer-binding site (SRPBS). That is, the primer extension product generated after the second PCR contains, now, longer primer-binding sites for the long forward primer (LFP) and the long reverse primer (LRP), thereby eliminating the short primer-binding sites (short forward primer-binding site (SFPBS) and short reverse primer-binding site (SRPBS)).
  • short primer-binding sites short forward primer-binding site (SFPBS) and short reverse primer-binding site (SRPBS)
  • long primer-binding sites long forward primer-binding site (LFPBS) and long reverse primer-binding site (LRPBS)
  • the template for primer extension is second primer extension product, which was generated during the second PCR cycle, which is shown in FIG. 3B. That is, the template for primer extension in the third PCR cycle includes the template/insert (shown as a dotted black line), and including a long reverse primer (LRP) on its 5’ end (shown in red and purple) and a long forward primer-binding site (LFPBS) on its 3’ end (shown as a dotted light blue and dark blue line).
  • LRP long reverse primer
  • LFPBS long forward primer-binding site
  • the long forward primer (LFP) (shown in dark blue and light blue) acts as the primer, upon which extension will occur, and the long forward primer (LFP), and the entire length of the long forward primer (LFP) will anneal/hybridize to a primer-binding site (z.e., the long forward primer-binding site (LFPBS).
  • LFPBS long forward primer binding site
  • the long forward primer (LFP) anneals/hybridizes completely to the long forward primerbinding site (LFPBS), which is ⁇ 15 nucleotides, and shown as a dotted dark blue and light blue line, as shown in FIG. 3C.
  • the third primer extension product includes the long forward primer (LFP), as well as the reverse complement of the template/insert (shown as a dashed black line), and a long reverse primer-binding site (LRPBS) (shown as a dashed purple and red line), as shown in FIG. 3C. That is, during the third primer extension reaction, the template/insert is copied into its reverse complement (depicted as a dashed black line), and the long reverse primer-binding site (LRPBS) is generated from the long reverse primer (LRP) in the template (depicted as a dashed purple and red line), as shown in FIG. 3C.
  • LFP long forward primer
  • LRPBS long reverse primer-binding site
  • FIG. 3A, FIG. 3B, and FIG. 3C show the first few cycles of PCR, where there are short primer-binding sites, flanking a template, and employing long primers.
  • the short primer-binding sites have been replaced by long primer-binding sites. That is, by the conclusion of the second PCR cycle, the short primer-binding sites are gone, replaced by long primer-binding sites, allowing for higher T m , better annealing and priming, and improved PCR yields of amplicons.
  • This disclosure provides for long forward primers and long reverse primers for amplification of a tempi ate/target nucleic acid, where the template/target nucleic acid has a short primer-binding region.
  • Long forward primers and long reverse primers were designed to have two regions: (1) a region for annealing/hybri dizing to a region of the template/target nucleic acid; and (2) a region that does not anneal/hybridize to a region of the template/target nucleic acid region.
  • the long primers are longer than the length of the primer-binding site (z.e., the site upon which the long primers are to anneal/hybridize).
  • the long primers can act as templates, and the template (having short primer-binding sites) acts as a primer. This is depicted in FIG. 4. That is, by employing long primers, it is possible to have priming and primer extension in two directions, simultaneously (simultaneous bi-directional primer extension).
  • the long forward primer (LFP) (shown in dark blue and light blue) anneals to the short forward primer-binding site (SFPBS) (shown in light blue).
  • SFPBS short forward primer-binding site
  • the T m of annealing is lower, but primer concentration is high, and the annealing/priming occurs at lower efficiency.
  • the long forward primer (LFP) anneals, in part, to the short forward primer-binding site (SFPBS) (shown in light blue), which is ⁇ 15 nucleotides, as shown in FIG. 3A, and also FIG. 4.
  • a region of the 5’ end of the long forward primer (LFP) does not anneal/hybridize to any portion of the short forward primer-binding site (SFPBS), and appears as a single-stranded 5’ tail (shown in dark blue), as shown in FIG. 3A, and also FIG. 4.
  • the T m of annealing is lower, but primer concentration is high, which allows for annealing to take place (albeit, with lower efficiency for this cycle).
  • Primer extension is then initiated and completed, generating a first primer extension product, depicted as a dashed line (black and purple), as shown in FIG. 3A, and also FIG. 4
  • the 3’ end of the targeted template is extendable (z.e., 3 ’-OH)
  • priming occurs from the 3’ end of the targeted template, while using the long forward primer (LFP) as a template, as shown in FIG. 4 (depicted by the short forward primer-binding site, in light blue, having an arrowhead at its 3’ end).
  • LFP long forward primer
  • SFPBS short forward primerbinding site
  • extension can occur off it, resulting in synthesis of the reverse complement of the 5’ tail of the long forward primer (depicted by a dark blue dotted line), as seen in FIG. 4.
  • the 3’ end of the template extends on the long forward primer (LFP), thereby generating a long forward primer-binding site (LFPBS) as an extension product, as shown in FIG. 4.
  • LFPBS long forward primer-binding site
  • subsequent priming with the long forward primer (LFP) on this product will be high efficiency.
  • This type of priming and extension off the short primerbinding site can occur with the long reverse primer (LRP) and the short reverse primer-binding site (SRPBS) as well (not shown).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés d'amplification d'un acide nucléique matrice/cible, l'acide nucléique matrice/cible d'origine ayant des sites de liaison d'amorce courts (par exemple, ≤ 15 nucléotides), en utilisant des amorces longues. Des amorces sens longues et des amorces antisens longues ont deux régions : (1) une région de recuit/hybridation à une région de l'acide nucléique matrice/cible; et (2) une région qui ne s'hybride pas/s'hybride à une région de la région d'acide nucléique matrice/cible. En d'autres termes, les amorces longues sont plus longues que le site de liaison de l'amorce (c'est-à-dire le site sur lequel les amorces longues doivent s'hybrider). L'utilisation de ces amorces longues permet de surmonter les problèmes et les obstacles de séquences de liaison d'amorce courtes.
PCT/EP2024/071686 2023-08-03 2024-07-31 Utilisation d'amorces longues à queue en 5' pour améliorer les performances d'amplification lors du ciblage de sites de liaison d'amorce courts Pending WO2025027080A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363517453P 2023-08-03 2023-08-03
US63/517,453 2023-08-03

Publications (1)

Publication Number Publication Date
WO2025027080A1 true WO2025027080A1 (fr) 2025-02-06

Family

ID=92214434

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/071686 Pending WO2025027080A1 (fr) 2023-08-03 2024-07-31 Utilisation d'amorces longues à queue en 5' pour améliorer les performances d'amplification lors du ciblage de sites de liaison d'amorce courts

Country Status (1)

Country Link
WO (1) WO2025027080A1 (fr)

Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984003285A1 (fr) 1983-02-22 1984-08-30 Molecular Biosystems Inc Oligonucleotides a brin unique et a sequence definie comprenant des groupes rapporteurs, procede pour effectuer leur synthese chimique, et nucleosides utiles dans une telle synthese
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4996143A (en) 1985-12-23 1991-02-26 Syngene, Inc. Fluorescent stokes shift probes for polynucleotide hybridization
US5035996A (en) 1989-06-01 1991-07-30 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5565322A (en) 1991-11-07 1996-10-15 Nanogen, Inc. Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to donor energy transfer system
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5683896A (en) 1989-06-01 1997-11-04 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
WO1997046714A1 (fr) 1996-06-04 1997-12-11 University Of Utah Research Foundation Controle de l'hybridation pendant la pcr
WO1997046712A2 (fr) 1996-06-04 1997-12-11 University Of Utah Research Foundation Systeme et procede d'execution et de suivi de processus biologiques
US5994056A (en) 1991-05-02 1999-11-30 Roche Molecular Systems, Inc. Homogeneous methods for nucleic acid amplification and detection
US6001611A (en) 1997-03-20 1999-12-14 Roche Molecular Systems, Inc. Modified nucleic acid amplification primers
WO2003093509A1 (fr) * 2002-05-01 2003-11-13 Seegene, Inc. Methodes et compositions permettant d'ameliorer la specificite de l'amplification par pcr
US7741467B2 (en) 2005-10-05 2010-06-22 Roche Molecular Systems, Inc. Non-fluorescent energy transfer
US20210040550A1 (en) * 2018-01-25 2021-02-11 The University Of Nottingham Detection of nucleic acid sequences

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984003285A1 (fr) 1983-02-22 1984-08-30 Molecular Biosystems Inc Oligonucleotides a brin unique et a sequence definie comprenant des groupes rapporteurs, procede pour effectuer leur synthese chimique, et nucleosides utiles dans une telle synthese
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4996143A (en) 1985-12-23 1991-02-26 Syngene, Inc. Fluorescent stokes shift probes for polynucleotide hybridization
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
US4800159A (en) 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5035996A (en) 1989-06-01 1991-07-30 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
US5683896A (en) 1989-06-01 1997-11-04 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
US5945313A (en) 1989-06-01 1999-08-31 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US6171785B1 (en) 1991-05-02 2001-01-09 Roche Molecular Systems, Inc. Methods and devices for hemogeneous nucleic acid amplification and detector
US5994056A (en) 1991-05-02 1999-11-30 Roche Molecular Systems, Inc. Homogeneous methods for nucleic acid amplification and detection
US5849489A (en) 1991-11-07 1998-12-15 Nanogen, Inc. Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system
US6162603A (en) 1991-11-07 2000-12-19 Nanogen, Inc. Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system
US5565322A (en) 1991-11-07 1996-10-15 Nanogen, Inc. Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to donor energy transfer system
WO1997046707A2 (fr) 1996-06-04 1997-12-11 University Of Utah Research Foundation Systeme et procedes de suivi d'un processus acp de l'adn par fluorescence
WO1997046712A2 (fr) 1996-06-04 1997-12-11 University Of Utah Research Foundation Systeme et procede d'execution et de suivi de processus biologiques
WO1997046714A1 (fr) 1996-06-04 1997-12-11 University Of Utah Research Foundation Controle de l'hybridation pendant la pcr
US6001611A (en) 1997-03-20 1999-12-14 Roche Molecular Systems, Inc. Modified nucleic acid amplification primers
WO2003093509A1 (fr) * 2002-05-01 2003-11-13 Seegene, Inc. Methodes et compositions permettant d'ameliorer la specificite de l'amplification par pcr
US7741467B2 (en) 2005-10-05 2010-06-22 Roche Molecular Systems, Inc. Non-fluorescent energy transfer
US20210040550A1 (en) * 2018-01-25 2021-02-11 The University Of Nottingham Detection of nucleic acid sequences

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Diagnostic Molecular Microbiology: Principles and Applications", 1993, AMERICAN SOCIETY FOR MICROBIOLOGY,
ALTSCHUL ET AL.: "Basic local alignment search tool", J. MOL. BIOL., vol. 215, 1990, pages 403 - 410, XP002949123, DOI: 10.1006/jmbi.1990.9999
ALTSCHUL ET AL.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: 10.1093/nar/25.17.3389
FRANK VANDENBUSSCHE ET AL: "A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR", PLOS ONE, vol. 11, no. 10, 10 October 2016 (2016-10-10), pages e0164463, XP055552544, DOI: 10.1371/journal.pone.0164463 *
GISH ET AL.: "Identification of protein coding regions by database similarity search", NATURE GENET, vol. 3, 1993, pages 266 - 272
MADDEN ET AL.: "Applications of network BLAST server", METH. ENZYMOL, vol. 266, 1996, pages 131 - 141, XP001006313, DOI: 10.1016/S0076-6879(96)66011-X
VANDENBUSSCHE FRANK: "S1 Table. Primers and probes used throughout this study. primer/probe a sequence (5'-3') b", 10 October 2016 (2016-10-10), XP093216002, Retrieved from the Internet <URL:https://doi.org/10.1371/journal.pone.0164463.s001> *
ZHANG ET AL.: "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation", GENOME RES, vol. 7, 1997, pages 649 - 656, XP055970184, DOI: 10.1101/gr.7.6.649

Similar Documents

Publication Publication Date Title
US12460267B2 (en) Compositions and methods for detection of Mycoplasma genitalium
EP3655554B1 (fr) Compositions et procédés de détection de babesia
US11773458B2 (en) Compositions and methods for detection of BK virus
EP3068897B1 (fr) Détection d&#39;un polymorphisme mononucléotidique en utilisant une amorce de chevauchement et une sonde de fusion
EP4004240A1 (fr) Compositions et procédés pour la détection du virus epstein-barr (ebv)
US20240240273A1 (en) Compositions and Methods for Detecting Hepatitis Delta Virus by a Dual-Target Assay
EP3494227B1 (fr) Oligonucléotide auxiliaire pour améliorer l&#39;efficacité de l&#39;amplification et de la détection/quantification d&#39;acides nucléiques
US10370731B2 (en) Compositions and methods for detection of hepatitis C virus genotype 3
WO2025027080A1 (fr) Utilisation d&#39;amorces longues à queue en 5&#39; pour améliorer les performances d&#39;amplification lors du ciblage de sites de liaison d&#39;amorce courts
US11028451B2 (en) Compositions and methods for detection of Mycobacterium tuberculosis
EP3963102A1 (fr) Utilisation de ditp pour une amplification préférentielle/sélective d&#39;arn par rapport à des cibles d&#39;adn sur la base de la température de séparation de brin

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24751668

Country of ref document: EP

Kind code of ref document: A1