HK40032105B - A method to quantify telomere length and genomic motifs - Google Patents

Description

Methods for quantifying telomere length and genomic motifs

Cross-reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/856,449, filed June 3, 2019, which is incorporated herein by reference in its entirety.

Background of the Invention

Molecular inverted probes, or MIPs, are single-stranded DNA probes with 5' and 3' ends on the same strand that target (complement) a target template. In all previous applications, the principle of MIP assays utilizes the formation of a circularized product via a template-dependent ligation event, which is specific to the template sequence in the sample and used to query the sample's sequence content. To remove non-specific MIPs, after the ligation step, all non-circular products are removed, for example, by a combination of DNA-digesting enzymes such as exonuclease I and exonuclease III.

However, classic MIP probes and their usage methods (e.g., for nucleic acid detection and quantification) are not suitable for tandem repeat sequences, such as telomeres. Assays designed for classic MIP probes are based on hybridization of the 5' and 3' ends of the MIP to specific invariant sites on the template DNA, where the 5' and 3' sequences hybridize to different target sequences on the template DNA. In classic MIP-based methods, the 5' and 3' ends are adjacent to each other when binding to the same template, allowing for direct ligation and circularization, or they are separated by one or more nucleotides, allowing for circularization after 3' end extension (also known as "gap filling") and subsequent ligation. In all these cases, the circularized products of these reactions are subsequently separated from linear, i.e., unligated MIPs and non-circularized ligated MIPs before detection and/or quantification.

Because the 5' and 3' ends of a MIP targeting tandem repeat sequences will necessarily target the same repeat sequence, such probes are unsuitable for traditional MIP-based methods. This is because they would allow the MIP ends to bind randomly to any of the many repeat sequences within a given repeat array, with the 5' and 3' ends separated by a highly variable number of inserted nucleotides within the template DNA, resulting in different sets of products. Given this product diversity, and the fact that classical MIP-based assays involve removing all linear products before detection or quantification, classical MIP-based methods will lead to highly inaccurate and unreliable results when used with tandem repeat sequences. Therefore, MIP-based queries for telomere length or other tandem repeat lengths have not previously shown success.

The ability to measure the length of telomeres or other variable-length tandem repeat sequences can be used for a variety of purposes, including as a biomarker of biological aging. Human telomeres consist of 6-base-pair repeating motifs located at the ends of each chromosome. Telomeres shorten during cell division, and their rate of loss can be influenced by disease and health conditions. For example, their shortening rate can be influenced by lifestyle factors such as diet, smoking, physical activity and alcohol consumption, as well as stress and psychosocial factors (Epel et al., 2004; Starkweather et al., 2013). Therefore, telomere length measurements can be used to indicate an individual's biological age and the types of lifestyle changes an individual can make to enjoy better health.

Several methods have been developed to measure telomere length, but each method has its own set of limitations and constraints. For example, the classic method for measuring telomere length is telomere restriction fragmentation assay (TRF; Kimura et al., 2010), which involves restrictive digestion of telomeres from peripheral blood leukocytes followed by Southern blotting. However, TRF is not well-suited for handling large DNA sample sizes (Samani et al., 2001; Valdes et al., 2005), and the results are susceptible to factors such as the type of gel electrophoresis used, the amount of DNA available, the quality of the image capture device, and the computer software used for analysis (Samani et al., 2001; Valdes et al., 2005).

Another method for measuring telomere length is the T/S assay, which is based on real-time quantitative PCR and compared with a reference sample. However, studies have shown that the initially provided coefficient of variation is underestimated, and that DNA quality, the PCR machine used, and the analysis software can affect the results (Aubert et al., 2012; Cunningham et al., 2013; Nussey et al., 2014). Other assays used include quantitative fluorescence in situ hybridization (Q-FISH) and flow cytometry FISH (Flow-FISH); these methods allow for high levels of accuracy but are limited by the requirement for freshly drawn blood samples for analysis, which is impractical for applications such as epidemiological studies.

Therefore, there is a need for new, reliable methods for measuring the length of telomeres or other tandem repeat sequences, which can be applied on a large scale, for example, in epidemiological studies or routine hospital laboratory practices. This invention addresses these and other needs.

Invention Overview

In one aspect, the present invention provides a single-stranded DNA probe for determining telomere length and/or copy number of a tandem repeat sequence, wherein the probe comprises: i) a 5' homologous region extending to the 5' end of the probe and comprising a nucleotide sequence complementary to the tandem repeat sequence; ii) a linker region; and iii) a 3' homologous region extending to the 3' end of the probe and comprising a nucleotide sequence complementary to the tandem repeat sequence, such that the 5' homologous region and the 3' homologous region are capable of binding to the same strand of template DNA comprising the tandem repeat sequence; wherein, when the 5' homologous region and the 3' homologous region bind to the same strand of template DNA, the 3' homologous region is immediately adjacent to the 3' of the 5' homologous region within a single repeat unit on the template, and thus the 3' end of the 3' homologous region and the 5' end of the 5' homologous region are separated by a nucleotide gap on the template DNA less than one complete repeat unit, the nucleotide gap less than one complete repeat unit comprising up to two different bases.

In some embodiments of the invention, the tandem repeat sequence is a telomere sequence. In some embodiments, the telomere is a human telomere. In some embodiments, the nucleotide sequences of the 5' and 3' homologous regions are 100% complementary to the tandem repeat sequence. In some embodiments, the length of each of the 5' and 3' homologous regions is 15-25 nucleotides. In some embodiments, the length of each repeat unit of the tandem repeat sequence is 2-10 nucleotides. In some embodiments, the adapter region comprises one or more sequence elements selected from universal primer sequences, probe-specific primer sequences, TaqMan probe sequences, and tag sequences.

In some embodiments of the invention, when bound to a single repeat unit on template DNA, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region are separated by one or two nucleotides. In some embodiments, the one or two nucleotides comprise the base G. In some embodiments, when bound to a single repeat unit on template DNA, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region are separated by three nucleotides. In some embodiments, the three nucleotides comprise two different bases, wherein the two different bases are A and G. In some embodiments, the 5' homologous region comprises the nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the 3' homologous region comprises the nucleotide sequence of any one of SEQ ID NO:6-8. In some embodiments, the probe comprises the nucleotide sequence of any one of SEQ ID NO:1-3.

In another aspect, the present invention provides a kit for determining the copy number of variable tandem repeat sequences within a genome, wherein the kit comprises: a first single-stranded DNA probe as described herein, wherein the 5' and 3' homologous regions of the first DNA probe are complementary to tandem repeat sequences in the genome whose copy number varies between individuals; and a second single-stranded DNA probe as described herein, wherein the 5' and 3' homologous regions of the second DNA probe are complementary to tandem repeat sequences in the genome whose copy number is stable between individuals; wherein at most two bases contained in the nucleotide gap of the first probe are also contained in the nucleotide gap of the second probe.

In some embodiments, the kit further comprises a reaction mixture containing a DNA polymerase, an enzyme with ligase activity, and deoxyribonucleoside triphosphates containing up to two bases corresponding to the nucleotide gaps of the first and second probes. In some embodiments, the DNA polymerase is T4 DNA polymerase. In some embodiments, the ligase activity is provided by Amp ligase. In some embodiments, the reaction mixture does not contain exonuclease. In some embodiments, the kit further comprises genomic DNA of the test sample. In some embodiments, the kit further comprises universal primers complementary to sequences within the adapter regions of the first and/or second probes, two or more probe-specific primers, and/or two or more TaqMan probes.

In some embodiments of the kit of the present invention, the tandem repeat sequence recognized by the 5' and 3' homologous regions of the first probe is a telomere sequence. In some embodiments, the telomeres are human telomeres. In some embodiments, the tandem repeat sequence recognized by the 5' and 3' homologous regions of the second probe is a short tandem repeat of 4 bp. In some embodiments, the nucleotide sequence of the 5' and 3' homologous regions of the first probe is 100% complementary to a tandem repeat sequence in the genome whose copy number varies between individuals. In some embodiments, the nucleotide sequence of the 5' and 3' homologous regions of the second probe is 100% complementary to a tandem repeat sequence in the genome whose copy number is stable between individuals. In some embodiments, the length of the 5' and/or 3' homologous regions of the first and/or second probes is 15 to 25 nucleotides. In some embodiments, the repeat unit of the tandem repeat sequence recognized by the first and/or second probes is 3-6 nucleotides in length.

On the other hand, the present invention provides a method for determining the length of variable-length tandem repeat regions in an individual's genome, wherein the method comprises: i) providing a first single-stranded DNA probe as described herein, wherein the 5' and 3' homologous regions of the first DNA probe are complementary to tandem repeat sequences whose copy number varies between individuals in the genome; and ii) providing a second single-stranded DNA probe as described herein, wherein the 5' and 3' homologous regions of the second DNA probe are complementary to tandem repeat sequences whose copy number is stable between individuals in the genome; wherein at most two bases contained in the nucleotide gap of the first probe are also contained in the nucleotide gap of the second probe; iii) in a manner conducive to the determination of the length of a variable-length tandem repeat region in an individual's genome; Under the conditions of extension of the 3' end of the probe and ligation to the 5' end of the probe bound to the same template, a biological sample from an individual is contacted with the first and second probes in the presence of DNA polymerase, ligase activity, and at most two deoxyribonucleoside triphosphates corresponding to the bases contained in the nucleotide gaps of the first and second probes; iv) quantifying the circularized and ligated linear probe products of the first and/or second probes generated in step iii); and v) determining the normalized abundance of the ligation sites of the first and/or second probes using the relative amounts of the circularized and ligated linear probe products of the first and/or second probes, which is an indicator corresponding to the tandem repeat length of the probes in the individual genome.

In some embodiments of the method of the present invention, the nucleotide sequences of the 5' and 3' homologous regions of the first and second probes are 100% complementary to the first and second tandem repeat sequences, respectively. In some embodiments, the length of the 5' and 3' homologous regions of the probes is 15-25 nucleotides. In some embodiments, the variable tandem repeat sequence region is a telomere. In some embodiments, the telomere is a human telomere. In some embodiments, the adapter region of each probe contains one or more sequence elements selected from universal primer sequences, probe-specific primer sequences, TaqMan probe sequences, and tag sequences. In some embodiments, when bound within a single repeat unit on a tandem repeat sequence, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region of the probe are separated by a 1- or 2-nucleotide gap, wherein the 1- or 2-nucleotide gap comprises a single base, and wherein only one deoxyribonucleoside triphosphate corresponding to the single base is provided in step iii). In some embodiments, the single base is G. In some embodiments, when bound within a single repeat unit on a tandem repeat sequence, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region of the probe in step i) or step ii) are separated by a 3-nucleotide gap, wherein the 3 nucleotides comprise two bases, and wherein two deoxyribonucleoside triphosphates corresponding to the two bases are provided in step iii). In some embodiments, the two bases are A and G.

In some embodiments of the method of the present invention, the 5' homologous region of the probe in step i) comprises the nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the 3' homologous region of the probe in step i) comprises the nucleotide sequence of any one of SEQ ID NO:6-8. In some embodiments, the probe in step i) comprises the nucleotide sequence of any one of SEQ ID NO:1-3. In some embodiments, a large excess of the probes in steps i) and ii) is provided relative to the amount of genomic DNA in the biological sample. In some embodiments, no exonuclease is added during steps i) to v). In some embodiments, the only exonuclease added during steps i) to v) is exonuclease I, and the level of exonuclease I does not exceed 20 units/50 ng of genomic DNA and is present for a maximum of 1 hour. In some embodiments, the amount of circularized and ligated linear probe product is determined using a method selected from quantitative PCR, digital PCR, and sequencing. In some embodiments, only the first single-stranded probe is used, while another method is used to quantify the amount of input sample DNA.

Brief description of the attached diagram

Figures 1A-1C. Formation of circularized MIP products in conventional MIP assays and novel ligated and linear MIP (LL-MIP) products used in this invention. Figure 1A shows a simple circularized MIP in a conventional MIP assay. Figure 1B shows other types of ligated LL-MIP products. Figure 1C shows the sequence of an exemplary LL-MIP product after cloning a PCR product, demonstrating the ligation of six MIP probes together in this LL-MIP.

Figure 2. The principle that the number of junctions represents telomere length or the number of genomic motifs. The abundance of junctions is an indicator of telomere length.

Figure 3. Gel showing the LL-MIP product after PCR. Lanes 1, 3, 4, and 5 show bands that are ~100 bp (MIP length). The size markers on the left show the 100 bp intervals.

Figure 4. Comparison of existing MIP measurements and the method for measuring telomere length in this invention.

Figure 5. Correlation between reported telomere length (kbp) measured by TRF and the abundance ratio of junctions representing telomere motifs relative to a reference sample, the ratio being determined by Δ-ΔCt values.

Figure 6 shows examples and data where the method of the present invention has a proportional (linear) response to the amount of DNA template added.

Figure 7. Examples and data used in efficiency (slope) corrected Δ-ΔCt quantification. This Δ-ΔCt value can be used as another measure of telomere length. In this example, a short MIP is used in the hybridization reaction.

Figure 8. Schematic workflow for quantifying tandem repeats after hybridization and ligation reactions. Each tandem repeat represents a unit of length of the target tandem repeat. The abundance of tandem repeats is quantified using methods such as qPCR. The normalized abundance can then be used as a biomarker for the length of the tandem repeat. It can also be converted to units of length (e.g., kbp in the case of telomeres) using a calibrated sample, such as the regression equations shown in the examples and Figure 5.

Figure 9. The index value of telomere length is not affected by the different starting DNA amounts used for hybridization.

Figure 10. Calibration of index values obtained from the method of this invention to TL (in kbp).

Figure 11. Two-dimensional clustering diagram of ddPCR used to quantify the linkage sites of two MIPs, in which channel 1 fluorescence (FAM, telomere linkage site) is plotted relative to channel 2 (HEX, α-satellite linkage site).

Invention Details

1. Introduction

This invention provides methods and compositions for determining tandem repeat length by quantifying the abundance of corresponding repeat motifs in a sample. One particular type of tandem repeat of interest herein is telomeres. The methods and compositions involve the use of molecular inverted probes (MIPs) specifically designed for tandem repeat sequences.

The methods and compositions of the present invention provide MIPs specifically designed for regions containing tandem repeats in the genome and utilize the diversity of ligation products generated in MIP-based assays on such regions, including circularized and ligated linear products. The methods quantify the circularized and ligated linear products to allow for reliable and rapid assessment of copy numbers of tandem repeat sequences (such as telomere sequences) that vary between individuals. In particular, in a preferred method, the present invention relates to the use of MIPs for tandem repeat sequences, wherein the 5' and 3' ends of the MIP contain identical sequences, which are typically 100% complementary to the template DNA sequence when bound within a single repeat unit on template DNA, and wherein they are designed to bind to template DNA strands with a small gap (e.g., 1-3 nucleotides) between the 3' and 5' ends of the MIP. Furthermore, the gap between the 3' and 5' ends of the present invention typically contains only one or two different nucleotides, allowing the use of one or two dNTPs in the methods, and thus ensuring that ligation of the ends occurs only within a single repeat unit of the tandem repeat, and not across multiple repeats. In a preferred embodiment, the method omits the step of removing linear products before detection and/or quantification, such as a step based on exonucleases, and instead quantifies the connection points of circularized and ligated linear products to determine the abundance of a specific repetitive motif in the sample as an indicator parameter of telomere length or the length of another genomic motif.

2. Definition

As used herein, the term “about” in relation to, for example, the size of a polynucleotide, percentage of homology, or any other quantitative measurement, means that there may be a small degree of variation within the provided values. For example, “about” means that the value may differ from the stated value by, for example, +/- 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and their polymers. Unless otherwise specified, the term covers nucleic acids containing known analogs of natural nucleotides, having similar binding properties to reference nucleic acids, and being metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a specific nucleic acid sequence also implicitly covers variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by producing a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and/or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

A molecular inverted probe (MIP) is a nucleic acid probe that hybridizes to the complementary sequence of a target nucleic acid of interest. The 5' end of the MIP hybridizes to the 5' end of the target from the 3' end of the MIP, meaning the 5' and 3' ends bind adjacently within a single repeat. When hybridizing with the target, the MIP forms a loop from one end to the other, such that the ends are "inverted" relative to each other, i.e., the 3' end is located at the 5' end of the MIP. The MIP sequences at both ends are configured to hybridize with the target sequence and are referred to herein as the 5' and 3' homologous regions. Between the 5' and 3' homologous regions is the adapter region, which is essentially not complementary to the target DNA sequence and therefore does not hybridize with the template DNA. This adapter region contains one or more functional elements, such as primer binding sites, tag sequences, etc. Typically, the MIP requires 5' phosphorylation for ligation to occur.

As used in this article, "circularization" refers to the joining of the 3' end of a nucleic acid to its 5' end, resulting in a continuous, uncut circular structure or loop. "Linked linear MIP," "LL-MIP," or "linked linear probe" refers to two or more MIPs linked together, where the 3' end of one MIP has been extended (through "gap filling") and joined to the 5' end of a different MIP that binds adjacent to the same DNA template (e.g., within a single repeat unit on the template at 3').

As used herein, the term "complementary" refers to hybridization or Watson-Crick base pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are typically A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are considered complementary when, after optimal alignment and comparison, the nucleotides of one strand with appropriate nucleotide insertions or deletions pair with the nucleotides of the other strand at least about 80%, typically at least about 90% to 95%, and more preferably about 98% to 100%. Alternatively, complementarity exists when the RNA or DNA strand hybridizes with its complement under selective hybridization conditions. Typically, selective hybridization occurs when there is at least about 65% complementarity, preferably at least about 75%, and more preferably at least about 90% complementarity in a segment of at least 14 to 25 nucleotides. See M. Kanehisa Nucleic Acids Res. 12:203 (1984). Typically, the homologous region of a MIP has 100% complementarity with the target nucleic acid of interest, although mismatches may be included in some implementations.

"Gap filling" is the reaction described herein, in which a gap is filled by polymerase action between the 5' and 3' ends of a molecularly inverted probe that hybridizes to a complementary target nucleic acid. Gap filling is also referred to herein as "extension" of the 3' end of the MIP probe, wherein the extension covers the gap and allows for ligation between the extended 3' end and the bound 5' end. In a preferred embodiment, the filled gap consists of 1-3 nucleotides or is confined within a single repeat motif unit.

The term "homologous region" as used herein, such as "3' homologous region" or "5' homologous region," refers to those portions of a molecularly inverted probe that are complementary to the target nucleic acid of interest. A MIP typically has two homologous regions (HRs), one at or near the 5' end of the probe and one at or near the 3' end. In a preferred embodiment, the HRs of the present invention are adapted to hybridize with target nucleic acids of interest containing tandem repeats, such that they are separated by a 1-3 nucleotide gap within a single repeat unit on the target DNA sequence. In a preferred embodiment, the homologous region is 100% complementary to the target sequence; however, in some embodiments, mismatches may exist within a given homologous region, such as one, two, three, or more mismatches.

As used herein, the term "hybridization" refers to a process in which two single-stranded polynucleotides non-covalently combine to form a stable double-stranded polynucleotide; triple-strand hybridization is also theoretically possible. The resulting (usually) double-stranded polynucleotide is called a "hybrid." Hybridization is typically performed under stringent conditions, for example, with a salt concentration not exceeding about 1 M and a temperature of at least 25 °C. Hybridization can be performed in the presence of reagents such as about 0.1 mg/ml of herring sperm DNA or about 0.5 mg/ml of (acetylated) BSA. Because other factors can affect the stringency of hybridization, including base composition and the length of the complementary strand, the combination of parameters is more important than the absolute measurement of any one of them alone. Further guidance on hybridization conditions applicable to various assays can be found, for example, in Michael R. Green & Joseph Sambrook, Molecular Cloning: A Laboratory Manual, (4th edition, 2012).

"Polymerase chain reaction" or "PCR" refers to the in vitro amplification of a specific DNA sequence by simultaneous primer extension of the complementary strand of DNA, as is well known in the art. PCR is a reaction used to prepare multiple copies or replicas of a target nucleic acid flanked by primer binding sites. Such a reaction comprises one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing the primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Typically, the reaction is cycled in a thermal cycler at different temperatures optimized for each step. The specific temperature, the duration of each step, and the rate of change between steps depend on many factors well known to those skilled in the art, such as those illustrated in references: McPherson et al. (eds.), PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in conventional PCR using Taq DNA polymerase, double-stranded target nucleic acids can be denatured at temperatures >90°C, primers are annealed in the range of 50–75°C, and primers are extended in the range of 72–78°C. The term “PCR” encompasses various forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, etc. “Multiplex PCR” refers to PCR in which multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously performed in the same reaction mixture, e.g., Bernard et al., Anal. Biochem., 273:221-228 (1999) (two-color real-time PCR). Typically, a different set of primers is used for each sequence being amplified. “Quantitative PCR” or “qPCR” refers to PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Quantitative PCR includes both absolute and relative quantification of such target sequences. Quantitative measurements are performed using one or more reference sequences, which may be measured separately from or together with the target sequences. For a sample or specimen, the reference sequence can be endogenous or exogenous; in the latter case, the reference sequence may include one or more competing templates. Typical endogenous reference sequences include segments of transcripts of genes such as β-actin, GAPDH, _β2 -microglobulin, and ribosomal RNA. Quantitative PCR techniques are well known to those skilled in the art, as illustrated in the following references: Freeman et al., Biotechniques, 26:112-126 (1999); Becker-Andre et al., Nucleic Acids Research, 17:9437-9447 (1989); Zimmerman et al., Biotechniques, 21:268-279 (1996); Diviacco et al., Gene, 122:3013-3020 (1992); Becker-Andre et al., Nucleic Acids Research, 17:9437-9446 (1989).

As used herein, the term "primer" refers to a single-stranded oligonucleotide that, under suitable conditions (e.g., buffer and temperature), in the presence of four different nucleosides of triphosphate and polymerization reagents (e.g., DNA polymerase, RNA polymerase, or reverse transcriptase), serves as the starting point for template-guided DNA synthesis. In any given case, the length of a primer depends on, for example, its intended use and is typically in the range of 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form a sufficiently stable and specific hybridization complex with the template. Primers do not need to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with such a template. The primer site is the template region that hybridizes with the primer. A primer pair is a set of primers consisting of a 5' upstream primer that hybridizes to the 5' end of the sequence to be amplified and a 3' downstream primer that hybridizes to the complementary element at the 3' end of the sequence to be amplified.

"Sample" means a quantity of material from a biological, environmental, medical, animal, bacterial, plant, or patient source from which detection or measurement of a target nucleic acid is sought. Typically, a sample is a lysate of an organism's cellular tissue. Generally, in the context of this invention, a sample includes material containing nucleic acids. Biological samples can be fluids, solids (e.g., feces), or tissues of animals, including humans, as well as liquid and solid food and feed products and ingredients (e.g., dairy products, vegetables, meat and meat by-products) and waste. Biological samples can include material taken from a patient, including but not limited to cultures, blood, saliva, cerebrospinal fluid, pleural fluid, milk, lymph, sputum, semen, needle aspirates, etc. Biological samples can be obtained from all different families of livestock as well as from wild or wild animals, including but not limited to animals such as ungulates, bears, fish, rodents, etc. Biological samples can also be obtained from plants such as corn, rice, wheat, lettuce, and pepper.

As used in this article, "target nucleic acid of interest" refers to nucleic acids in a sample that presumably include target sequences of interest, such as regions containing tandem repeats, like telomeres, or ATGG-containing regions of the genome. Regarding MIPs, target sequences of interest include those sequences complementary to homologous regions of the MIP.

Tandem repeats are nucleotide sequences in which short nucleotide sequence units, 2–60 nucleotides in length, are repeated multiple times, with the repeating units directly adjacent to each other. The exact nucleotide sequence of each repeating unit is called a repeat motif. Common examples of repeat motifs (also referred to as repeating units in this document) include [CA], [CAG], [GATA], etc. Tandem repeats are classified into microsatellites and microsatellites based on their length. Typically, microsatellite repeat motif units are short and less than 10 nucleotides. Dinucleotide repeats (e.g., [CA] _n repeats) are among the most well-known microsatellites or short tandem repeats in the human genome. Repeat motifs in microsatellites range in length from 10 to 60 nucleotides. As an example, the repeat motif [TTAGGG] in human telomeres can be repeated hundreds or thousands of times within the telomere at the end of each chromosome. Therefore, [TTAGGG] is a sequence motif specific to human telomere repeats, and [TTAGGG] _n is used to indicate the repeating nature of the motif in telomeres. It should be understood that a tandem repeat sequence can refer to one or two strands of DNA containing tandem repeats. Tandem repeats can vary in the genome, such as in the case of telomeres. This means that the number of repeat units, or copy number, or the total size of the genomic region carrying the repeat can vary to any degree between individuals or between different cells or cell types within an individual, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 times or more. Tandem repeats in the genome can also be “stable” or relatively “constant,” such as in the case of ATGG repeats, which means that the number of repeat units, or copy number, or the total size of the genomic region carrying the repeat does not vary between individuals, or between different cells or cell types within an individual, by more than, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% or more.

"Hybridization conditions" are the conditions expected to result in specific hybridization between complementary sequences. For example, regarding perfectly matched complementary targets, when the test nucleic acid also hybridizes with the probe at least 50% (e.g., quantified under the same hybridization conditions), that is, when a perfectly matched probe binds to a perfectly matched complementary target with a signal-to-noise ratio at least about 5x-10x higher than that observed for hybridization with any unmatched target nucleic acid, the test nucleic acid is considered to have specifically hybridized with the probe nucleic acid.

As used herein, in the context of describing two or more polynucleotide sequences, the terms "identical" or "percentage of identity" refer to two or more identical sequences or specified subsequences. Two sequences that are "substantially identical" have at least 60% identity when compared and aligned to the maximum correspondence within a comparison window or specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, as measured using sequence comparison algorithms or by manual alignment and visual inspection (if no specific region is specified). Regarding polynucleotide sequences, this definition also refers to the complement of the test sequence.

For sequence comparisons, a reference sequence is typically used, and the test sequence is compared to this reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. Default program parameters can be used, or optional parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters. For nucleic acid and protein sequence comparisons, the BLAST 2.0 algorithm and default parameters discussed below are used.

The “comparison window” used in this article includes any of the following segments selected from 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150 consecutive positions, wherein after the two sequences are best aligned, the sequence can be compared with a reference sequence of the same number of consecutive positions.

The algorithm used to determine sequence identity and sequence similarity percentage is the BLAST 2.0 algorithm, described in Altschul et al., (1990) J.Mol.Biol.215:403-410. The software used for BLAST analysis is publicly available on the website of the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short characters of length W in the query sequence. These short characters match or satisfy a certain positive threshold score T when compared to characters of the same length in a database sequence. T is called the neighboring character score threshold (Altschul et al., ibid.). These initial neighboring character hits serve as seeds for initiating the search to find longer HSPs containing them. Character hits are then extended in both directions along each sequence until the cumulative alignment score increases. For nucleotide sequences, a cumulative score is calculated using parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of a character hit in each direction is terminated when: the cumulative alignment score drops by X from its maximum realized value; the cumulative score reaches 0 or is below 0 due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to: word length (W) 28, expected value (E) 10, M = 1, N = -2, and two-strand comparison (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs statistical analysis on the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in the comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

3. Tandem repeating targeted molecular inverted probe

The MIP of this invention is a single-stranded DNA probe comprising three regions: a 5' homologous region located in the 5' portion of the MIP, a 3' homologous region located in the 3' portion of the MIP, and a central adapter region located between the 5' and 3' homologous regions. As described in more detail below, the 5' and 3' homologous regions are designed to be homologous to the same strand of the template DNA containing tandem repeat sequences, such that the 3' end can bind to the template adjacent to the 5' end (i.e., in the 3' direction of the template DNA), allowing the 3' end to extend and connect to the adjacent 5' end via DNA polymerase. However, it should be understood that due to the nature of the repeat sequences within the template DNA, the 5' and 3' ends can bind to any repeat unit within the tandem repeat, and therefore can be separated by multiple repeat units when binding. In contrast, the adapter region forms a loop between the hybridized 3' and 5' homologous regions and does not bind to the template DNA.

Homologous regions are typically 15-25 nucleotides long, for example, 18-22 nucleotides long, and are usually 100% complementary to the tandem repeat sequence within the template (i.e., they are 100% identical to the nonhybridized strand of the template DNA), although less than 100% complementarity, such as 90%, 95%, 96%, 97%, 98%, or 99%, or containing, for example, one, two, three, or more mismatched homologous regions, may also be used. The 5' and 3' regions typically extend to the corresponding ends of the MIP; that is, the 5' end of the 5' homologous region is also the 5' end of the MIP, and the 3' end of the 3' homologous region is also the 3' end of the MIP. In cases where the complementarity to the template tandem repeat sequence is less than 100%, the mismatched nucleotides typically do not contain the terminal 5' and 3' nucleotides of the MIP (i.e., the terminal nucleotides that will be extended and/or ligated by DNA polymerase in this method). Compared to other assays (e.g., real-time PCR telomere T/S ratio assays), the hybridization sequences at the 5' and 3' ends of the MIP of the present invention are preferably 100% identical to the segments of the telomere repeat motif [TTAGGG] _n without mismatched bases.

The central adapter region can vary in size, but its length is typically about 30-200 nucleotides, for example, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides, or any integer value corresponding to this range. In addition to having sufficient length to allow the 5' and 3' homologous regions to form a loop for binding the same template DNA, the central adapter region may also contain any of a variety of functional sequences for, for example, separating, purifying, detecting, cleaving, or quantifying the product of the method described in this invention. For example, the adapter region may contain universal primer sequences, MIP-specific primer sequences, probe sequences (e.g., TaqMan probe sequences), tag ID or barcode sequences, and restriction enzyme recognition or cleavage sites.

tandem repeats and homologous regions

The compositions and methods of the present invention can be used to detect the copy number or length of any region in the genome containing tandem repeat sequences, including regions where the total length or size, i.e., the copy number of the repeat, varies between individuals, such as telomeres. Any such sequence can be evaluated using the methods and compositions of the present invention. In many embodiments, the methods and compositions are used to determine the exact or approximate copy number, i.e., the total length, of telomeres, such as human telomeres. In many embodiments, tandem repeats whose size or copy number is substantially invariant between individuals and are therefore referred to as “stable” or “constant,” such as 4-bp short tandem repeat [ATGG] sequences in the human genome, are also evaluated.

Tandem repeats can be analyzed regardless of the length of a single repeat unit or the copy number of tandem repeats in the genome. For example, a 6-nucleotide human telomere sequence (TTAGGG) can be repeated hundreds or thousands of times, covering, for example, 1-11 kb in the genome. However, it is also possible to assess the copy number of tandem repeat sequences with fewer repetitions, such as tens to hundreds of repetitions in the genome, or the copy number of tandem repeat sequences with more repetitions, such as tens of thousands of repetitions in the genome.

The methods of the present invention can be used to assess the total length of telomeres or other tandem repeats in an individual, for example, to assess on a single basis whether telomeres are longer or shorter than a standard reference length, for example, to determine whether it indicates the presence or absence of a condition associated with altered telomere length, and they can also be used to assess the evolution of telomeres or other tandem repeats in a single individual, for example, to assess the efficacy of treatment regimens on, for example, telomere length.

The length of the individual repeat sequence units within the tandem repeats evaluated using the method of this invention can be of any size, such as 2-20 nucleotides, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more. In some embodiments, the repeat sequence is 6 nucleotides, as in human telomeres, or 4 nucleotides, as in human ATGG or AAGG repeats. In some embodiments, repeats that expand with certain disease states are evaluated, for example, CGG triplet repeats that expand with Fragile X syndrome or CAG triplet repeats that expand with Huntington's disease.

In addition to assessing variable tandem repeats such as telomeres, the methods and compositions can also be used to assess the length or copy number of relatively constant or stable repetitive sequences between individuals, for example, as an internal control when assessing the size of variable regions in the genome, such as telomeres. The size of such constant sequences preferably varies between individuals, for example, by an average of less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or less, or by an average relative level variation between individuals not exceeding, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold. In some embodiments, the constant sequences used in the methods of the present invention exist in the genome at large copy numbers, for example, containing hundreds or thousands of tandem repeats, such that they exist at a level similar to that of variable repeat sequences (e.g., telomeres). The use of such high copy number control sequences provides a significant advantage over conventional methods for assessing tandem repeats (e.g., telomere length), which tend to use non-repetitive sequences as controls.

For variable and constant repeats, the method can be used to assess the total size of repeating regions in the genome, for example, the length of the region reaching, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher precision, or the approximate or exact number of repeats reaching, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher precision.

In one implementation, the ATGG sequence is used as a constant control repeat sequence. Other tandem repeats that can be used include AAGG, GAATG, AATGG, ACTCC, CAGC, AACGG, AACAT, ACAGAG, AGGGTC, AAAAT, AAAAG, GAGAGG, and AACC. Any of these tandem repeats can be used, provided that their quantity is relatively stable in the genome of an individual of the species under study (e.g., in humans when assessing human telomere length) and that a suitable MIP can be designed to target the tandem repeat using the methods described herein.

5' and 3' cognate regions

The 5' and 3' homologous regions of the MIP contain nucleotide sequences homologous to tandem repeat sequences within the template DNA. In a preferred embodiment, the 5' and 3' regions are 100% homologous to the tandem repeat sequences, although in some embodiments, the 5' and/or 3' regions may contain mismatches, for example, one, two, three, or more mismatches within a given homologous region relative to the tandem repeat sequence. The 5' homologous region begins at the 5' end of the MIP and extends, for example, 15-25 nucleotides into the MIP, and the 3' homologous region begins at the 3' end of the MIP and extends, for example, 15-25 nucleotides into the MIP. The 5' and 3' homologous regions are complementary to the same strand of the template DNA, i.e., they both contain the same repeat sequence, for example, containing two, three, four, five, six, or more repeat units (e.g., an 18-base-pair homologous region targeting human telomeres would contain three 6-base-pair repeats). The 5' and 3' regions are designed such that the 3' homologous region can bind to the template at the 5' of the 5' homologous region within the same repeat unit on the template, but is separated by a small number of nucleotides (e.g., 1-3 nucleotides) on the template, so that ligation only occurs after the 3' end is extended with DNA polymerase to add 1-3 nucleotides into the gap (i.e., "gap filling").

When designing the MIP used in this invention, particularly the 5' and 3' homologous regions, an important consideration is the gap separating the 5' and 3' ends of the MIP when the two ends hybridize adjacently within a single repeat unit on the same DNA template. Typically, for the methods of this invention, the 5' and 3' ends of the MIP will be separated by a small number of nucleotides, such as 1, 2, or 3 nucleotides. It should be understood that the size of the gap between the 5' and 3' ends of the MIP can vary between different MIPs used in a given assay; that is, the MIP used to assess the copy number of variable tandem repeat sequences (e.g., telomeres) can be, for example, 1, 2, or 3 nucleotides, and the control MIP used to assess the copy number of constant tandem repeat sequences (e.g., ATGG) can be, for example, 1, 2, or 3 nucleotides, wherein the number of nucleotides in the two gaps is independent of each other.

It should be understood that the 5' and 3' homologous regions can be designed to hybridize with either strand of the template DNA, as long as the two regions share the same sequence, thus making them both target the same strand.

When bound within a single repeat unit of the template DNA strand, another consideration for the number of nucleotides separating the 3' and 5' ends of the MIP is the identity of the bases corresponding to the nucleotides within the nick. In a preferred embodiment of designing telomere-targeting MIPs, regardless of the number of nucleotides within the nick, the nucleotides will contain one or two bases, such that the extension step of the method of the present invention can be carried out in the presence of those deoxyribonucleoside triphosphates (dNTPs) corresponding only to one or two bases within the nick. In this way, as shown below, one or two dNTPs present in the reaction mixture are sufficient to allow extension across the nick within a single given repeat unit, but not sufficient to allow extension across multiple repeat units requiring, for example, three or four dNTPs.

For illustrative purposes, since the human telomere sequence motif is GGGTTA, the template (complementary) DNA strand for a MIP targeting this sequence will be [AATCCC] _n or a segment representing a length of 3 motifs...CCCAATCCCAATCCCAAT.... Therefore, the 5' end of a MIP targeting this sequence can be read as 5'-GTTAGGGTTAGGGTTAGGTT-, and the 3' end can be read as -TAGGGTTAGGGTTAGGGTTA-3'. In this way, if both ends bind adjacently to the same template, i.e., the 3' end is exactly the 3' of the 5' end within a single repeat, the G at the 5' end of the probe can hybridize up to the last (sixth base) C in the AATCC C repeat motif on the template, and the A at the 3' end of the probe can hybridize adjacently down to the T (third base) within the same repeat (i.e., from left to right, the third nucleotide terminates within AAT CCC). Thus, the two ends are separated by the first two Cs (the fourth and fifth bases) in the motif, so if DNA polymerase adds two Gs to the 3' end of the MIP, and if the second added G is then linked to the G at the 5' end of the MIP, the two ends can thus be joined together. In this case, when the two ends belong to the same MIP, the MIP will be circularized. Alternatively, if the two ends belong to different MIPs, a joined linear MIP will be produced, which contains two MIPs joined together.

Similarly, for human ATGG 4-base-pair repeats that can be used as control MIPs to assess copy number of constant or stable repetitive sequences in the genome, the template would be [CCTA] _n or ....TACCTACCTACC.... In this case, the 5' end of the MIP could be 5'-ATGGATGGATGGATGGATGGATGG-, and the 3' end of the MIP could be GGATGGATGGATGGATGGAT-3'. Therefore, if the two ends bind to the same template adjacent to each other, the A at the 5' end can hybridize up to the T in one of the repeat units within the template (i.e., from right to left, up to the seventh nucleotide in CCTACC T A), and the T at the 3' end can hybridize down to the A within the template (i.e., from left to right, up to the fourth nucleotide in CCT A CCTA). In this case, the two ends of the MIP will also be separated by two Cs within a single repeat, so if the DNA polymerase adds two Gs to the 3' end of the MIP, and if the second G is then linked to the A at the adjacent 5' end, the two ends can be joined.

In the two examples above, namely, using a MIP targeting human telomere sequences and a MIP targeting ATGG repeats, all nucleotides within the gap between the 3' and 5' ends of the two MIPs within a single repeat include the C base on the template. In this way, DNA polymerase-mediated extension of the 3' end of the MIP can proceed in the presence of dGTP alone, without other dNTPs in the reaction mixture. Since this utilizes two MIPs, multiplex reactions can be performed, where both MIPs are used simultaneously in the same reaction with the same genomic DNA template. Furthermore, if only dGTP is used, this also means that extension can only occur within a single repeat, and extension across multiple repeats (i.e., if the 3' and 5' ends bind to templates of more than one repeat unit separated from each other) is impossible, as this requires not only dGTP but also dATP and dTTP. Therefore, in many embodiments of the methods and compositions of the present invention, only one or two dNTPs are used or included.

In one embodiment, the only dNTP included in the reaction is dGTP. In other embodiments, only two nucleotides are included, dGTP and dATP. However, it should be understood that MIPs can be designed such that any combination of one or two dNTPs can be used, provided that one or more dNTPs used allow extension within (but not across) the tandem repeat unit of the MIP targeting telomeres.

Connector area

The adapter region, located between the 5' and 3' homologous regions, does not hybridize with the template DNA, i.e., it is not complementary to the template DNA. It serves both a spacer function and includes any of a number of elements that allow for, for example, the identification, separation, cleavage, or amplification of the probe. This spacer function allows the probe to assume a circular (loop) conformation when the 5' and 3' homologous regions bind to the same template DNA. Examples of such sequence elements include primer binding sites common to all MIPs used in a given assay, such as universal forward primer binding sites; probe-specific primer binding sites, such as reverse primer binding sites specific to each MIP; probe binding sequences, such as sequences bound by probes used for quantitative amplification reactions (e.g., TaqMan probes); tag or barcode sequences; and cleavage or restriction sites, which can be used, for example, to linearize the circularized MIP prior to the amplification reaction.

The linker region can be of any size, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more nucleotides, and can include any number of elements, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more elements.

The tag sequence or barcode contained in the adapter region can be a unique sequence that can encode any desired information, such as a sample identification sequence, target information sequence, sample acquisition time encoding sequence, etc., thereby allowing their identification by, for example, sequencing or hybridization characteristics. By including such a sequence, MIP products from multiple samples can be definitively detected in a single detection event. The tag element can include information such as within a “barcode” nucleic acid sequence, which can be read by, for example, a sequencing program or specific hybridization. Optionally, one or more tag elements can be affinity portions that can specifically bind the MIP to an identifiable array location. For example, the tag can be queried by an array of nucleic acid probes with polynucleotide probes complementary to various tag sequences. For example, the tag can contain a sequence complementary to a capture probe of a branched DNA (bDNA) program. The bDNA capture probes can be arranged in an array on a solid support, each location corresponding to a specific tag and sample. Optionally, the tag can correspond to beads with appropriate capture and signaling elements, for example, for detection in a FACS flow apparatus or by imaging with a charge-coupled device.

The cleavage site can be introduced, for example, by incorporating any of a number of modified nucleotides or bases into a linker region for cleavage, and/or by configuring a linker to be cleaved by, for example, uracil N-glycosylation enzymes or other suitable enzymes known to those skilled in the art, such as restriction enzymes.

Other nucleic acid derivatives may also be incorporated into the MIP of the present invention, for example, to prevent their digestion, particularly when they are exposed to biological samples that may contain nucleases. As used herein, nucleic acid derivatives are non-naturally occurring nucleic acids or units thereof. Nucleic acid derivatives may contain non-naturally occurring elements, such as non-naturally occurring nucleotides and non-naturally occurring backbone linkages.

Nucleic acid derivatives may contain backbone modifications, such as, but not limited to, phosphate-thioester bonds, phosphodiester-modified nucleic acids, phosphate-thioester modifications, combinations of phosphodiester and phosphate-thioester nucleic acids, methylphosphonates, alkylphosphonates, phosphate esters, alkyl phosphate-thioesters, aminophosphate esters, carbamate esters, carbonate esters, triphosphate esters, acetamidate esters, carboxymethyl esters, methyl phosphate-thioesters, dithiophosphate esters, p-ethoxy groups, and combinations thereof. The backbone composition of nucleic acids can be homogeneous or heterogeneous.

Nucleic acid derivatives may contain substitutions or modifications in the sugar and/or base. For example, they may comprise nucleic acids having a backbone sugar covalently linked to a low molecular weight organic group (e.g., a 2'-O-alkylated ribose group) other than a hydroxyl group at the 3' position and a phosphate group at the 5' position. Nucleic acid derivatives may include non-ribose sugars, such as arabinose. Nucleic acid derivatives may contain substituted purines and pyrimidines, such as C-5 propyne-modified bases, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-thiouracil, and pseudoisocytosine. In some embodiments, substitutions may include one or more of the following: sugar/base, a group linked to the base (including biotin, fluorescent groups (fluorescein, cyanin, rhodamine, etc.)), a chemically reactive group (including carboxyl, NHS, thiols, etc.), or any combination thereof.

In some implementations, other methods are used to enhance the formation of LL-MIP products, such as pre-forming internal self-loops by using locked nucleic acid bases in the linker region of the MIP. Locked nucleic acids (LNAs) contain one or more nucleotide building blocks in which additional methylene bridges anchor the ribose moiety in a C3’-endo (β-D-LNA) or C2’-endo (α-L-LNA) conformation. LNAs can be synthesized using commercial nucleic acid synthesizers and standard phosphoramide chemistry.

4. Isolation of biological samples and genomic DNA

To implement the method of the present invention, one or more MIP probes are hybridized, for example in a multiplex reaction, to a biological sample containing genomic DNA from an individual for which the length (e.g., approximate or precise copy number) of tandem repeat regions (such as telomeres) needs to be determined. In some embodiments, samples are taken from individuals who have characteristics, lifestyle factors, or conditions associated with altered telomere size, such as telomere loss, such as aging and various age-related conditions, cognitive decline, mental disorders such as depression, schizophrenia, stress-anxiety, cancer, cardiovascular disease, diabetes, and various lifestyle factors such as diet, smoking, physical activity, and alcohol consumption. The method can be performed in such individuals, or in individuals considered susceptible to such characteristics, factors, or conditions, for purposes such as screening or diagnostic purposes (i.e., in a one-off assessment comparing telomere length to a reference telomere length), or to monitor response to a given treatment regimen (i.e., where telomeres of a given individual are repeatedly measured over time to monitor their progression).

Genomic DNA from any source can be used in this invention, such as genomic DNA from peripheral blood cells. Other cell types, such as specific cell types like cancer cells, can also be used to monitor telomere length in specific cell types. Suitable samples include, but are not limited to, biological samples such as tissues and body fluids. For example, samples may be obtained from, for example, blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat and semen, skin, organs, etc.

Isolates of target nucleic acids are obtained from samples using methods known in the art. Isolation of nucleic acids from biological samples typically involves processing the biological sample in a manner that extracts genomic nucleic acids present in the sample and makes them available for analysis. Any isolation method that produces extracted/isolated genomic nucleic acids can be used in the practice of this invention.

Nucleic acids are typically extracted using techniques such as those described in Sambrook, J., Fritsch, E R., and Maniatis, T. (1980) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory. Other methods include: salting out DNA extraction (P. Sunnucks et al., Genetics, 1996, 144:747-756; S.M. Aljanabi and I. Martinez, Nucl. Acids Res. 1997, 25:4692-4693), trimethylammonium bromide DNA extraction (S. Gustincich et al., BioTechniques, 1991, 11:298-302), and guanidine thiocyanate DNA extraction (J.B.W. Hammond et al., Biochemistry, 1996, 240:298-300). Several protocols have been developed for extracting genomic DNA from blood.

There are also many kits available for extracting DNA from tissues and body fluids, and from sources such as BDBiosciences Clontech (Palo Alto, Calif.), Epicentre Technologies (Madison, Wis.), Gentra Systems, Inc. (Minneapolis, Minn.), MicroProbe Corp. (Bothell, Wash.), Organon Teknika (Durham, N.C.), Qiagen Inc. (Valencia, Calif.), Autogen (Holliston, Mass.); Beckman Coulter (B (Rea, Calif.), (AutoGenFlex STAR robot with QiagenFlexiGene chemistry is commercially available. For example, Autogen manufactures the FlexStar Automated Extraction Kit for use with QiagenFlexiGene Chemistry, and Beckeman Coulter manufactures the Agencourt GenFind Kit for bead-based extraction chemistry. User guides detailing the protocols followed are typically included in all these kits, for example, Qiagen literature on its PureGene extraction chemistry entitled "Qiagen PureGene Handbook," 3rd edition, dated June 2011.)

After obtaining cells from the sample, cells are preferably lysed to isolate genomic nucleic acids. The cell extract can undergo further steps to drive nucleic acid separation, such as differential precipitation, column chromatography, extraction with organic solvents, etc. The extract can then be further treated, for example, by filtration and/or centrifugation and/or with a dissociative salt such as guanidine isothiocyanate or urea, or with an organic solvent such as phenol and/or HCl ₃ , to denature any contaminating and potentially interfering proteins. Genomic nucleic acids can also be resuspended in a hydration solution, such as an aqueous buffer. Genomic nucleic acids can be suspended in, for example, water, Tris buffer, or other buffers. In some embodiments, genomic nucleic acids can be resuspended in Qiagen DNA hydration solution or other Tris-based buffers at approximately pH 7.5.

The size of the obtained genomic nucleic acids can vary depending on the type of extraction method used. The integrity and size of the genomic nucleic acids can be determined, for example, by pulsed-field gel electrophoresis (PFGE) using agarose gels.

In some embodiments, genomic DNA is fragmented prior to use in the methods of the present invention. Nucleic acids (including genomic nucleic acids) can be fragmented using any of a variety of methods, such as mechanical fragmentation, chemical fragmentation, and enzymatic fragmentation. Methods for nucleic acid fragmentation are known in the art and include, but are not limited to, DNase digestion, sonication, mechanical shearing, etc. (J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2. sup. and Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; P. Tijssen, "Hybridization with Nucleic Acid Probes--Laboratory Techniques in Bioc "Hemistry and Molecular Biology (Parts I and II)," 1993, Elsevier; C.P. Ordahl et al., Nucleic Acids Res., 1976, 3:2985-2999; P.J. Oefner et al., Nucleic Acids Res., 1996, 24:3879-3889; Y.R. Thorstenson et al., Genome Res., 1998, 8:848-855). US Patent Publication 2005/0112590 provides a general overview of various fragmentation methods known in the art.

In many implementations, genomic DNA is digested with restriction enzymes, such as enzymes having restriction sites not found in any sequence targeted by the MIP used in the assay. Restriction endonucleases recognize specific sequences within double-stranded nucleic acids and typically cleave both strands within or near the recognition site to fragment the nucleic acid. Based on the composition and cofactor requirements of naturally occurring restriction endonucleases, the nature of their target sequences, and the position of their DNA cleavage sites relative to the target sequence, they are classified into four groups (Types I, II, III, and IV). Bickle T A, Kruger D H (June 1993). "Biology of DNA restriction". Microbiol. Rev. 57(2):434-50; Boyer H W (1971). "DNA restriction and modification mechanisms in bacteria". Annu. Rev. Microbiol. 25:153-76; Yuan R (1981). "Structure and mechanism of multifunctional restriction endonucleases". Annu. Rev. Biochem. 50:285-319. All types of enzymes recognize specific short DNA sequences and perform endonuclease cleavage to produce specific fragments with a terminal 5'-phosphate. Enzymes differ in their recognition sequences, subunit composition, cleavage sites, and cofactor requirements. Williams RJ(2003). "Restriction endonucleases: classification, properties, and applications". Mol. Biotechnol. 23(3): 225-43.

In one embodiment, before inactivating the enzyme (by incubating at 90°C for 20 minutes), centrifuging the DNA (e.g., at 7 krpm for 10 minutes), and collecting the supernatant, the DNA is digested with AluI (e.g., from New England Biolabs), for example, digesting 50 ng of DNA with 10 units of AluI at 37°C for 2 hours. The DNA can then be stored, for example, at 4°C until used in the method of the present invention.

In another embodiment of the invention, the methods and compositions of the invention can be used to assess, i.e., quantify, the degree of methylation at the genome scale. For example, in a DNA sample pre-digestion step, a methylation-sensitive restriction enzyme can be added to the pre-digested DNA sample. The results of the two reactions from the same sample are then compared, i.e., in the presence of a methylation-sensitive restriction enzyme (whose ability to cleave DNA depends on the presence of methylated nucleotides) and in the presence of a methylation-insensitive restriction enzyme (isoschistoses) that recognizes the same restriction site. For example, enzyme pairs HpaII (methylation-sensitive) and MspI can be used. For this approach, additional MIPs designed to target CpG islands can be used in conjunction with a genome reference MIP (such as the 4-base pair (ATGG) MIP described herein).

In some embodiments, the method of the present invention also provides denaturation of genomic DNA to make it single-stranded for hybridization with the MIP probes of the present invention. Denaturation can be produced by a fragmentation method selected as described above. For example, those skilled in the art will recognize that genomic nucleic acids can be denatured during fragmentation by pH-based cleavage or via a nick-generating endonuclease. Denaturation can occur before, during, or after fragmentation. Furthermore, the use of pH or heating during the fragmentation step can produce denatured nucleic acid fragments. See, for example, McDonnell, “Antisepsis, disinfection, and sterilization: types, action, and resistance”, pg. 239 (2007).

Thermal denaturation is the process by which double-stranded deoxyribonucleic acid (DNA) unwinds and separates into single strands by breaking hydrogen bonds between bases. Thermal denaturation of nucleic acids with unknown sequences typically uses sufficiently high temperatures, such as 95-98°C, to ensure denaturation even of nucleic acids with very high GC content in the absence of any chemical denaturing agents. Optimizing the conditions used for nucleic acid denaturation (e.g., time, temperature, etc.) is within the capabilities of those skilled in the art.

Denaturation of nucleic acids using pH is also well known in the art, and such denaturation can be accomplished using any method known in the art (such as introducing nucleic acids into high or low pH, low ionic strength, and/or heat), which disrupts base pairing, causing the double-stranded helix to dissociate into single strands. For pH-based denaturation methods, see, for example, Dore et al., Biophys J. 1969 November; 9(11):1281-1311; A.M. Michelson, The Chemistry of Nucleosides and Nucleotides, Academic Press, London and New York (1963). Nucleic acids can also be denatured by electrochemical means, such as applying a voltage to the nucleic acids in solution via electrodes.

Hybridization, extension and connection

It should be understood that aspects of the present invention may involve altering the amount of genomic nucleic acid and the amount of MIP probes to achieve customized results. In some embodiments, the amount of genomic nucleic acid used per subject ranges from 1 ng to 10 g (e.g., 500 ng to 5 μg). However, higher or lower amounts may be used (e.g., less than 1 ng, greater than 10 μg, 10-50 μg, 50-100 μg or higher). In some embodiments, for each tandem repeat of interest, the amount of probe used for each assay may be optimized for a specific application. In some embodiments, the ratio (molar ratio, e.g., measured as a concentration ratio) of the MIP probe to a genomic equivalent (e.g., a diploid genomic equivalent) ranges from 1 x ^10⁶ to ¹ x 10¹² or 1e⁶ to 1e¹². However, lower, higher, or intermediate ratios may be used, particularly as a function of the abundance of repetitive motifs in each genome.

In the method of this invention, to ensure maximum coverage of each repeat within the repeat array (e.g., at telomeres), the MIP is typically present in excess relative to the genomic DNA. The ratio of the aforementioned MIP probe to its genomic equivalent is higher than that used in conventional MIP assays. For example, this ratio is as high as 1000x or more compared to conventional MIP methods, to saturate potential binding sites (e.g., repetitive sequences) within the genome and to maximize the number of ligation products generated (whether circular or linear). In one embodiment, 50 ng of genomic DNA (e.g., 5 μl of a 10 ng/μl solution) is used, with 2 μl of a 10 nM stock solution of each MIP added. In this way, in the presence of DNA polymerase activity and appropriate dNTPs, and in the presence of ligase activity, a large number of ligation products reflecting the number of repeats present in the genome will be generated. The use of excessive MIP relative to genomic DNA contrasts with the traditional teachings of classic MIP assays, in which using high levels of MIP relative to genomic DNA only increases the likelihood of generating nonspecific and (undesired) linear products and decreases the likelihood of generating (desired) cyclized products.

In the presence of reagents and enzymes, the MIP and genomic DNA are incubated, allowing the 3' MIP to extend for hybridization, and the extended 3' end is subsequently ligated to the 5' end, which binds to the same template within the same tandem repeat unit. In one embodiment, a first hybridization step is performed, comprising the following components: genomic DNA, MIPs targeting the tandem sequence of interest (e.g., MIPs targeting telomeres and MIPs targeting ATGG repeats), appropriate buffer (e.g., 10x Amp ligase buffer), and water to the desired final volume. The hybridization step can be performed according to standard methods, for example using a thermal cycler with the following temperature cycles: 1. 95°C for 10 minutes; 2. 72°C for 1 minute with a slow temperature change rate (e.g., 0.1°C/s); 3. 56°C for 5 minutes with a slow temperature change rate (e.g., 0.1°C/s); 4. Proceed to step 2, 10x; and 5. 56°C for 16 hours.

Following hybridization, a gap-filling and ligation step is performed, wherein, for example, the following reagents are added to the hybridization mixture: ligase (e.g., 5 U of Amp ligase; e.g., from Lucigen), DNA polymerase (e.g., 0.6 U of T4 polymerase; e.g., from New England Biolabs), appropriate deoxyribonucleoside triphosphate (e.g., dGTP, e.g., 2.5 μl of 2.5 mM stock solution), BSA (e.g., 1X), and buffer (e.g., Amp ligase buffer) and water to the appropriate final buffer concentration and total volume. Gap-filling and ligation are then performed according to standard methods, e.g., using a thermal cycler with the following temperature cycles: 37°C for 30 minutes; 75°C for 20 minutes.

The above experimental conditions are provided for illustrative purposes, and it should be understood that the experimental conditions and/or properties, amounts, or presence or absence of different reagents used can vary in all reaction steps of the present invention, for example, regarding the temperature, duration, or number of cycles of hybridization, gap filling/ligation, and amplification steps, and regarding the reagents used. Such optimization of reaction conditions is entirely within the capabilities of those skilled in the art. In some embodiments, higher concentrations of MIPs and/or dNTPs than those described above are used in the reaction.

As described elsewhere in this document, the method of the present invention typically omits the exonuclease step (compared to conventional MIP-based assays), although it may include short exonuclease treatments to remove unligated MIPs or for quality control or comparison purposes.

5. Products and control measures generated using the new experimental design

As described above, when hybridization, extension, and ligation reactions are performed only in the presence of those deoxynucleotides required to cover the gap between the 3' and 5' ends of the same template within a single repeat, two types of products can be produced. The first product, produced when the 5' and 3' ends belong to the same MIP, is a cyclized ligation product. Such a cyclized product corresponds to the expected product of a previous MIP assay. The second product, produced when the 5' and 3' ends belong to different MIPs, is a ligated linear product (or "LL-MIP"). In classical MIP assays, for example, all linear products are removed by exonuclease activity, and only the cyclic product is quantified, detected, or analyzed.

The method of this invention produces several types of products, including circularized MIPs described in various classical MIP assays and novel ligation products called LL-MIPs, which contain two or more MIPs. Specifically, due to the large number of MIP binding sites present within long tandem repeats, and due to the high concentration of MIPs used in the method of this invention relative to the concentration of genomic DNA, multimeric forms are produced, in which 3, 4, 5, 6, 7, 8, 9, 10, or more MIPs are linked together. These products together reflect the copy number of tandem repeats in the genome. Just as a circularized probe reflects a ligation event (the ligation point in Figure 1), similarly, an LL-MIP containing two MIPs also reflects a ligation point, and both represent a length unit of the tandem repeat. Furthermore, LL-MIPs containing more than two MIPs reflect multiple ligation events and thus multiple length units of the target tandem repeat (e.g., telomeres). Typically, the number of ligation events in a multimeric LL-MIP containing n MIPs is n-1. Therefore, in many embodiments of the invention, all linked products obtained by the method, including cyclic products and linear products comprising two or more individual MIPs linked together, are retained and quantified to determine the overall size, i.e., copy number, of tandem repeat sequences such as telomeres. The overall abundance of the linkage points or the copy number of such motifs indicates the length of telomeres or other tandem repeats (Figure 8).

Telomere repeat motifs [GGGTTA] consist of three types of nucleotides: G, T, and A. While the binding sites at the 5' and 3' ends of the MIPs are randomly distributed, some can bind to the same motif, as described above, while others can span multiple repeats. For telomere length quantification, it is necessary to perform subsequent reaction steps and count only those MIP ligations occurring within the same individual motif. This can be achieved by limiting the number of dNTPs used in the nick-filling reaction step to a maximum of two dNTPs. In one embodiment, only one dNTP (dGTP) is used in the nick-filling reaction. However, it should be understood that this requirement is not critical for other reference (stable) tandem repeats. For example, when the [AAGG] tandem repeat is used as the reference tandem repeat in a telomere length quantification reaction, dATP and dGTP can be provided in the nick-filling reaction.

6. Separation, detection, and/or quantification of products.

As described elsewhere in this document, in previous MIP-based assays, all linear products were removed by enzymatic methods (using, for example, a combination of exonucleases, including exonuclease III) or via affinity binding (using, for example, a biotin-tagged MIP with an open 5' end). This is inappropriate in the context of measuring telomere length or other tandem repeat sequences because it will incorrectly reduce the calculated size (i.e., copy number) of telomeres or other repeat sequences. This is because telomere length is proportional to the total number of junctions, regardless of whether these junctions are within the cyclized product or within the LL-MIP.

Therefore, in one embodiment, exonucleases are not used in the method of the present invention to quantify the size of telomeres or other tandem repeats. However, in some embodiments, only exonuclease I is used, and only at limited levels (e.g., less than 1, 2, 3, 4, 5, 10, 15, or 20 units) and for a limited duration (e.g., up to 1 hour, or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes) to remove free (i.e., unligated) MIP probes without degrading ligated linear MIPs.

The abundance of linkage sites in the linkage products (including cyclized and linear products (LL-MIP)) obtained using the method of this invention can be quantified in any of a variety of ways, including relative quantification by qPCR with or without a reference sample and/or efficiency correction; absolute quantification by qPCR using a precisely measured amount of clonal MIP product as a calibrator sample; digital PCR, such as droplet digital PCR; or counting the number of various MIP products by sequencing, such as high-throughput sequencing.

In one embodiment, PCR, such as qPCR using TaqMan probes or other standard methods, is used to quantify the abundance of linkage sites in a circularized and ligated linear MIP. For example, qPCR can be performed using primers common to all MIPs used in the reaction (e.g., a universal forward primer) as well as MIP-specific primers (e.g., reverse primers specific to each MIP). In one embodiment, the universal forward primer is longGC_M13_F (ggcgcatggcTCA CAC AGG AAA CAGCTA TGA C). When used to determine telomere length, in one embodiment, the telomere-MIP-specific reverse primer is longGC_Link_R_teltail (gcgcatgtgaATC GGG AAG CTG AAG TAA CC), and the ATGG-MIP-specific reverse primer is longGC_Link_R_teltail (gcgcatgtgaATC GGG AAG CTG AAG TAA CC). In addition, when performing the assay in TaqMan, probes specific to each MIP can be used, such as Taq-3-Telo (for telomere MIPs; /56-FAM/TA GGG TTA G/ZEN/G GTT AGG GTT/3IABkFQ) and Taq-3-ATGG (for ATGGMIPs; /5HEX/GG ATG GAT G/ZEN/G ATG GAT GGA T/3IABkFQ).

Such reactions can be performed according to standard methods, for example, using the following reagents: MIP product (e.g., 5 μl); PCR premix (e.g., 7.5 μl of 2X stock solution); universal forward primer (e.g., primer M13_F; 0.375 μl of 20 μM stock solution); reverse primer for MIP 1 (e.g., telomere reverse primer; 0.375 μl of 20 μM stock solution); reverse primer for MIP 2 (e.g., ATGG reverse primer; 0.375 μl of 20 μM stock solution); TaqMan probe for MIP 1 (e.g., Taqman-telo; 0.3 μl of 10 μM stock solution); TaqMan probe for MIP 2 (e.g., Taqman-4bp; 0.3 μl of 10 μM stock solution); and water added to a final volume of 15 μl.

Quantitative PCR can be performed on a thermal cycler, such as the Roche LC480 thermal cycler, using standard temperature cycles, for example: 1.95°C for 10 minutes; 2.95°C for 10 seconds; 3.65°C for 30 seconds; 4.72°C for 10 seconds; 5. Proceed to step 2, 40x.

However, it should be understood that the amount of each reagent (e.g., each primer) and other reaction conditions (e.g., the temperature cycle used) can be varied to obtain optimal results; such optimization of reaction conditions is entirely within the capabilities of those skilled in the art.

In some implementations, next-generation sequencing (NGS) is used to evaluate MIP products, which is capable of determining the nucleic acid sequence of, for example, a probe or probe product in a multi-parallel manner. Exemplary NGS technologies include, for example, sequencing by synthesis (Illumina, Inc., San Diego, Calif.), real-time single-molecule sequencing with zero-mode waveguides (Pacific Biosciences of California, Inc., Menlo Park, Calif.), pyrosequencing, ion semiconductor sequencing (Thermo Fisher Scientific Corporation, Carlsbad, Calif.), and sequencing by ligation (Thermo Fisher Scientific Corporation, Carlsbad, Calif.). Typically, for genome sequencing via NGS, full-length nucleic acids (e.g., gDNA) are split into “template” fragments. The template is then captured or immobilized at typically spatially separated detector locations, allowing hundreds to billions of sequencing reactions to be performed simultaneously. In some cases, the template must be amplified before sequencing to allow sufficient signal.

7. Determine the copy number of tandem repeat sequences.

Once the ligated MIP product has been quantified using, for example, qPCR, the length of the variable tandem repeat region in the genome has been determined. This determination can be performed in any of a variety of ways, such as by calculating parameters based on the relationship between the abundance of targets (e.g., telomeres) obtained by qPCR, digital PCR, or high-throughput sequencing of the ligated MIP product and stable control tandem repeats. Such methods are outlined and illustrated in the following examples.

In one implementation, Δ-Δ-Ct is determined by calculating the Δ-Δ-Ct of two qPCRs (i.e., the qPCR for the target MIP and the qPCR for the control MIP) and using a reference sample. For example, Δ-Δ-Ct is determined for each sample by determining the Ct of each qPCR, i.e., the difference between the Ct of the variable tandem repeat sequence in the sample and the Ct of the stable control repeat sequence, and Δ-Δ-Ct is calculated by determining the difference between the Δ-Δ-Ct of the sample and the Δ-Δ-Ct of the reference sample after averaging the different Δ-Δ-Cts of a given sample (if, for example, qPCR is performed in duplicate, triplicate, etc.). The reference sample can be any suitable sample where the size of the variable tandem repeat region is known and/or appropriate depending on the purpose of the determination discussed; for example, for a method of determining the telomere length of a patient with or suspected of having cancer, the reference sample can be from a healthy individual without cancer. Once the Δ-Δ-Ct of each sample is determined, the total length of the tandem repeat sequence in each sample can be determined. For example, assuming an efficiency value of 2, the ratio of the number of connection points in a variable tandem repeat sequence (e.g., telomeres) to the number of connection points in a control tandem repeat sequence (e.g., an ATGG-containing tandem repeat sequence) is 2^(Δ-ΔCt). The Δ-Δ-Ct and ratio for each sample can be used as novel biomarkers for tandem repeats, such as telomere length.

Several other methods for quantifying PCR products in qPCR reactions include relative and absolute quantification methods. In one implementation, serial dilutions of a reference sample are quantified to obtain Ct values to calculate the overall assay efficiency. Alternatively, the reference sample can be used for all qPCR batches, and samples are compared to the same reference sample to remove batch effects due to inter-plate variations in qPCR Ct values. These methods (referred to herein as Δ-Δ-Ct methods and efficiency-corrected Δ-Δ-Ct methods) can be applied to quantify the linkage points of two MIPs.

Furthermore, absolute quantification of qPCR can be performed if a calibrator with a known (given) amount of linkage sites is used in the same qPCR with the test sample. This method is referred to as absolute quantification of qPCR results. In other embodiments, other methods for quantifying linkage site abundance can be used, including digital PCR and sequencing.

In addition, as described in the following examples section, further assays can be performed to determine the overall efficiency of qPCR performed with different MIPs, as well as the linearity (e.g., different starting DNA concentrations) and specificity of the reaction. Furthermore, the products can be amplified by, for example, conventional PCR and cloned and sequenced, or separated by gel electrophoresis, to assess, for example, the size and properties of the obtained LL-MIP products. Such procedures can be performed by the kit manufacturer, for example, during the kit development phase.

The overall efficiency of an assay can be calculated by combining the efficiencies of the MIP hybridization-gap-filling-ligation reaction and the qPCR reaction, given a specific MIP. This calculated overall efficiency can be used to correct for the slope difference of the Ct values of the two (or more) MIPs used in the assay, allowing for an index value corresponding to the relative ratio corrected for the efficiency (slope). This value can be used to express the length of the tandem repeats as a ratio compared to a reference sample. For example, given a sample with a given telomere length, such as the cancer cell line shown in Figure 5, other samples can use their ratios as independent variables (x-axis) and read their telomere length values based on the regression line in Figure 5 (y-axis).

Example

The invention will be described in more detail through specific embodiments. The following embodiments are provided for illustrative purposes only and are not intended to limit the invention in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results.

Example 1: A novel MIP-based method for determining telomere length or other variable tandem repeat sequences.

In this embodiment, telomere repeat length was investigated using a telomere motif MIP probe, which contains telomere repeat sequences (GGGTTA) separated by adapter sequences at both its 5' and 3' ends. The sequence targeting length of the telomere repeat motifs at both ends of the telomere MIP can range from, for example, 15 to 25 base pairs.

To normalize the amount of input DNA in the sample, a second MIP is used to quantify the number of diploid genomic equivalents in the sample DNA. In another embodiment, other methods may also be used to represent diploid genomic equivalents in the sample DNA. For example, single-copy genes can be quantified by qPCR as an indicator for this purpose.

The precise matching/complementarity provided by the preferred MIP of this invention, for example, 100% complementarity between the 5' and 3' homologous regions and the target sequence of the template DNA, makes it feasible to detect repetitive motifs (such as telomeres), and common tandem repeat sequences, such as the common 4-base-pair sequence ATGG, can be used as normalization controls. ATGG or other similar 4-bp tandem repeats are suitable control repeats for such normalization because they are abundant in the human genome and exist at roughly similar (stable) levels per diploid genome equivalent in samples taken from different individuals.

The MIP used to quantify the 4-base-pair tandem repeat consists of 4-base-pair repeat motifs (ATGG in this example) separated by a linker sequence at its 5' and 3' ends. Similar to telomere-targeting MIPs, the sequence length (i.e., the 5' and 3' homologous regions) of the ATGG repeat motifs at both ends of the 4-base-pair motif MIP can range from, for example, 15 to 25 base pairs.

Since telomeres and 4-base pair repeating motifs are tandem repeats, with the same 6-base pair or 4-base pair motif being tandem repeats, this special sequence configuration poses a new challenge to conventional MIP determination methods, which are based on the specific detection of cyclized MIPs after ligation.

Because the 3' and 5' ends of a MIP can hybridize with the same repeating target motif, conventional MIP assays cannot control the hybridization distance between the two ends of the same MIP molecule. In other words, the gap between the hybridization positions of the 3' and 5' ends is random and cannot be determined; it can be within a single repeating motif or it can span multiple repeats.

Therefore, for the specific purpose of quantifying the length of telomeres or other tandem repeat sequences, quantification of ligation products as an indicator of telomere length will be problematic if the gap distance between the 3' and 5' ends of the MIP hybridized with the template DNA is random, as some ligation products will represent one motif unit while others will represent two, three, or six or even more motif units. For example, if multiple motifs hybridized at two ends are separated from each other, quantification will underestimate the abundance of telomeres and therefore underestimate telomere length, as multiple repeat units will be counted as one repeat unit. Therefore, the ideal assay should provide a ligation signal for each motif repeat (Figure 2). This is achieved in various ways in the methods and compositions of the present invention, particularly by using (1) specially designed MIP probes and (2) a limited number of deoxyribonucleotide triphosphates (dNTPs) in the gap-filling/extension step. In one embodiment, the gap between the 3' and 5' ends of the telomere MIP is “GG”. Similarly, in one embodiment, the gap between the 3' and 5' ends of the 4-base-pair (ATGG) tandem repeat MIP is “GG”.

This MIP design, where both the telomere MIP and the 4-base-pair repeating MIP have GG nicks, allows the use of dGTP alone in the MIP extension step without the addition of other dNTPs. During the extension process, only dGTP is present in the reaction mixture, so only MIP molecules that hybridize to each other at their ends—that is, MIP molecules with a 2-nucleotide GG nick separating their ends—are ligated. In contrast, when the nick between the 5' and 3' ends of the telomere MIP is larger than 2 nucleotides, i.e., they are separated by more than one repeating motif unit, such that the nick includes "GGGTTAGG", "GGGTTAGGGTTAGG", or (GGGTTA)nGG, where 'n' is any integer, the ends cannot be extended and cannot be ligated.

Similarly, when the gap between the 3' and 5' ends of a 4-base pair (ATGG) tandem repeat MIP is greater than 2 nucleotides, i.e. they are separated by more than one motif (repetition) unit, such that the gap includes "GGATGG", "GGATGGATGG", or (GGAT)nGG, where n' is any integer, any MIP with multiple separate units hybridized at the ends cannot complete its extension and cannot be joined.

In one implementation, another type of telomere MIP (referred to as MIP-TelF) is used, where the gap between the 3' and 5' ends is “AGG”. In this case, the MIP elongation reaction involves only two deoxyribonucleoside triphosphates, namely dATP and dGTP, in order to restrict the formation of the ligation product to hybridization within a single repeat unit at its 3' and 5' ends and prevent those MIPs from elongating across multiple repeat units.

The sequence of MIP-TelF: 5'/Phos/GTT AGG GTT AGG GTT AGG GTT ACTTCAGCTTCCCGATCCGACGGTAGTGTTCA CAC AGG AAA CAG CTA TGA CTAG GGT TAG GGT TAG GGT T.

The repetitive nature of the motif unit leads to the formation of a linked and linear MIP product (referred to as LL-MIP) after ligation (Figure 1-3). In previous MIP assays, such linear MIPs were completely removed by enzymatic methods (e.g., using a combination of exonucleases, including exonuclease III targeting linear double-stranded DNA) or by affinity-based methods (e.g., biotin-tagged MIPs with an open 5' end).

However, this is inappropriate for measuring telomere length because it would incorrectly reduce the motif count (i.e., the calculated copy number) of telomeres or other tandem repeat sequences. In fact, we found that a significant proportion of telomere MIPs formed LL-MIPs upon binding to the telomere motifs of the sample template (Figures 1-3). These LL-MIPs were cloned and sequenced to confirm that their sequences contained multiple MIPs and that their target sequences were linked together (Figure 1C).

In a preferred embodiment of the method of the present invention, both quantitatively cyclized MIPs (conventional MIP products) and LL-MIPs (a new form of MIP product specifically retained herein, including LL-MIPs comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more individual MIPs linked together) are simultaneously cyclized. Thus, each connection point or signal represents a specific unit of telomere length (i.e., a repeating unit within a tandem repeat).

Quantification can be performed using various methods, including but not limited to:

1. Relative quantification by qPCR with or without reference samples and/or efficiency correction;

2. Absolute quantification was performed by qPCR using precisely measured amounts of cloned MIP products as a reference sample;

3. Digital PCR, such as digital PCR via droplets.

4. Sequencing, for example, high-throughput sequencing to count the number of various MIP products.

Example 2: Hybridization, extension, and ligation reactions

DNA samples pretreated with restriction enzymes

Add up to 10 units of restriction enzyme, such as AluI (New England Biolab), to a 50 ng DNA sample and incubate the sample at 37°C for 1 hour. Then inactivate the enzyme at 80°C for 20 min. After centrifuging at 7,000 rpm for 10 min and collecting the supernatant, the digested sample can be kept at 4°C.

MIP hybridization steps

A MIP probe targeting human telomere repeats with a GG notch was used, as well as a second MIP targeting the ATGG repeat motif, also with a GG notch. 5' phosphorylation was required for all MIP probes.

Probes targeting telomere repeat motifs (with notches "GG"):

MIP-TelF+A:/5Phos/GT TAG GGT TAG GGT TAG GGT TAC TTC AGC TTC CCG ATCCGA CGG TAG TGT TCA CAC AGG AAA CAG CTA TGA CTA GGG TTA GGG TTA GGG TTA

Probes targeting the 4bp (ATGG) repeat motif (with a "GG" notch):

MIP-ATGG:/5Phos/AT GGA TGG ATG GAT GGA TGG ATG GCT TCA GCT TCC CGATCC GAC GGT TAG GTT CAC ACA GGA AAC AGC TAT GAC GGA TGG ATG GAT GGA TGG AT

Both probes were designed to leave a “GG” gap between the 3’ and 5’ ends when they bind adjacently to the template DNA; these two nucleotide gaps must be filled by DNA polymerase before template-dependent ligation can occur.

Perform the hybridization procedure overnight as follows (values in parentheses are the stock concentration):

The hybridization scheme was performed in a thermal cycler using the following temperature cycles, but it can also be performed using other suitable equipment well known in the art:

After the hybridization procedure, add the following reagents to the above 25 μl to obtain a total of 30 μl (values in parentheses are stock concentrations):

The following are the notch filling and connection temperature schemes:

37℃ for 30 minutes, then

Set the temperature to 75℃ for 20 minutes.

In this embodiment, exonuclease treatment is not performed, but it may be added herein as an optional step. As highlighted elsewhere in this disclosure, this method is specifically designed to detect circularized MIPs (also referred to as typical or conventional MIP products) and novel LL-MIP products. Therefore, exonuclease treatment is not required.

However, for quality control or comparative purposes, such as those performed by the kit manufacturer, an exonuclease digestion step may be added. This is also done in some instances to demonstrate that the use of exonucleases adversely affects assay performance. In some embodiments, a limited amount of exonuclease 1 (e.g., 20 U for 50 ng of genomic DNA) may be added for a limited time, e.g., up to 1 hour, to remove unligated probes but not to eliminate ligated linear products.

Example 3: Quantitative analysis of cyclized MIP and LL-MIP products

The total number (or abundance) of linkage sites in circulated MIP and LL-MIP products is a new indicator for calculating telomere length. Quantification can be performed in any of a variety of ways, including, for example, by (1) quantitative PCR (qPCR) using relative or absolute quantification methods, (2) digital PCR, (3) sequencing, or (4) any other suitable quantification method.

In a typical implementation, three or four primers are used to amplify and quantify the abundance of the junction between the telomere MIP and the genomic reference MIP (e.g., 4-bp MIP-ATGG). In this example, a total of three primers are used because the forward primer (called longGC_M13_F) is universal for both the telomere MIP and the genomic reference (control) 4bp-MIP-ATGG, and two additional primers specific to either the telomere-specific probe or the control ATGG probe are used. A 5’ GC-rich tail is added to all three primers.

Universal primers and MIP-specific primers used:

longGC_M13_F:ggcgcatggcTCA CAC AGG AAA CAG CTA TGA C

longGC_Link_R_tel tail:gcgcatgtgaATC GGG AAG CTG AAG TAA CC

longGC_Link_R_ATGG:gcatggcgcaATC GGG AAG CTG AAG CCA tccat

In this implementation, two TaqMan probes are used in qPCR to distinguish ligation products formed by two types of MIP probes.

Taq-3-Telo:/56-FAM/TA GGG TTA G/ZEN/G GTT AGG GTT/3IABkFQ/

Taq-3-ATGG:/5HEX/GG ATG GAT G/ZEN/G ATG GAT GGA T/3IABkFQ/

The PCR reaction tube preparations (total volume 15 μl) are as follows:

qPCR was performed in a Roche LC480 thermal cycler using the following PCR temperature cycling protocol:

Variations and extensions of typical experimental procedures

Depending on the characteristics of the sample template or target motif, variations of the typical experimental steps presented above can be implemented to improve measurement accuracy and specificity.

Such changes include, but are not limited to:

1. Further increase the MIP level, such as 1.5x, 2x, 4x, etc. of the given concentration mentioned above.

2. Further increase the concentration of dGTP or the specific nucleoside triphosphate provided, such as 2x, 4x, etc. of the concentrations mentioned above.

3. In other implementation schemes, alternative methods can be used instead of the second MIP (mutually invoked protein) of the query control tandem repeat as an indicator of the presence of diploid genomic equivalents in the sample. For example, qPCR of single-copy genes or other genetic markers can be performed directly on the input sample to normalize the telomere MIP results.

Example 4: Results

The current metric for telomere length is derived from the number (abundance) of junctions formed by telomere MIPs and the number (abundance) of junctions formed by genomic reference MIPs (i.e., the 4-bp (ATGG) MIPs used in this example). In this example, Δ-Δ-Ct from two qPCRs was used. Ct values from qPCRs of adult male samples were used as reference samples (the last three samples in the table). Three cancer cell lines were studied: MCF-7, HepG2, and K562. Control samples from the Roche Telomere Length Assay Kit (catalog number 1220913600, Roche Diagnostics GmbH, Germany) and female samples were also included. All reactions were performed in triplicate because three different samples and one reaction failed for the K562 cell line. ΔCT (the difference between FAM, telomere repeats, and HEX, and the 4bp repeat Ct value) was calculated, and the mean value for each sample was obtained. Then, the ΔCt of each sample is subtracted from the ΔCt of the reference sample obtained from male subjects to calculate the Δ-Δ-Ct value. Assuming an efficiency value of 2, the ratio of telomere motif attachment sites to genomic 4-base pair (ATGG) motif attachment sites can be calculated using 2^(Δ-ΔCt).

Δ-ΔCt, or the ratio per sample (column 7 in Table 1 below), can be used as a new indicator of telomere length.

Telomere length has been reported in the literature for several cancer cell lines, such as those measured by terminal restriction fragment (TRF) assays. Figure 5 shows a high correlation between TRF and relative quantitative ratios. The regression line in Figure 5 is used to read telomere length (y-axis) from any given ratio (x-axis). Based on the regression line shown in Figure 5, the ratio for the female sample is 1.39, and the estimated telomere length is 8.98 kbp.

Table I

Example 5: Confirmation of the linear response of the present invention

The same reference samples from the male samples in Examples 2-4 were diluted to a series of different DNA concentrations, resulting in varying amounts (abundance) of telomere repeats in the samples. These were prepared as samples with these total DNA amounts: 50 ng, 37.5 ng, 25 ng, 12.5 ng, and 6.25 ng. Triples of each of these five concentrations were used for the telomere assay of this invention. That is, a total of 15 separate reactions were performed. The results are shown in Table II below.

The input DNA amount was log-transformed, and the results are shown on the X-axis in Figure 6. In this relative quantification analysis, the threshold cycles (Ct values, also referred to as Cq in other references) of qPCR for the two MIP targets were plotted against the input DNA amount.

Quantification of the abundance of two targets (telomeres and ATGG MIP linkage sites) by qPCR showed good linearity between the amount of input DNA and the corresponding threshold cycles. The coefficients of determination (r²) were 0.96 and 0.98.

The slopes of the two regression lines were -2.96 and -2.75, respectively. The overall efficiency of the analysis can be calculated in a manner similar to that used for calculating qPCR efficiency with serially diluted samples. However, this overall combined efficiency measured by MIP combines the efficiencies of two individual reactions (i.e., the MIP hybridization-gap filling-ligation reaction and the qPCR quantification reaction).

The overall efficiency was calculated to be 10^(-1/slope). Therefore, the efficiencies for MIP determination of telomere length and 4-base pair (ATGG) repeating motifs were 2.2 and 2.3, respectively.

Table II shows the results of obtaining different amounts of input DNA by serially diluting a single sample.

Example 6: Confirmation of the specificity of the determination of the target motif

Water blank control samples and DNA from *Caenorhabditis elegans* (C. elegans, commonly known as roundworm) (whose telomere repeat motifs differ from those in humans) were both used to demonstrate that the present invention can distinguish between target motifs from nonspecific DNA (*C. elegans*) or water blanks.

Table III

Three *C. elegans* DNA samples (50 ng each) were used in the assay. The standard protocol in Examples 2-3 was used.

Table IV

The results showed that the abundance of junctions in the water control was only about 1/32 of that in a typical human sample (50 ng) used for this telomere assay (the difference in Ct values over more than 5 cycles, e.g., 24.32 - 18.88 = 5.44). Therefore, this method showed 30x specificity for targeting genomic motifs. Similar results were observed in *C. elegans*, compared to the water control.

Example 7: Confirmation of Linked and Linear MIP (LL-MIP) Products

Six samples were used in the telomere MIP protocol of Examples 2-3, except that the TaqMan probe was not added in step 5. Instead, conventional PCR was performed. A higher hybridization temperature (69.3°C) was used, and only one pair of primers (i.e., longGC_M13_F and longGC_Link_R_tel tail) targeting the two types of telomere MIP ligation products were present in this conventional PCR reaction. The PCR products were then separated by size on a 4% agarose gel. The smallest band (~100 bp) represents a typical circularized MIP product with one ligation point. Then, the band ~200 bp represents an LL-MIP with two MIPs ligated together, and so on.

The gel electrophoresis results are shown in Figure 3. The left side shows markers with 100 bp intervals. The effects of omitting the exonuclease digestion step and using different amounts of exonuclease I (New England Biolab) are illustrated. The results show that the exonuclease digestion step can be omitted. Similar results were obtained when using less than 20 units of exonuclease I for no more than 1 hour. If this amount and/or duration is exceeded, LL-MIP degradation (PCR products longer than ~100 bp) was observed. The addition of other exonucleases (such as exonuclease III or exonuclease VII in conventional MIP assays) causes degradation of the LL-MIP product and adversely affects the assay performance of this invention.

Table V

Example 8: Cloning the PCR product from Example 4 to confirm that LL-MIP represents the target genomic motif.

PCR products obtained from conventional PCR, similar to that in Example 7, were used for cloning and DNA sequencing to confirm that the LL-MIP products were specific to the target genomic motif. A TA cloning kit (Invitrogen, USA) for directly cloning PCR products was used to directly clone PCR products generated by primers (longGC_M13_F and longGC_Link_R_tel tail).

Positive clones with inserts were selected for sequencing, and those clones with LL-MIP inserts were longer than the expected insert size of ~100 bp for conventional circularized MIP products. The insert sizes of the two longer clones were longer than 400 bp. One insert represents an LL-MIP with six MIPs linked together, as schematically shown in Figure 1B. The target genome motif is shown as TAACCC, which is the exact complementary sequence to the GGGTTA telomere motif.

The sequencing results of the clone insert are shown in Figure 1C.

Example 9: Design of an alternative MIP probe with fewer hybridization bases (17 bp) with the target motif (Figure 7).

Another set of MIPs targeting telomeres and 4bp motifs was also designed and successfully tested. The sequences of the two MIPs are as follows:

ShortMIPtelo:/5Phos/G GT TAG GGT TAG GGT TAC TTC AGC TTC CCG ATC CGACGG TAG TGT TCA CAC AGG AAA CAG CTA TGA C GG TTA GGG TTA GGG TTA

ShortMIP4bp:/5Phos/G ATG GAT GGA TGG ATG GCT TCA GCT TCC CGA TCC GACGGT TAG GTT CAC ACA GGA AAC AGC TAT GAC TGG ATG GAT GGA TGG AT.

Both MIPs hybridized to their respective target repeats, with hybridization ends of 17 base pairs in length. Other conditions were the same as typical experimental procedures, except for the use of a lower hybridization temperature of 50°C. A reference human DNA sample was used as a template. Different input amounts of template DNA were used as test samples to examine the linearity and accuracy of the telomere measurement as an indicator of the amount of telomere motifs present in the sample. Four samples with input DNA of 50 ng, 37.5 ng, 25 ng, and 12.5 ng were evaluated. A water blank control was also included. All samples were performed in triplicate to evaluate assay accuracy.

This pair of short MIPs significantly enhanced the difference between the typical amount of DNA used in the assay (50 ng) and water as a template control. The Ct differences for telomere motifs and 4 bp (ATGG) motifs were >8.5 cycles and ~6 cycles, respectively. These differences correspond to >60-fold specificity.

Table VI uses a short MIP design to show the results of obtaining different amounts of input DNA by serially diluting a single sample (the results are plotted in Figure 7).

As described in the examples above, the overall efficiency of the two MIP assays can be calculated. Similar to the relative quantification method in efficiency-corrected qPCR, the overall efficiency is used in the MIP assays to correct for the slope difference of the Ct values of the two motifs. Therefore, the relative ratio of the overall efficiency (slope) correction can be analyzed. First, the efficiencies (slopes) of the two regression lines in Figure 7 are calculated. The slopes for the telomere motif and the 4bp (ATGG) motif are -3.06 and -2.46, respectively.

Then, based on the two regression lines, the Ct value of the 37.5 ng sample was used as the baseline sample. The efficiency was calculated as 10^(-1/slope).

The relative ratio of the efficiency (slope) correction of telomere junctions to 4bp (ATGG) motif junctions relative to the abundance of the reference sample is equal to:

[Efficiency of telomere motifs^(ΔCt between reference and test samples)]/[Efficiency of 4bp motifs^(ΔCt between reference and test samples)]

Using this index value, namely the efficiency (slope) corrected relative ratio, telomere length is expressed as a ratio to a reference sample. The average of triplicate samples ranges from 0.985 to 1.038. This is an example of the new index for telomere length generated by this novel method. It represents the ratio of telomere length to a reference sample. For example, the telomere length of a 50 ng sample is 0.99x that of the reference sample (37.5 ng sample). The expected value is 1 because all samples were derived from the same human subject that was also used as the reference sample. The results show that the efficiency (slope) corrected relative ratio eliminates any bias effects caused by differences in the amount of input DNA.

Example 10: Determining telomere length index values based on the abundance of telomere MIPs and genomic reference MIPs Its calculation method

Several methods can be used to quantify the exact amount of PCR products in qPCR, including relative and absolute quantification methods. In another embodiment (see the examples above), serial dilutions of a reference sample can be quantified to obtain Ct values to calculate the overall assay efficiency. The reference sample can be used in all qPCR batches and samples and compared with the same reference sample to remove batch effects due to inter-plate variations in qPCR Ct values. These methods are referred to as Δ-Δ-Ct methods and efficiency-corrected Δ-Δ-Ct methods. They are used herein to quantify the linker point of two MIPs.

Furthermore, absolute quantification of qPCR can be performed if a calibrator with a known (given) number of linkage sites is used together with the test sample in the same qPCR. This method is called absolute quantification of qPCR results.

In other implementations, other methods for quantifying linkage site abundance can be used, including digital PCR and sequencing.

Example 11: Further expansion for quantitative epigenetic markers

In the DNA sample pre-digestion step, the addition of a methylation-sensitive restriction enzyme to the pre-digested DNA sample allows for the quantification of the degree of methylation on a genome-wide scale. The results of two reactions from the same sample are compared: a methylation-sensitive restriction enzyme (whose ability to cleave DNA depends on the presence of methylated nucleotides) and a methylation-insensitive restriction enzyme (isoschistoses) that recognizes the same restriction sites. For example, enzyme pairs HpaII (methylation-sensitive) and MspI can be used. Novel MIPs designed to target CpG islands are used in conjunction with genomic reference MIPs, such as the 4 bp (ATGG) MIP used in this paper.

In one implementation, a specific target genomic region of interest is first isolated using a target enrichment method, many of which are known in the art. For example, array-based capture or in-solution capture are useful capture methods that can be used to isolate the target genomic region of interest. After target enrichment, the sample can be processed to quantify repeat motifs to detect motif amplification (length). This method can be used, for example, to detect trinucleotide repeat amplification associated with various diseases.

Example 12: Alternative Implementation Scheme

The experimental procedures, MIP targets, probes, primers, and enzymes used in telomere length estimation may vary. Such variations include:

(a) Shorter hybridization step duration, such as 2 hours of hybridization

(b) A simpler hybridization temperature scheme

(c) Use other stable short tandem repeats as targets for the second MIP. For example, the [AAGG]n tandem repeat can be used as a target. The corresponding MIP for [AAGG]n is: 5’-GAA GGAA GGAA GGAA GGAA GGGGCGCTTCAGCTTCCCGATCCGACGGTAGTGTTCA CAC AGG AAA CAG CTA TGA CAA GGAA GGAA GGAA GGAA GGAA GG-3’. The terminal phosphorylation and hydroxylation requirements are the same as in typical schemes.

(d) Other enzymes with similar properties to those in the typical scheme can be used.

(e) In experiments conducted during the scale-up production phase, the duration of the hybridization step can be reduced to 16 hours.

(f) Use additional probes and primers as shown in Table VII.

Table VII. Additional probes and primers

Example 13: Alternative implementation scheme for hybridization, extension, and ligation reactions

DNA samples were pretreated with restriction enzymes.

50-500 ng of DNA can be used in a single sample to quantify telomere length (TL). Add up to 10 units of two restriction enzymes, such as AluI and DdeI (New England Biolabs), to the DNA sample and incubate the sample at 37°C for 1 hour. Then inactivate the enzymes at 80°C for 20 min. After centrifugation at 7,000 rpm for 10 min and collection of the supernatant, the digested sample can be kept at 4°C.

MIP hybridization steps

The same MIP probe targeting human telomere repeats as in Example 2 is used, wherein the MIP probe targets (AAGG) repeat motifs.

Probes targeting telomere repeat motifs (with notches "GG"):

MIP-TelF+A:/5Phos/GT TAG GGT TAG GGT TAG GGT TAC TTC AGC TTC CCG ATCCGA CGG TAG TGT TCA CAC AGG AAA CAG CTA TGA CTA GGG TTA GGG TTA GGG TTA.

Another probe targeting (AAGG) repeating motifs (with a notch of "GAA"):

MIP-AAGGpure:/5Phos/GG AAGG AAGG AAGG AAGG AAGG TCG ATC CGA CAG CTTCCG TAG CGG TTT CAC ACA GGA AAC AGC TAT GAC TCA CAG AAGG AAGG AAGG AAGG AAGGAAG.

The hybridization mixture was subjected to the following hybridization procedure overnight: (values in parentheses are stock concentrations):

Hybridization schemes were performed in a thermal cycler at the following temperatures, but other suitable devices known in the art can also be used:

After the hybridization procedure, add 5 μl of the hybridization mixture to 15 μl of the notch filling and ligation reaction reagent (shown below) and preheat it to 50 °C.

Gap filling and connection steps

Prepare the gap-filling and ligation reaction reagents with dGTP and dATP at 37°C, or preheat and maintain them at 37°C after preparation (values in parentheses are stock concentrations):

Preheat 5 μl of the hybridization mixture to 50 °C for 30 minutes, then add 15 μl of preheated notch filling and ligation reagent.

Then, the notch filling and ligation reactions are performed as follows to produce a MIP product with ligation sites for two types of MIP probes:

37℃ for 30 minutes, then

45℃, for 5 minutes, then

95℃, for 10 minutes, then

Keep at 4℃.

Quantification of connection point steps

The gap-filled and ligated MIP product can be used to quantify the ligation sites, serving as a measure of the number of genomic motifs. In this implementation, qPCR was used, but other techniques can easily be employed.

Four primers and two TaqMan probes were used in the qPCR.

Long GC_Telo_F GGTCCGAGCCAGC TAT GAC TAG GGT TAG GG

LongGC_Link_R_tel GCGCATGTGA ATC GGG AAG CTG AAG TAA CC

LongGC_M13F+6GCTGCCTCGC AGG AAA CAG CTA TGA CTC ACA G

LongGC_AAGG_R GGTGCGTCGC GCT GTC GGA TCG ACC TTC CTT

Taqman_long_Telo/56-FAM/TA GGG TTA GGG TTA GGG TTA GGGT/3IABkFQ/

Taqman_AAGG/5HEX/AAG GAA GGA AGG AAG GAA GG/3IABkFQ/

Prior to qPCR, all nick-filled and ligated products of samples, standards, and calibrators were diluted 100-fold with water. Therefore, all samples, standards, and calibrators had the same dilution factor.

The qPCR reaction tubes were prepared (total volume 15 μl) as follows:

qPCR was performed in a Roche LC480 thermal cycler according to the following PCR temperature cycling protocol:

5. Go to step 2, 40 cycles.

Example 14: The linear response and index values of telomere length are not affected by different starting DNA amounts used for hybridization. Confirmation

The reference sample was diluted to a series of different DNA concentrations to ensure varying amounts (abundance) of telomere repeats and AAGG repeats in the sample. These were prepared as samples, and the following total DNA amounts were added to the hybridization reaction: 160 ng, 80 ng, 40 ng, and 20 ng. Each of these four amounts was repeated in the telomere assay protocol described in Example 13. The results are shown in Table VIII below. The mean index value of telomere length obtained using Δ-Ct (the difference between the two Ct values of the two MIP probes) was 0.945, with a coefficient of variation of 6.32%.

The input DNA amount was logarithmically transformed, and the results are shown on the X-axis in Figure 9. In this relative quantification analysis, the threshold cycles (Ct values, also referred to as Cq in other references) of qPCR for the two MIP targets (MIP-TelF+A and MIP-AAGGpure) were plotted against the input DNA amount (logarithmic bar).

Quantification of the abundance of two targets (representing the linker sites formed by MIP-TelF+A and MIP-AAGGpure, respectively) by qPCR showed good linearity between the amount of input DNA and the corresponding threshold cycle (Ct value). The coefficients of determination (r²) were 0.999 and 0.997, respectively.

The slopes of the two regression lines were -3.05 and -3.021, respectively. The overall efficiency of the assay can be calculated using serially diluted samples in a manner similar to that used to calculate qPCR efficiency. However, the overall combined efficiency of the MIP assay incorporates the efficiency of all reaction steps involved as described in Example 13, namely the MIP hybridization step, the nick-filling-ligation step, and the quantitative junction site step. In contrast, the qPCR efficiency typically mentioned in a typical qPCR assay only describes the efficiency of the qPCR itself.

The overall efficiency was calculated as 10^(-1/slope) of the results in Figure 9. Therefore, the MIP assay efficiency for both MIP-TelF+A (telomere probe) and MIP-AAGGpure (AAGG probe) was 2.1. This is close to the ideal efficiency value of 2.

Using these near-ideal total efficiency values, telomere length can be easily calibrated using samples with given or known telomere length values (in kbp). These reference samples and samples with unknown telomere lengths (TLs) are measured together using the method of this invention, and the index values obtained by quantifying the junction of the reference and unknown samples can then be converted to TLs in kbp.

As shown in the previous embodiments, the index value can be obtained by one of the following calculation methods: the Δ-Ct method, the Δ-Δ-Ct method, and the efficiency-corrected Δ-ΔCt method.

Table VIII Results of Example 13 with different amounts of input DNA

Example 15: Converting the index values of the connection points to TL in kbp using a reference sample

By using one or more reference samples with a given known TL, the index values generated by the assay of this invention can be converted to TL in kbp. In experiments using a procedure similar to that of Example 13, four samples with known TLs were measured. Their index values and raw data from qPCR for the connection points of the two probes are shown in Table IX.

The TL index value is based on an efficiency-corrected relative ratio of the abundance of telomere (MIP-TelF+A) junctions to AAGG motif (MIP-AAGGpure) junctions, as given in Example V. Reference samples in this batch had corresponding Ct values of 23.7 and 24.2, respectively, and reaction efficiencies of 1.88 and 1.85, respectively.

The correlation between the TL values of these reference samples and the index values is shown in Figure 10. The determined correlation (r²) is close to 1. The TL values of the reference samples cover typical values of TL in human samples.

Based on the regression formula shown in Figure 10, the TL of other samples with unknown TL can be derived by linear regression.

Table IX Quantitative Results of Connection Points in Reference Samples

Example 16: Quantification of the junction sites of two MIP probes using digital droplet PCR (ddPCR)

The ligation points in the MIP products formed after the gap-filling and ligation steps of Examples 2, 13, or similar embodiments can be quantified by digital droplet PCR to determine the index values used to assess telomere length.

This implementation uses MIP products generated via the following MIP probes;

Probes targeting telomere repeating motifs:

MIP-TelF:/5Phos/GT TAG GGT TAG GGT TAG GGT TAC TTC AGC TTC CCG ATCCGA CGG TAG TGT TCA CAC AGG AAA CAG CTA TGA CTA GGG TTA GGG TTA GGG TT

Probes targeting centromere α-satellite sequences:

MIP-Centv2:5'/Phos/gtc TAG GTT TGA TGT GAA GAT Ata ccc gCT TCA GCTTCC CGA TCC GAC GGT agg ttT CAC ACA GGA AAC AGC TAT GAC tca cag aaA ACG TTCTGA GAA TGC

After the gap filling and ligation steps, the MIP product was diluted 50-fold and 5 μl was used in each ddPCR reaction.

Four primers and two TaqMan probes were used in ddPCR.

M13_MIP_L2:GCGGGCAGGGCGGCtctagaTCACACAGGAAA CAGCTATGAC

MIP_LinkC-2Ls:GGCCCTACCGTCGGATCGGGAAGC

M13_Cent_v2-s:GGCCTATGACTCACAGAAAACGTTCTGAG

Linker_v2-s:CTACCGTCGGATCGGGAAG

TaqMan probe:

Taqman-3-Telo:/56-FAM/TAG GGT TAG GGT TAG GGT T/3IABkFQ/

Taq-cent:/5HEX/GTC TAG GTT TGA TGT GAA GAT ATA CCC G CTT/3IABkFQ/

Perform digital PCR (ddPCR) with the following reagents in a total reaction volume of 20 μl (values in parentheses are stock concentrations):

The ddPCR thermal cycling protocol is as follows:

The digital counts of telomere junctions formed by MIP-TelF (Figure 11) were 551 and 567 copies/μl (two replicates per sample), and the digital counts of centromere α-satellite junctions formed by MIP-Centv2 were 95 and 97 copies/μl (two replicates per sample). Therefore, the index values for these replicates were 5.8 and 5.85, respectively.

Sequence information

SEQ ID NO:1 (MIP-TelF)

5'/Phos/GT TAG GGT TAG GGT TAG GGT TAC TTC AGC TTC CCG ATC CGA CGGTAG TGT TCACAC AGG AAA CAG CTA TGA CTA GGG TTA GGG TTA GGG TT

SEQ ID NO:2(MIP-TelF+A)

5’Phos/GT TAG GGT TAG GGT TAG GGT TAC TTC AGC TTC CCG ATC CGA CGG TAGTGT TCA CAC AGG AAA CAG CTA TGA CTA GGG TTA GGG TTA GGG TTA

SEQ ID NO:3 (ShortMIPtelo)

5’Phos/G GT TAG GGT TAG GGT TAC TTC AGC TTC CCG ATC CGA CGG TAG TGTTCA CAC AGG AAA CAG CTA TGA C GG TTA GGG TTA GGG TTA

SEQ ID NO:4 (5' homologous region of MIP-TelF, MIP-TelF+A)

GT TAG GGT TAG GGT TAG GGT TA

SEQ ID NO:5 (5' homologous region of ShortMIPtelo)

G GT TAG GGT TAG GGT TAG

SEQ ID NO:6 (3' homologous region of MIP-TelF)

TA GGG TTA GGG TTA GGG TT

SEQ ID NO:7 (3' homologous region of MIP-TelF+A)

TA GGG TTA GGG TTA GGG TTA

SEQ ID NO:8 (3' homologous region of ShortMIPtelo)

GG TTA GGG TTA GGG TTA

MIP probe targeting the 4-bp motif of (AAGG)n sequence

SEQ ID NO:9(AAGG:MIP-2019AAGGpure)

/5Phos/ggaaggaaggaaggaaggaaggTCGATCCGACAGCTTCCGTagCGgttTCACACAGGAAACAGCTATGACtcacagaaggaaggaaggaaggaaggaag

Other primers used in qPCR

SEQ ID NO:10(Long GC_Telo_F G)

GGTCCGAGCCAGC TAT GAC TAG GGT TAG GG

SEQ ID NO:11(longGC_Link_R_tel tail)

gcgcatgtgaATC GGG AAG CTG AAG TAA CC

SEQ ID NO:12(longGC_M13F+6)

GCTGCCTCGC AGG AAA CAG CTA TGA CTC ACA G

SEQ ID NO:13(longGC_AAGG_R)

GGTGCGTCGC GCT GTC GGA TCG ACC TTC CTT

Taqman probe

SEQ ID NO:14(Taqman_long_Telo(FAM))

5’FAM-TA GGG TTA GGG TTA GGG TTA GGGT

SEQ ID NO:15(Taqman_AAGG(HEX))

5’HEX-AAG GAA GGA AGG AAG GAA GG

SEQ ID NO:16(longGC_M13_F)

ggcgcatggcTCA CAC AGG AAA CAG CTA TGA C

SEQ ID NO:17(longGC_Link_R_ATGG)

gcatggcgacaATC GGG AAG CTG AAG CCA tccat

SEQ ID NO:18 (Taq-3-Telo (for telomere MIP))

/56-FAM/TA GGG TTA G/ZEN/G GTT AGG GTT/3IABkFQ

SEQ ID NO:19(Taq-3-ATGG (for ATGG MIP))

/5HEX/GG ATG GAT G/ZEN/G ATG GAT GGA T/3IABkFQ

SEQ ID NO:20(MIP-ATGG)

/5Phos/AT GGA TGG ATG GAT GGA TGG ATG GCT TCA GCT TCC CGA TCC GAC GGTTAG GTT CAC ACA GGA AAC AGC TAT GAC GGA TGG ATG GAT GGA TGG AT

SEQ ID NO:21(ShortMIP4bp)

/5Phos/G ATG GAT GGA TGG ATG GCT TCA GCT TCC CGA TCC GAC GGT TAG GTTCAC ACA GGA AAC AGC TAT GAC TGG ATG GAT GGA TGG AT

SEQ ID NO:22 (Query for MIP of [AAGG]n)

5’-GAA GGAA GAA GAA GAA GGGGCGCTTCAGCTTCCCGATCCGACGGTAGTGTTCA CACAGG AAA CAG CTA TGA CAA GGAA GAA GAA GGAA GG-3’

SEQ ID NO:23(MIP-AAGGpure)

/5Phos/GG AAGG AAGG AAGG AAGG AAGG TCG ATC CGA CAG CTT CCG TAG CGGTTT CAC ACA GGA AAC AGC TAT GAC TCA CAG AAGG AAGG AAGG AAGG AAGG AAG

SEQ ID NO:24(MIP-Centv2)

5'/Phos/gtc TAG GTT TGA TGT GAA GAT Ata ccc gCT TCA GCT TCC CGA TCCGAC GGT agg ttT CAC ACA GGA AAC AGC TAT GAC tca cag aaA ACG TTC TGA GAA TGC

SEQ ID NO:25(M13_MIP_L2)

GCGGGCAGGGCGGCtctagaTCACACAGGAAACAGCTATGAC

SEQ ID NO:26(MIP_LinkC-2Ls)

GGCCCTACCGTCGGATCGGGAAGC

SEQ ID NO:27(M13_Cent_v2-s)

GGCCTATGACTCACAGAAAACGTTCTGAG

SEQ ID NO:28(Linker_v2-s)

CTACCGTCGGATCGGGAAG

SEQ ID NO:29(Taq-cent)

/5HEX/GTC TAG GTT TGA TGT GAA GAT ATA CCC G CTT/3IABkFQ/

SEQ ID NO:30 (Example LL-MIP product sequence in Figure 1)

GCGCATGTGAATCGGGAAGCTGAAGTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAGTCATAGCTGTTTCCTGTGTGAACACTACCGTCGGATCGGGAAGCTGAAGTAACCCTAACCCTAACCCTA ACCCTAACCCTAACCCTAGTCATAGCTGTTTCCTGTGTGAACACTACCGTCGGATCGGGAAGCTGAAGTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAGTCATAGCTGTTTCCCGTGTGAACACT ACCGTCGGATCGGGAAGCGAAGTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAGTCATAGCTGTTTCCTGTGTGAACACTACCGTCGGATCGGAAGCTGAATAACCCTAACCCTAACCCTAACCCTAACCCT AACCCTAACCCTAGTCATAGCTGTTTCCTGTGTGAACACTACCGTCGGATCGGGAAGCTGAAGTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAGTCATAGCTGTTTCCTGTGTGAGCCATGCGCC

Although the invention has been described in considerable detail by way of illustration and examples for purposes of clarity, those skilled in the art will understand that certain changes and modifications may be made within the scope of the appended claims. Furthermore, each reference provided herein is incorporated herein by reference in its entirety as if it were incorporated individually.

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<213> Artificial Sequence

<400> 10

ggtccgagcc agctatgact agggttaggg 30

<210> 11

<211> 30

<212> DNA/RNA

<213> Artificial Sequence

<400> 11

gcgcatgtga atcgggaagc tgaagtaacc 30

<210> 12

<211> 32

<212> DNA/RNA

<213> Artificial Sequence

<400> 12

gctgcctcgc aggaaacagc tatgactcac ag 32

<210> 13

<211> 31

<212> DNA/RNA

<213> Artificial Sequence

<400> 13

ggtgcgtcgc gctgtcggat cgaccttcct t 31

<210> 14

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> FAM

<400> 14

tagggttagg gttagggtta gggt 24

<210> 15

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> HEX

<400> 15

aaggaaggaa ggaaggaagg 20

<210> 16

<211> 32

<212> DNA/RNA

<213> Artificial Sequence

<400> 16

ggcgcatggc tcacacagga aacagctatg ac 32

<210> 17

<211> 34

<212> DNA/RNA

<213> Artificial Sequence

<400> 17

gcatggcgac aatcgggaag ctgaagccat ccat 34

<210> 18

<211> 19

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> 6-FAM

<220>

<221> misc_feature

<222> (9)..(9)

<223> ZEN

<220>

<221> misc_feature

<222> (19)..(19)

<223> IABkFQ

<400> 18

tagggttagg gttagggtt 19

<210> 19

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> HEX

<220>

<221> misc_feature

<222> (9)..(9)

<223> ZEN

<220>

<221> misc_feature

<222> (20)..(20)

<223> IABkFQ

<400> 19

ggatggatgg atggatggat 20

<210> 20

<211> 94

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> Phosphorylation

<400> 20

atggatggat ggatggatgg atggcttcag cttcccgatc cgacggttag gttcacacag 60

gaaacagcta tgacggatgg atggatggat ggat 94

<210> 21

<211> 84

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> Phosphorylation

<400> 21

gatggatgga tggatggctt cagcttcccg atccgacggt taggttcaca caggaaacag 60

ctatgactgg atggatggat ggat 84

<210> 22

<211> 95

<212> DNA/RNA

<213> Artificial Sequence

<400> 22

gaaggaagga aggaaggaag gggcgcttca gcttcccgat ccgacggtag tgttcacaca 60

ggaaacagct atgacaagga aggaaggaag gaagg 95

<210> 23

<211> 99

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> Phosphorylation

<400> 23

ggaaggaagg aaggaaggaa ggtcgatccg acagcttccg tagcggtttc acacaggaaa 60

cagctatgac tcacagaagg aaggaaggaa ggaaggaag 99

<210> 24

<211> 102

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> Phosphorylation

<400> 24

gtctaggttt gatgtgaaga tatacccgct tcagcttccc gatccgacgg taggtttcac 60

acaggaaaca gctatgactc acaggaaacg ttctgagaat gc 102

<210> 25

<211> 42

<212> DNA/RNA

<213> Artificial Sequence

<400> 25

gcgggcaggg cggctctaga tcacacagga aacagctatg ac 42

<210> 26

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 26

ggccctaccg tcggatcggg aagc 24

<210> 27

<211> 29

<212> DNA/RNA

<213> Artificial Sequence

<400> 27

ggcctatgac tcacagaaaa cgttctgag 29

<210> 28

<211> 19

<212> DNA/RNA

<213> Artificial Sequence

<400> 28

ctaccgtcgg atcgggaag 19

<210> 29

<211> 31

<212> DNA/RNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (1)..(1)

<223> HEX

<220>

<221> misc_feature

<222> (31)..(31)

<223> IABkFQ

<400> 29

gtctaggttt gatgtgaaga tatacccgct t 31

<210> 30

<211> 556

<212> DNA/RNA

<213> Artificial Sequence

<400> 30

gcgcatgtga atcgggaagc tgaagtaacc ctaaccctaa ccctaaccct aaccctaacc 60

ctaaccctag tcatagctgt ttcctgtgtg aacactaccg tcggatcggg aagctgaagt 120

aaccctaacc ctaaccctaa ccctaaccct aaccctagtc atagctgttt cctgtgtgaa 180

cactaccgtc ggatcgggaa gctgaagtaa ccctaaccct aaccctaacc ctaaccctaa 240

ccctaaccct agtcatagct gtttcccgtg tgaacactac cgtcggatcg ggaagcgaag 300

taaccctaac cctaacccta accctaaccc taaccctagt catagctgtt tcctgtgtga 360

acactaccgt cggatcggaa gctgaataac cctaacccta accctaaccc taaccctaac 420

cctaacccta gtcatagctg tttcctgtgt gaacactacc gtcggatcgg gaagctgaag 480

taaccctaac cctaacccta accctaaccc taaccctaac cctagtcata gctgtttcct 540

gtgtgagcca tgcgcc 556

Claims (45)

1. A single-stranded DNA probe for determining telomere length and/or copy number of tandem repeat sequences, said probe comprising: i) A 5’ homologous region that extends to the 5’ end of the probe and contains a nucleotide sequence complementary to the tandem repeat sequence; ii) Joint area; and iii) A 3' homologous region extending to the 3' end of the probe and containing a nucleotide sequence complementary to the tandem repeat sequence, such that the 5' and 3' homologous regions can bind to the same strand of template DNA containing the tandem repeat sequence; wherein When the 5' homologous region and the 3' homologous region bind to the same strand of the template DNA, the 3' homologous region is immediately adjacent to the 3' end of the 5' homologous region within a single repeat unit on the template, and thus the 3' end of the 3' homologous region and the 5' end of the 5' homologous region are separated by a nucleotide gap on the template DNA smaller than a complete repeat unit. The nucleotide gap, which is less than one complete repeat unit, contains at most two different bases. 2. The probe of claim 1, wherein the tandem repeat sequence is a telomere sequence. 3. The probe of claim 2, wherein the telomere is a human telomere. 4. The probe of claim 1, wherein the nucleotide sequences of the 5' homologous region and the 3' homologous region are 100% complementary to the tandem repeat sequence. 5. The probe of claim 1, wherein the length of the 5' homologous region and the 3' homologous region is 15-25 nucleotides each. 6. The probe of claim 1, wherein each repeat unit of the tandem repeat sequence is 2-10 nucleotides in length. 7. The probe of claim 1, wherein the adapter region comprises one or more sequence elements selected from universal primer sequences, probe-specific primer sequences, TaqMan probe sequences, and tag sequences. 8. The probe of claim 1, wherein when bound to a single repeat unit on the template DNA, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region are separated by one or two nucleotides. 9. The probe of claim 8, wherein one or two nucleotides comprise the base G. 10. The probe of claim 1, wherein when bound to a single repeat unit on the template DNA, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region are separated by three nucleotides. 11. The probe of claim 10, wherein the three nucleotides comprise two different bases, and wherein the two different bases are A and G. 12. The probe of claim 1, wherein the 5' homologous region comprises the nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:5. 13. The probe of claim 1, wherein the 3' homologous region comprises the nucleotide sequence of any one of SEQ ID NOs: 6-8. 14. The probe of claim 1, wherein the probe comprises the nucleotide sequence of any one of SEQ ID NOs:1-3. 15. A kit for determining the copy number of variable tandem repeat sequences in the genome, comprising first and second single-stranded DNA probes, wherein each of the first and second single-stranded DNA probes comprises: i) A 5’ homologous region that extends to the 5’ end of the DNA probe and contains a nucleotide sequence complementary to the tandem repeat sequence; ii) Joint area; and iii) A 3’ homologous region extending to the 3’ end of the DNA probe and containing a nucleotide sequence complementary to the tandem repeat sequence, such that the 5’ homologous region and the 3’ homologous region can bind to the same strand of template DNA containing the tandem repeat sequence; When the 5' homologous region and the 3' homologous region bind to the same strand of the template DNA, the 3' homologous region is immediately adjacent to the 3' end of the 5' homologous region within a single repeat unit on the template DNA. Therefore, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region are separated by a nucleotide gap on the template DNA smaller than one complete repeat unit, the nucleotide gap containing at most two different bases. The 5' and 3' homologous regions of the first single-stranded DNA probe are complementary to the first tandem repeat sequence whose copy number varies among individuals. The 5' and 3' homologous regions of the second single-stranded DNA probe are complementary to its second tandem repeat sequence, the copy number of which remains unchanged between individuals. The nucleotide gap of the first single-stranded DNA probe contains at most two bases that are also contained in the nucleotide gap of the second single-stranded DNA probe. 16. The kit of claim 15, further comprising a reaction mixture comprising DNA polymerase, an enzyme comprising ligase activity, and deoxyribonucleoside triphosphates comprising up to two bases corresponding to the nucleotide gaps of the first and second single-stranded DNA probes. 17. The kit of claim 16, wherein the DNA polymerase is T4 DNA polymerase. 18. The kit of claim 16, wherein the ligase activity is provided by Amp ligase. 19. The kit of claim 16, wherein the reaction mixture does not contain exonuclease. 20. The kit of claim 15, further comprising genomic DNA of the test sample. 21. The kit of claim 15, further comprising universal primers, two or more probe-specific primers, or two or more TaqMan probes that are complementary to sequences within the adapter region of the first and/or second single-stranded DNA probe. 22. The kit of claim 15, wherein the tandem repeat sequence identified by the 5' and 3' homologous regions of the first single-stranded DNA probe is a telomere sequence. 23. The kit of claim 22, wherein the telomeres are human telomeres. 24. The kit of claim 15, wherein the tandem repeat sequence identified by the 5' and 3' homologous regions of the second single-stranded DNA probe is a short tandem repeat of 4 bp. 25. The kit of claim 15, wherein the nucleotide sequences of the 5' and 3' homologous regions of the first single-stranded DNA probe are 100% complementary to tandem repeat sequences in the genome whose copy number varies between individuals. 26. The kit of claim 15, wherein the nucleotide sequences of the 5' and 3' homologous regions of the second single-stranded DNA probe are 100% complementary to a tandem repeat sequence in the genome with a copy number stable between individuals. 27. The kit of claim 15, wherein the length of the 5' and/or 3' homologous region of the first and/or second single-stranded DNA probe is 15 to 25 nucleotides. 28. The kit of claim 15, wherein the repeat unit of the tandem repeat sequence recognized by the first and/or second single-stranded DNA probe is 3-6 nucleotides in length. 29. A non-diagnostic method for determining the length of variable-length tandem repeat regions in an individual's genome, the method comprising: i) Provide a first single-stranded DNA probe and a second single-stranded DNA probe, each of the first and second single-stranded DNA probes comprising: (1) A 5’ homologous region that extends to the 5’ end of the DNA probe and contains a nucleotide sequence complementary to the tandem repeat sequence; (2) Joint area; and (3) A 3’ homologous region that extends to the 3’ end of the DNA probe and contains a nucleotide sequence complementary to the tandem repeat sequence, such that the 5’ homologous region and the 3’ homologous region can bind to the same strand of template DNA containing the tandem repeat sequence. When the 5' homologous region and the 3' homologous region bind to the same strand of the template DNA, the 3' homologous region is immediately adjacent to the 3' end of the 5' homologous region within a single repeat unit on the template DNA. Therefore, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region are separated by a nucleotide gap on the template DNA smaller than one complete repeat unit, the nucleotide gap containing at most two different bases. The 5' and 3' homologous regions of the first single-stranded DNA probe are complementary to the first tandem repeat sequence whose copy number varies across individual genomes. The 5' and 3' homologous regions of the second single-stranded DNA probe are complementary to its second tandem repeat sequence, the copy number of which remains unchanged across individual genomes. The nucleotide gap of the first single-stranded DNA probe contains at most two bases that are also contained in the nucleotide gap of the second single-stranded DNA probe; ii) Under conditions conducive to the extension of the 3' end of the DNA probe and its ligation to the 5' end of the DNA probe bound to the same template, in the presence of DNA polymerase, ligase activity, and at most two deoxyribonucleoside triphosphates corresponding to the bases contained in the denucleotide nicks of the first and second single-stranded DNA probes, a biological sample from the individual is contacted with a plurality of the first single-stranded DNA probes and a plurality of the second single-stranded DNA probes to extend the 3' ends of the first and second single-stranded DNA probes and ligate them to the 5' ends of the first and second single-stranded DNA probes, respectively, thereby producing first and second circularized and ligated linear probe products; iii) Quantifying the first and second cyclized and connected linear probe products generated in step ii); and iv) Normalize the amount of the first circularized and ligated linear probe product relative to the amount of the second circularized and ligated linear probe product to determine the copy number of the first tandem repeat sequence in the individual genome. 30. The method of claim 29, wherein the nucleotide sequences of the 5' and 3' homologous regions of the first and second single-stranded DNA probes are 100% complementary to the first and second tandem repeat sequences, respectively. 31. The method of claim 29, wherein the length of the 5' and 3' homologous regions of the probe is 15-25 nucleotides. 32. The method of claim 29, wherein the first tandem repeat sequence region is a telomere. 33. The method of claim 32, wherein the telomeres are human telomeres. 34. The method of claim 29, wherein the adapter region of each probe comprises one or more sequence elements selected from universal primer sequences, probe-specific primer sequences, TaqMan probe sequences, and tag sequences. 35. The method of claim 29, wherein when bound within a single repeat unit on the tandem repeat sequence, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region of the probe are separated by a gap of one or two nucleotides, wherein the one or two nucleotides comprise a single base, and wherein only one deoxyribonucleoside triphosphate corresponding to the single base is provided in step ii). 36. The method of claim 35, wherein the single base is G. 37. The method of claim 29, wherein when bound within a single repeat unit on the tandem repeat sequence, the 3' end of the 3' homologous region and the 5' end of the 5' homologous region of the probe in step i) are separated by a 3-nucleotide gap, wherein the 3 nucleotides comprise 2 bases, and wherein in step ii) two deoxyribonucleoside triphosphates corresponding to the 2 bases are provided. 38. The method of claim 37, wherein the two bases are A and G. 39. The method of claim 29, wherein the 5' homologous region of the probe in step i) comprises the nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:5. 40. The method of claim 29, wherein the 3' homologous region of the probe in step i) comprises the nucleotide sequence of any one of SEQ ID NOs: 6-8. 41. The method of claim 29, wherein the probe in step i) comprises the nucleotide sequence of any one of SEQ ID NOs:1-3. 42. The method of claim 29, wherein a large excess of the probe in step i) is provided relative to the amount of genomic DNA in the biological sample. 43. The method of claim 29, wherein no exonuclease is added during steps i) to iv). 44. The method of claim 29, wherein the only exonuclease added during steps i) to iv) is exonuclease I, and wherein the level of exonuclease I does not exceed 20 units/50 ng genomic DNA and is present for a maximum of 1 hour. 45. The method of claim 29, wherein the amount of circularized and ligated linear probe product of the probe is determined using a method selected from quantitative PCR, digital PCR, and sequencing.