Improved detection of microsatellite instability

Abstract

Improved methods and compositions for detecting microsatellite instability (MSI) are provided herein.

Description

Background Art

[0001] Microsatellites are repetitive regions consisting of 1 to 6 nucleotides in length and are scattered throughout the genome. Microsatellite instability (MSI) appears as a change in repeat length, i.e., elongation or shortening, in one or both alleles. MSI is caused by defects in the mismatch repair system, the causes of which are germline mutations (Lynch syndrome), or sporadic genetic or epigenetic changes within somatic mismatch repair genes. MSI is an important diagnostic tool for screening Lynch syndrome. This is because almost all patients' tumors with mutations in mismatch repair genes have an MSI phenotype.

[0002] Mismatch repair (MMR) deficiency is associated with a high proportion of mutant neoantigens in cancer, and these mutant neoantigens render cancer sensitive to immune checkpoint blockade regardless of the primary tissue (Le et al. (2017) Science 357:409). Therefore, MSI is a predictive biomarker for the response to immune checkpoint therapy in many cancers (e.g., glioblastoma, colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, breast cancer). The FDA has expedited the approval of pembrolizumab (Keytruda) for pediatric and adult patients with solid tumors deficient in MMR. In early-stage CRC, patients with dMMR have better survival rates, indicating that MSI also has prognostic value (Merok et al. (2013) Annals Oncology 24:1274; Roth et al. (2012) J. Natl. Cancer Inst. 104:1635).

Summary of the Invention

[0003] Disclosed herein are oligonucleotides, kits, assays, and methods for detecting microsatellite instability (MSI).

[0004] In one aspect, kits containing genomic DNA from an individual (e.g., tumor samples from a human) are provided as kits for detecting microsatellite instability (if present) in a sample. In some embodiments, the kit includes a) a drop-off probe attached to a quencher with a first label (e.g., a phosphor), which has a sequence complementary to a wild-type microsatellite repeat sequence in genomic DNA, and at least one complementary additional nucleotide at the 5' end of the wild-type microsatellite repeat sequence (e.g., 1, 2, 3, 4, 5, 6, 3-7, 7, 8, 9, 10, or more) and at least one complementary additional nucleotide at the 3' end (e.g., 1, 2, 3, 4, 5, 6, 3-7, 7, 8, 9, 10, or more), and b) a reference probe attached to a quencher with a second label (e.g., a phosphor), which has a sequence complementary to a reference sequence in genomic DNA that is not a microsatellite repeat sequence.

[0005] In some embodiments, depending on what microsatellites are being detected, the drop-off probe is at least 10–80 or 25–50 nucleotides long. For example, in some embodiments, the drop-off probe is the length of a normal, most common microsatellite repeat sequence plus the length of additional nucleotides at the 5' and 3' ends of the drop-off probe that are complementary to the genome sequence and adjacent to the microsatellite repeat sequence. In some embodiments, the reference probe is similar in size to the drop-off probe or has a similar melting temperature to the drop-off probe.

[0006] In some embodiments, the reference probe binds to a conserved / invariant genomic sequence in the vicinity of the drop-off probe (e.g., within 1-5, 5-10, 10-50, 50-100, or 100-500 nucleotides from the binding site of the drop-off probe).

[0007] In some embodiments, the kit further includes a set of amplification primers located at the 5' and 3' ends of the binding sites of the drop-off probe and the reference probe (i.e., the wild-type microsatellite repeat sequence and the reference sequence in the genomic DNA). In some embodiments, the set of primers is designed to amplify both binding sites (e.g., if those binding sites are relatively close to each other in the genome). In some embodiments, the kit further includes a heat-stable DNA polymerase. In some embodiments, the kit further includes a control (e.g., DNA with known microsatellite instability (positive control), or DNA with a normal (wild-type) microsatellite sequence (negative control)).

[0008] In some embodiments, the quencher on the drop-off probe is attached inside the drop-off probe (e.g., inside the microsatellite repeat sequence). In some embodiments, the quencher is attached within 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides from the first label. In some embodiments, the drop-off probe has an internal quencher and at least 4, 5, 6, 7, 8, 9, or 10 complementary additional nucleotides at the 5' end of the microsatellite repeat sequence.

[0009] In some embodiments, the kit further includes a quencher oligonucleotide that is complementary to at least a portion of the drop-off probe and contains a quencher. In some embodiments, the quencher is located at the 3' end of the quencher probe or within 5 nucleotides of the 3' end. In some embodiments, the melting temperature of the quencher probe and drop-off probe (the temperature at which 50% of the double-stranded complex melts into single-stranded DNA) is 20–60°C, particularly 42–55°C, or 46–52°C, or about 50°C. In some embodiments, the melting temperature is lower than the temperature selected for annealing in the PCR reaction. In some embodiments, the quencher oligonucleotide is contiguous with the reference probe, so the reference probe includes a sub-sequence that is not complementary to the microsatellite repeat sequence (e.g., adjacent to the microsatellite repeat sequence) and a complementary sub-sequence. In some embodiments, the reference probe contains at least 6–20, 8–15, 5, 6, 7, 8, 9, 10, or more nucleotides complementary to the drop-off probe, with the quencher located near the 3' end.

[0010] In some embodiments, the drop-off probe forms a hairpin structure at 20–60°C, or below 60°C, or below 55°C, or below 50°C, or below a temperature selected for annealing in the PCR reaction. In some embodiments, the drop-off probe contains at least one mismatch with the genomic sequence adjacent to the microsatellite repeat sequence to improve hairpin formation (i.e., to increase the 5' and 3' nucleotide complementarity of the sequence complementary to the microsatellite repeat sequence).

[0011] In some embodiments, the kit includes multiple probes for detecting multiple microsatellite repeat sequences. In some embodiments, the kit includes at least two, three, four, five, six, seven, eight, nine, or ten of the above and herein-described drop-off probes (multiple drop-off probes) having sequences complementary to different wild-type microsatellite repeat sequences in genomic DNA. In some embodiments, the multiple drop-off probes are housed in separate containers or combined with two, three, four, or more drop-off probes in the same container. In some embodiments, each of the above multiple drop-off probes is attached to the same label (e.g., a label that emits the same fluorescence), and the corresponding multiple reference probe is attached to a different label. In such cases, the signal from the reference probe identifies the signal of each microsatellite repeat sequence detected in the multiple reaction. In some embodiments, the above multiple drop-off probes are attached to different labels than other drop-off probes in the same container, or to different labels than other drop-off probes in the same kit. In some embodiments, the kit further includes the above-mentioned and / or numerous reference probe and / or numerous primer sets, paired with each of the above-mentioned numerous drop-off probes. In some embodiments, the numerous reference probes are labeled differently from other reference probes and drop-off probes in the same container, or differently from other reference probes and drop-off probes in the same kit.

[0012] In yet another aspect, a reaction mixture is provided for detecting microsatellite instability when present in genomic DNA from an individual (e.g., tumor sample). In some embodiments, the reaction mixture comprises: a) a drop-off probe having a sequence complementary to a wild-type microsatellite repeat sequence in the genomic DNA, and having at least one nucleotide at the 5' end and at least one nucleotide at the 3' end of the wild-type microsatellite repeat sequence, and attached to a first label and quencher; b) a reference probe having a sequence complementary to a reference sequence in the genomic DNA that is not a microsatellite repeat sequence, and attached to a second label and quencher; c) a set of amplification primers located at the 5' and 3' ends of the binding sites of the drop-off probe and the reference probe; d) a thermostable DNA polymerase; and e) genomic DNA. The drop-off probe and reference probe may include any of the features described above and herein (e.g., internal quencher, duplicate sequence, etc.). In addition, the reaction mixture can be multiplexed using two or more drop-off probes, reference probes, and primer sets.

[0013] In yet another aspect, a method is provided for detecting microsatellite instability when present in a sample of genomic DNA from an individual or a group of individuals (e.g., a tumor sample). In some embodiments, the method includes: a) contacting the genomic DNA with the components of the kit described above and herein; b) amplifying and detecting the binding of a drop-off probe and a reference probe to the genomic DNA by performing dPCR (e.g., when a positive signal is detected from the first and second labels); and c) detecting microsatellite instability when the binding of the drop-off probe to the genomic DNA is less than a control value or threshold. In some embodiments, the control value is obtained from normal, disease-free tissue from the same individual or a group of individuals (to obtain a broader range of average values ​​for one population). In some embodiments, microsatellite instability is detected when the binding of the drop-off probe to the genomic DNA decreases, but the binding of the reference probe remains unchanged or does not decrease as much as the binding of the drop-off probe. In this specification, the drop-off probe and reference probe may include any of the features described above and herein (e.g., internal quencher, duplicate sequence, etc.). In addition, the reaction mixture may be multiplexed using two or more drop-off probes, reference probes, and primer sets.

[0014] Therefore, in some embodiments, this method includes: a) contacting genomic DNA with i) a drop-off probe having a sequence complementary to the wild-type microsatellite repeat sequence in the genomic DNA, and having at least one nucleotide at the 5' end and at least one nucleotide at the 3' end of the wild-type microsatellite repeat sequence, attached to a first label and quencher; ii) a reference probe having a sequence complementary to a reference sequence in the genomic DNA that is not a microsatellite repeat sequence, attached to a second label and quencher; iii) contacting a set of amplification primers located at the 5' and 3' ends of the binding sites of the drop-off probe and the reference probe; b) amplifying and detecting the binding of the drop-off probe and the reference probe to the genomic DNA by performing dPCR; and c) detecting microsatellite instability when the binding of the drop-off probe to the genomic DNA is less than a control value or threshold. In some embodiments, this method is performed in multiples using two, three, four, five, or more drop-off probes to detect microsatellite instability at the locations of numerous microsatellite repeat sequences.

[0015] In some embodiments, the method further includes directing or administering treatment to an individual with MSI (e.g., MSI at one, two, three, or more microsatellite repeat locations). In some embodiments, the treatment includes immune checkpoint therapy (e.g., drugs targeting PD-1 or PD-L1). In some embodiments, for example, for a patient already receiving oncology therapy, the treatment includes reducing the dose of the therapeutic agent being used. [Brief explanation of the drawing]

[0016] [Figure 1]Figure 1 shows a list of commonly detected microsatellites and their genomic locations. The repeating motif sequences are as follows: SEQ ID NO: 10 for BAT-25, SEQ ID NO: 11 for BAT-26, SEQ ID NO: 12 for NR-21, SEQ ID NO: 13 for NR-24, SEQ ID NO: 14 for MONO-27, SEQ ID NO: 15 for BAT-40, SEQ ID NO: 16 for D2S123, SEQ ID NO: 17 for S5S346, SEQ ID NO: 18 for D17S250, SEQ ID NO: 19 for PentaC, SEQ ID NO: 20 for PentaD, and SEQ ID NO: 21 for MYCL1.

[0017] [Figure 2] Figure 2 shows an example of results using the current method. Matching of a normal sample (top) and an MSI-positive tumor sample (bottom) was analyzed using an MSI analysis system. After amplifying 2 nanograms of genomic DNA, it was analyzed using an ABI PEISM 3100 Genetic Analyser with POP-4 polymer and a 36 cm capillary. The allele patterns of the normal and MSI-positive samples are shown. The presence of new alleles (indicated by arrows) in the MSI-positive sample that were not present in the normal sample indicates MSI.

[0018] [Figure 3] Figure 3 shows an example of a drop-off assay for detecting MSI deletions in the marker BAT25. In microsatellite-stable samples (MSS), the drop-off probe binds to the 25 T / A mononucleotide repeat sequence and several adjacent "anchor" nucleotides. In MSI deletion samples (e.g., 23 T / A mononucleotide repeats), the drop-off probe is no longer fully complementary to the genomic sequence, resulting in decreased affinity and less fluorescence from probe hydrolysis (i.e., hydrolysis separates the fluorophores and quenchers). In this example, the fluorophores (FAM or HEX) are at the 5' end of the probe and the quenchers are at the 3' end.

[0019] [Figure 4] Figure 4 shows the dPCR results from the probe design shown in Figure 3. The plot on the left shows the results for the BAT25 marker (top) and BAT26 marker (bottom) without a distant quencher oligonucleotide. These results indicate that the signal from the drop-off probe's phosphor is relatively large in the presence of MSI. The plot on the right shows the results for the BAT25 (top) marker and BAT26 marker (bottom) with a distant quencher oligonucleotide. These results indicate that the MSS signal and MSI signal are more clearly separated as a result of the drop-off probe's phosphor being more effectively quenched.

[0020] [Figure 5] Figure 5 shows a comparison of results from a detached 16-mer quencher oligonucleotide and results from a quencher oligonucleotide attached to a reference probe. The top plot shows results from an MSI assay lacking the quencher oligonucleotide. The bottom left plot shows results from an MSI assay using a detached 16-mer quencher oligonucleotide, which reduces background fluorescence from an unhydrolyzed drop-off probe, as can be seen in Figure 4. The center and bottom right plots show that quencher oligonucleotides attached to a reference probe (16-mer and 12-mer, respectively (SEQ ID NOs. 27 and 28, respectively)) are effective as detached quencher oligonucleotides.

[0021] [Figure 6]Figure 6 shows the design of a drop-off probe that forms a hairpin at 50°C or below (SEQ ID NO: 3). The left plot shows the results from a BAT25 MSI assay using a linear drop-off probe (SEQ ID NO: 3) without a quencher oligonucleotide. The right plot shows the results from a BAT25 MSI assay using a hairpin-shaped drop-off probe. In the case of the hairpin structure, quenching is more effective when the probe is hydrolyzed (i.e., not bound to the genomic microsatellite repeat sequence) because the quencher and the fluorophore are closer together.

[0022] [Figure 7] Figure 7 shows drop-off probes having quenchers at various positions relative to the 5'-label (i.e., located at the 3'-end or internally). The drop-off probe for the BAT25 MSI assay is SEQ ID NO: 4 (top), and the drop-off probe for the BAT26 MSI assay is SEQ ID NO: 5 (bottom). The results show that in both assays, having the quencher located internally in the drop-off probe is more effective in reducing background fluorescence from unhydrolyzed drop-off probes. Figure 7 also discloses drop-off probes having internal quenchers for BAT25 and BAT26 (SEQ ID NOs: 22 and 5, respectively).

[0023] [Figure 8]Figure 8 shows that the method described herein can detect small variations in the size of microsatellites. The left plot shows the results from the BAT26 MSI assay using quenching oligonucleotides. The MSS state in the MSI cell line or the described number of nucleotide deletions is shown for the BT-549 cell line, Raji cell line, Daudi cell line, Molt-4 cell line, Jurkat cell line, and HCT-116 cell line. The right graph shows the positive average fluorescence amplitude for the drop-off probe label (FAM). Some cell lines show different results, indicating the presence of alleles with different repeat lengths. In Figure 8, "16-mer polyA" is disclosed as SEQ ID NO: 27.

[0024] [Figure 9] Figure 9 shows that the method described herein is effective in detecting MSI in DNA that has undergone typical damage and fragmentation from FFPET samples. The left plot shows the results from non-tumor MSS FFPET samples for both the BAT25 (top) and BAT26 (bottom) markers, and the right plot shows the results from tumor MSI FFPET samples. The MSI assay was performed using quencher oligonucleotides to reduce background fluorescence.

[0025] [Figure 10] Figure 10 shows that the method described herein can be multiplexed. The leftmost figure shows the results of the BAT25 MSI assay from two MSI-positive cell lines, the middle figure shows the results of the BAT26 MSI assay, and the rightmost figure shows the results from the multiplex assay.

[0026] [Figure 11]Figure 11 shows the results from 2D plots of five MSI markers commonly detected in BT-549 (MSS) and RKO (MSI-H) cell lines. The markers are BAT-25, BAT-26, NR-21, NR-24, and Mono-27.

[0027] [Figure 12] Figure 12 shows that the NR-21 assay, designed to detect subtle differences in deletion length, is effective in distinguishing between two nucleotide differences in the MSI marker between the BT-549 cell line (top two figures) and the Daudi cell line (bottom two figures). [Modes for carrying out the invention]

[0028] I. Introduction This disclosure provides compositions and methods for detecting MSI in a more streamlined manner with reduced risk of contamination and lower costs compared to current methods. The methods disclosed herein utilize drop-off probes designed to bind to normal genomic microsatellite repeat sequences, which, upon binding, are amplified and hydrolyzed (cleaved, fragmented). This cleavage separates the label and quencher attached to the drop-off probe, thus generating a signal. In the case of MSI, the drop-off probe does not bind to the microsatellite sequence as effectively, and therefore is not cleaved to the same extent, and does not generate a signal of the same degree.

[0029] II. Definitions The term “biomarker” means any detectable marker used to distinguish individual samples (e.g., cancer samples from non-cancer samples). Biomarkers include modifications (e.g., DNA methylation, protein phosphorylation), differences in expression, mutations, or variants (e.g., single nucleotide variations, insertions, deletions, splice variants, fusion variants). Biomarkers can be detected in DNA samples, and / or RNA samples, and / or protein samples.

[0030] The term "multiplicative" refers to an assay in which two or more targets are detected, for example, within the same test tube, well, droplet, or microchamber.

[0031] Terms such as "container," "test tube," "well," and "chamber" refer to a containment space capable of holding reagents or assays. If a container is in a kit and holding reagents or used in an amplification reaction, it may be closed or sealed to prevent contamination or evaporation. If a container is used in an assay, it may be left open or accessible, at least while the assay is being performed.

[0032] The terms "individually detected" or "individually detected" in relation to marker genes or marker gene products indicate that each marker is detected in a multiple reaction; that is, each marker is associated with a different label (and is detected by a probe with a different label).

[0033] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to polymers of nucleotides (e.g., ribonucleotides and deoxyribonucleotides), including both naturally occurring nucleic acids (e.g., adenosine, guanidine, cytosine, uracil, thymidine) and non-natural (human-modified) nucleic acids. This terminology is not limited by the length of the polymer (e.g., the number of monomers). Nucleic acids can be single-stranded or double-stranded and generally contain a 5'-3' phosphodiester bond, although in some cases, nucleotide analogs may have other bonds. Monomers are typically called nucleotides. The terms "non-natural nucleotide" or "modified nucleotide" refer to nucleotides that contain any modified nitrogenous base, sugar, or phosphate group, or in which a non-natural moiety is incorporated into the structure. Examples of non-natural nucleotides include LNA, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and phosphor-labeled nucleotides.

[0034] In the context of this disclosure, terms such as “cell-free nucleic acid,” “cell-free DNA,” and “cell-free RNA” refer to non-tissue samples (e.g., liquid biopsies) derived from an individual and processed to remove most cells. Examples of non-tissue samples include blood and blood components, urine, saliva, tears, and mucus.

[0035] "LNA" stands for locked nucleic acid. LNA is a modified RNA nucleotide in which the ribose portion is modified with an additional crosslink connecting the 2' oxygen and 4' carbon atoms. LNA nucleotides can be mixed with DNA or RNA residues at any position in the oligonucleotide and can hybridize with DNA or RNA according to the Watson-Crick base pairing rules. The arrangement of the locked ribose enhances the hybridization properties (e.g., increases the melting temperature).

[0036] The term “primer” refers to a short nucleic acid (oligonucleotide) that serves as a starting point for polynucleotide chain synthesis by nucleic acid polymerase under appropriate conditions. The polynucleotide synthesis and amplification reactions are typically carried out at appropriate temperatures, including appropriate buffers and / or dNTPs and / or rNTPs and one or more optional cofactors. A primer typically contains at least one region that hybridizes to a target, and that region is at least substantially complementary to its target sequence (e.g., with 0, 1, or 2 mismatches). For the purposes of this disclosure, this region is typically about 4 to 10 nucleotides in length (e.g., 5 to 8 nucleotides). A “primer pair” refers to a forward primer and a reverse primer that are oriented in the opposite direction to the target sequence and cause amplification products to be produced under amplification conditions. The terms “forward” and “reverse” are arbitrarily assigned. Those skilled in the art will understand that the forward and reverse primers (primer pair) define the boundary of the amplification product. In some embodiments, multiple primer pairs rely on a single common forward or reverse primer. For example, multiple allele-specific forward primers can be considered part of a primer pair that shares the same common reverse primer, for instance, when multiple alleles are in close proximity to each other. A "primer set" or "primer configuration" can refer to a single primer pair, or two or more primer pairs designed to function simultaneously in a single multiple reaction.

[0037] In this specification, “probe” means any molecule that can selectively bind to a specific target biomolecule (e.g., a nucleic acid sequence of interest that hybridizes to the probe). The probe is labeled with at least one detectable non-nucleotide moiety. In some embodiments, the probe is labeled with a phosphor and a quencher.

[0038] The terms "complementary" or "complementarity" refer to the ability of one nucleic acid in a polynucleotide to form base pairs with another nucleic acid in a second polynucleotide. For example, the sequence AGT (AGU in RNA) is complementary to the sequence TCA (UCA in RNA). Complementarity can be partial or complete; in the former case, only some nucleic acids match according to the base pairing rules, and in the latter case, all nucleic acids match according to the base pairing rules. A probe or primer is considered "specific" to a target sequence if it is at least partially complementary to the target sequence. Depending on the circumstances, the degree of complementarity to the target sequence is typically greater for shorter nucleic acids such as primers than for longer sequences (e.g., greater than 80%, greater than 90%, greater than 95%, greater than 98%). In some embodiments, primers and / or probes are 100% complementary to the target sequence.

[0039] The expression "specifically amplifies" indicates that a primer set amplifies the target sequence at a statistically significantly higher level than the non-target sequence. The expression "specifically detects" indicates that a probe detects the target sequence at a statistically significantly higher level than the non-target sequence. In this field, it will be understood that specific amplification and detection can be determined using a negative control (e.g., a sample containing the same nucleic acid as the test sample but not the target sequence, or a sample lacking nucleic acid). For example, primers and probes that specifically amplify and detect the target sequence will have a Ct (e.g., at least 2, 3, 4, 5, 5-10, 10-20, or 10-30 cycles less than the background) that is easily distinguishable from the background (non-target sequence). The term "allele-specific" PCR means amplification of a target sequence using primers that specifically amplify a particular allele variant of the target sequence. Typically, the forward or reverse primer contains the exact complementary sequence of the allele variant at its position.

[0040] The terms "identical" or "% identical" in the context of two or more nucleic acids or polypeptides mean two or more sequences or subsequences that have the same nucleotides or amino acids when measured with default parameters using the BLAST or BLAST 2.0 sequence comparison algorithm, or when measured by manual alignment and visual inspection, or two or more sequences or subsequences that have a certain percentage of identical nucleotides or amino acids (e.g., approximately 60% identical across a particular region, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). See, for example, the NCBI website at ncbi.nlm.nih.gov / BLAST. Therefore, such sequences are said to be "substantially identical." Since % identical is typically determined by the best-aligned sequences, the definition applies to sequences with substitutions as well as deletions and / or additions. Algorithms commonly used in this field take gaps, etc., into account. Typically, the match is present in a region containing a sequence of at least 8 to 25 amino acids or nucleotides, or in a region of 50 to 100 amino acids or nucleotides, or over the entire length of the reference sequence.

[0041] Terms such as "isolate," "separate," and "purify" are not intended to be absolute. For example, the isolation of DNA or genomic DNA does not require the removal of 100% of non-DNA molecules. Those skilled in the art will recognize acceptable levels of purification in given circumstances.

[0042] The term "kit" means any manufactured product (e.g., package or container) that includes at least one reagent (such as a nucleic acid probe or probe pool) for specifically amplifying, capturing, tagging / converting, or detecting RNA or DNA, as described herein.

[0043] The term "amplification conditions" refers to the conditions in a nucleic acid amplification reaction (e.g., PCR amplification) that enable primer hybridization and template-dependent extension. The term "amplicon" or "amplification product" refers to a nucleic acid molecule that contains all or a fragment of the target nucleic acid sequence and is formed as the product of in vitro amplification by any appropriate amplification method. The expression "to produce an amplification product" means, when applied to primers, that the primer produces a specified amplification product under appropriate conditions (e.g., in the presence of nucleotide polymerase and NTP). Various PCR conditions are described in Chapter 14 of *PCR Strategies* (Innis et al., 1995, Academic Press, San Diego, California) and *PCR Protocols: A Guide to Methods and Applications* (Innis et al., Academic Press, New York, 1990).

[0044] The term "amplification product" refers to the product of an amplification reaction. Amplification products include the primers used to initiate polynucleotide synthesis in each round. "Amplicon" is the sequence targeted for amplification, and this term can also be used to refer to amplification products. The 5' and 3' boundaries of the amplicon are defined by the forward and reverse primers. Terms such as "reverse transcript product" and "RT product" refer to the cDNA molecule produced on an RNA template by the extension of RT primers by a polymerase with reverse transcription activity.

[0045] The terms “individual,” “subject,” and “patient” are interchangeable herein. An individual can be pre-diagnosis, post-diagnosis but pre-treatment, during treatment, or post-treatment. In the context of this disclosure, an individual is typically seeking medical care.

[0046] The term “sample” or “biological sample” means any composition that contains or is thought to contain nucleic acids. This term includes purified or isolated components of cells, tissues, or blood (e.g., DNA, RNA, proteins, cell-free portions, cell lysates). A sample may be, for example, FFPET from a tumor or metastatic lesion. Samples may also be from frozen or fresh tissue, or from liquid samples (e.g., blood or blood components (plasma or serum), urine, semen, saliva, sputum, mucus, tears, lymph, cerebrospinal fluid, mouth / throat lavage fluid, bronchoalveolar lavage fluid, material washed from a swab, etc.). Samples may also include components and constituents of in vitro cultures of cells obtained directly from an individual (including cell lines). Samples may also be partially processed from samples obtained directly from an individual (e.g., cell lysates, or blood depleted of red blood cells). Tumor samples may include tissue from a tumor, or samples may include DNA from a tumor (e.g., ctDNA in the blood of a cancer patient).

[0047] The phrase "obtaining a sample from an individual" means that a biological sample from an individual is provided for testing. This can be done directly from the individual or from a third party who has directly obtained a sample from that individual.

[0048] The phrase "to provide a therapy to an individual" means that the therapy is prescribed, recommended, or made available to that individual. The therapy can, in practice, be administered to the individual by a third party (e.g., by injection during hospitalization) or administered by the individual themselves.

[0049] A “control” sample or “control” value that serves as a baseline means a value that serves as a baseline (usually a known baseline) for comparison with a test sample or test condition. For example, a test sample may be taken from a test condition (e.g., from an individual suspected of having cancer) and compared to a sample from a known condition (e.g., from an individual without cancer (negative control), or from an individual known to have cancer or a target sequence of interest (positive control)). In the context of this disclosure, a test sample is typically from a cancer patient (e.g., a tumor sample). A control may also represent a mean or range collected from a large number of tests or results (e.g., a large number of individuals known to have MSS or MSI). For example, controls for the presence, and / or quality, and / or quantity of nucleic acids (e.g., internal controls) may include primers or probes that will detect sequences known to be present in the sample (e.g., housekeeping genes such as beta-actin, beta-globin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal proteins L37 and L38, PPIase, EIF3, eukaryotic translation elongation factor 2 (eEF2), DHFR, succinate dehydrogenase, etc.). In some embodiments, the internal control can be a sequence from a region of the same gene (e.g., within a different exon) that is not generally a variant. Additional known polynucleotides of a specified length may also be added, for example. An example of a negative control is a control without nucleic acids, or a control containing primers or probes specific to sequences (e.g., from a different species) that are not expected to be present in the sample. Those skilled in the art will understand that the selection of controls depends on the individual assay, for example, to ensure that the controls are appropriate for the cell type and organism. Those skilled in the art will recognize that any number of parameters can be evaluated as controls in a design. For example, controls can be designed to allow comparison of treatment benefits based on pharmacological data (e.g., half-life) or treatment measures (e.g., comparison of benefits and / or side effects). These controls can be designed for in vitro application.Those skilled in the art will understand which controls are important in a given situation and which controls allow for data analysis based on comparison with the control values. Controls are also useful in determining the significance of the data. For example, if the value of a given parameter varies widely among the controls, the variation in the test sample will not be considered significant.

[0050] Terms such as "label," "tag," and "detectable portion" refer to a composition that can be detected by any of the following means: spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Useful labels include, for example, fluorescent dyes (phosphors), luminescent agents, and radioisotopes (e.g., 32 P, 3 H), high electron density reagents, affinity-based moieties (e.g., poly-A tag (interacts with poly-T) or poly-T tag (interacts with poly-A), His tag (interacts with Ni), or streptavidin tag (can be separated using biotin)). Those skilled in the art will understand that the detectable labels to be bound to nucleic acids are not naturally occurring.

[0051] Unless otherwise specified, scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art. See, for example, Lackie, *Dictionary of Cell and Molecular Biology*, Elsevier (4th edition, 2007); Sambrook et al., *Molecular Cloning, A Laboratory Manual*, Cold Springs Harbor Press (Cold Springs Harbor, New York, 1989). The term "one" shall mean "one or more." The terms "contains" or "includes," when placed after the description of a process or element, shall mean that further processes or elements may be added, and such processes or elements shall not be excluded.

[0052] III. Microsatellite Instability (MSI) and Mismatch Repair (MMR)

[0053] Microsatellites are short DNA repeats (1–6 base pairs) scattered throughout the genome. Some normal variation exists in microsatellite length between individuals. However, microsatellite instability (MSI) causes differences in the length of one or both repeats in an individual's allele. This is due to defects in the mismatch repair (MMR) system.

[0054] Detecting MSI has value both for prediction and prognosis. MSI-H tumors are more likely to respond to immune checkpoint therapy (e.g., drugs targeting PD-1 or PD-L1, such as Keytruda). In addition, patients with MSI-H CRC have a better survival rate.

[0055] A panel consisting of five mononucleotide repeat markers is recommended for testing by the National Cancer Institute (Suraweera et al. (2002) Gastroenterology Vol. 123: p. 1804; Umar et al. (2004) J. Natl. Cancer Inst. Vol. 96: p. 261). Samples are called MSI-high (MSI-H) if two or more markers show differences in repeat length between a normal control sample and a tumor sample. Common MSI markers are shown in Figure 1 (Zhang et al. (2008) J. Mol. Diagnostics Vol. 10: p. 301).

[0056] Current methods for detecting MSI utilize capillary electrophoresis (CE) to identify size differences between normal and tumor samples, after amplifying the relevant region with fluorescent primers. Kits for one-well multiplex detection of five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24, MONO-27) (and two pentanucleotide markers for detecting potential sample mixing or contamination) are commercially available (Bacher et al. (2004) Disease Markers Vol. 20: p. 237). A typical readout is shown in Figure 2. This method suffers from significant cost, a cumbersome protocol, a risk of contamination (due to the open-tube design and post-PCR handling), and limited analytical sensitivity (requiring a 10% tumor cell concentration). Furthermore, results must be reviewed to remove peaks resulting from artifacts caused by bleed-through, stutter, or CE spikes.

[0057] IV. Nucleic Acid Samples Samples for detecting biomarkers can be obtained from any source thought to contain nucleic acids (e.g., tissue (including tumor tissue or FFPET tissue), blood, skin, swabs (e.g., oral, vaginal), urine, saliva, etc.).

[0058] Methods for isolating nucleic acids from biological samples are known, for example, described by Sambrook, and several kits are commercially available (e.g., High Pure RNA Isolation Kit, High Pure Viral Nucleic Acid Kit, MagNA Pure LC Total Nucleic Acid Isolation Kit, DNA Isolation Kit for Cells and Tissues, DNA Isolation Kit for Mammalian Blood, and High Pure FFPET DNA Isolation Kit, all available from Roche). In the context of the methods described herein, genomic DNA can be recovered and isolated.

[0059] V. Amplification and Detection For example, nucleic acid samples containing genomic DNA can be used for detection and quantification using nucleic acid amplification.

[0060] In some embodiments, the target nucleic acid is amplified using a thermostable polymerase that possesses both reverse transcription activity and DNA template-dependent activity. Representative enzymes include Tth DNA polymerase, C. therm polymerase system, and enzymes disclosed in US 2014 / 0170730 and US 2014 / 0051126.

[0061] Probes used as described herein may be labeled with a phosphor and, optionally, a quencher (e.g., TaqMan, LightCycler, Molecular Beacon, Scorpion, and Dual Labeled probes). Non-limiting examples of suitable phosphors include FAM, JOE, TET, Cal Fluor Gold 540, HEX, VIC, Cal Fluor Orange 560, TAMRA, Cyanine 3, Quasar 570, Cal Fluor Red 590, Rox, Texas Red, Cyanine 5, Quasar 670, and Cyanine 5.5. Non-limiting examples of suitable quenchers include TAMRA (for FAM, JOE, and TET), DABCYL, and BHQ1-3.

[0062] In some embodiments, microsatellite instability can be detected using digital PCR (dPCR). For example, using digital droplet PCR (ddPCR), it is possible to obtain absolute measurements of target nucleic acids in a sample even at very low concentrations. The dPCR method includes steps of digital dilution or droplet generation, PCR amplification, detection, and (potentially) analysis. The partitioning step, which generates droplets, involves generating multiple separate reaction volumes (e.g., droplets or partitions), each reaction volume containing the reagents necessary to carry out nucleic acid amplification. The PCR amplification step involves generating amplicons by subjecting the partitioned volumes to thermal cycling conditions suitable for amplification of the nucleic acid target. Detection involves identifying partitioned volumes containing the amplicon or fluorescent signal and partitioned volumes that do not contain it. The analysis step involves quantification, which involves obtaining, for example, the concentration, absolute amount, or relative amount (compared to another target) of the target nucleic acid in the sample. Commercially available dPCR systems can be found, for example, from Bio-Rad and ThermoFisher. Explanations of dPCR can be found, for example, in US 2014 / 0242582; Kuypers et al. (2017) J Clin Microbiol Vol. 55: p. 1621; and Whale et al. (2016) Biomol Detect Quantif Vol. 10: p. 15.

[0063] PCR detection systems are well-known in this field, and one suitable for the selected label can be chosen. Suitable detection systems for quantitative PCR include the cobas® system and the Light Cycler® system (Roche), and the PRISM 7000 and 7300 real-time PCR systems (Applied Biosystems). Six-channel detection is available with the CFX96 real-time PCR detection system (Bio-Rad) and Rotorgene Q (Qiagen), enabling higher-order multiplexing.

[0064] VI. MSI and Treatment The methods of this disclosure are useful for the detection of MSI and the treatment of MSI-H tumors and Lynch syndrome (hereditary mutations in mismatch repair genes). In some embodiments, targeted therapies are prescribed, provided, or administered to patients based on the presence or absence of disease-related biomarkers (such as at least one MSI). In particular, immune checkpoint therapies (e.g., therapies targeting PD-1 or PD-L1) may be prescribed, provided, or administered to patients with MSI-H tumors (MSI-positive at two or more microsatellite locations). Examples include pembrolizumab (Keytruda), nivolumab (Opdivo), atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi).

[0065] Some drugs are specifically designed for patients with certain biomarker profiles that can be investigated in combination with MSI (e.g., Tarceva and Tagrisso for certain EGFR mutations). With numerous new targeted therapies under development to address specific mutations, those skilled in the art are currently in the best position to select a targeted therapy for their individual needs.

[0066] In addition, cancer patients can benefit from standard chemotherapy. Therefore, in some embodiments, chemotherapy is prescribed, provided, or administered to patients based on the presence or absence of cancer-related biomarkers. Some chemotherapy regimens include CHOP (cyclophosphamide; doxorubicin; vincristine; prednisolone) or R-CHOP (which further includes rituximab and / or etoposide). This cocktail can be administered regularly over a set period or until a reduction in tumor size and / or symptoms is detected. For example, CHOP or R-CHOP may be administered every two or three weeks.

[0067] In patients with MSI-H, survival rates have been found to be better than in patients with MSS. In some embodiments, treatment is prescribed, provided, or administered in a manner that is adjusted, for example, to reduce side effects and prolong survival.

[0068] Regardless of the treatment method chosen, treatment typically begins with a low dose, and after identifying side effects, the dose is increased, for example, until side effects appear, or within the patient's tolerance, or until a clinical benefit is observed.

[0069] VII. Kits This specification provides kits for performing microsatellite instability (MSI).

[0070] In some embodiments, the kit includes sample collection containers (e.g., test tubes, vials, multiwell plates, multi-container cartridges).

[0071] In some embodiments, the kit includes reagents and / or components for purifying nucleic acids. For example, the kit may include lysis buffers (e.g., washing agents, chaotropic agents, buffers, etc.), enzymes or reagents for denaturing proteins or other undesirable materials in the sample (e.g., proteinase K), and enzymes for retaining nucleic acids (e.g., DNase and / or RNase inhibitors). In some embodiments, the kit includes components for separating nucleic acids (e.g., chromatographic matrices, which are solid or semi-solid matrices, magnetic beads, magnetic glass beads, glass fibers, silica filters, etc.). In some embodiments, the kit includes washing and / or elution buffers for purifying and releasing nucleic acids from solid or semi-solid matrices. For example, the kit may include components from MagNA Pure LC Total Nucleic Acid Isolation Kits, Mammalian DNA Isolation Kits, High Pure Isolation Kits or MagNA Pure RNA Isolation Kits (Roche), DNeasy Kits or RNeasy Kits (Qiagen), PureLink DNA Isolation Kits or PureLink RNA Isolation Kits (Thermo Fisher), etc.

[0072] In some embodiments, the kit includes reagents for detecting specific nucleic acids (e.g., target nucleic acids associated with MSI, cancer, or other diseases). For example, the kit may include microsatellite sequences, sequences adjacent to microsatellite sequences, or oligonucleotides that specifically bind to cancer-related biomarkers (such as certain miRNAs, mutations, or sequences known to change copy number in cancer). In some embodiments, the detection reagents are for qPCR, dPCR, or sequencing (Sanger or NGS).

[0073] The kit may further include reagents for amplification (e.g., reverse transcriptase, DNA polymerase, dNTPs, buffers, and / or other elements suitable for reverse transcription and / or amplification (e.g., cofactors or aptamers)). Typically, the reagent mixture is concentrated, and aliquots are added to the final reaction volume along with the sample (e.g., RNA or DNA), enzyme, and / or water. In some embodiments, the kit further includes reverse transcriptase (or an enzyme with reverse transcription activity) and / or DNA polymerase (e.g., the heat-stable DNA polymerases Taq, ZO5, and their derivatives).

[0074] In some embodiments, the kit further includes at least one control sample (e.g., nucleic acid from an MSS sample (or pooled sample), nucleic acid from a sample (or pooled sample) known to contain the target sequence). In some embodiments, the kit includes a negative control that, for example, lacks nucleic acid or the target nucleic acid sequence. In some embodiments, the kit further includes consumables (e.g., plates or test tubes for preparing nucleic acids, test tubes for collecting samples, plates, test tubes, or microchips for PCR or qRT-PCR). In some embodiments, the kit further includes either instructions for use, a reference to a website, or software. [Examples]

[0075] VIII. Examples Example 1: Design Strategy for Detecting MSI by Digital PCRWe attempted to detect nucleotide deletions in mononucleotide repeats. For this purpose, we designed probes and primers to detect the BAT25 and BAT26 markers (25 and 27 mononucleotide repeats, respectively). The MSI at these marker locations has a broad detection range of 1–13 nucleotides. We utilized a drop-off dPCR assay, i.e., an assay using a "drop-off" probe that binds to the wild-type sequence but has reduced affinity for the insertion or deletion sequence. The drop-off probe is used in combination with a reference probe. The reference probe is designed to hybridize to an invariant genomic sequence adjacent to or near the microsatellite sequence. A schematic diagram is shown in Figure 3. This diagram shows the positions of the drop-off probe and the reference (non-microsatellite) probe, as well as the positions of the fluorophores and quenchers on the probes. In principle, the quencher blocks fluorescence when the probe is intact, but the fluorophores emit a signal when the bound probe is hydrolyzed by polymerase during PCR.

[0076] The challenges include detecting only minor deletions (e.g., 1-2 nucleotides) while still detecting normal variations in allele length and DNA from damaged DNA (e.g., from FFPET samples).

[0077] Using a probe designed as shown in Figure 3 to detect differences between BAT25 markers (22 vs. 25 polyT (sequences 23 and 24, respectively)) and BAT26 markers (18 vs. 27 polyT (sequences 25 and 26, respectively)), we found inefficient quenching and a high baseline. This is partly due to the rigidity of the mononucleotide probe and inefficient quenching using a quencher located at the opposite end of the probe. The results are shown in Figure 4. This graph shows the signal from the phosphor of the drop-off probe on the vertical axis and the signal from the phosphor of the reference probe on the horizontal axis. Wild-type (non-MSI) signals appear in the upper right cluster, while MSI (Jurkat) signals are in the lower right cluster. The upper graph is for BAT25, and the lower graph is for BAT26 (27T (sequence 26)).

[0078] Example 2: Using a quencher oligonucleotide with a drop-off probe The drop-off probes for these markers contain a long stretch of poly-T, which forms a rigid structure. Therefore, the quencher does not approach the phosphor closely enough to effectively quench it. To improve the quenching of the non-binding probe, a poly-A quenching oligonucleotide was added. The drop-off probe and quenching oligonucleotide are shown below as SEQ ID NOs: 1 and 2, respectively. Drop-off probe: 5'-FAM-TGATTTTTTTTTTTTTTTTTTTTTTTTTTTGAG-BHQ2-3' Quenching oligo: 3'-BHQ2-AAAAAAAAAAAAAAAAA 16-20 -5'

[0079] In principle, the quencher oligo binds to the uncleaved probe at temperatures below 50°C (typical PCR annealing temperature), and hybridization improves quenching by keeping the additional quencher closer to the phosphor. Since this complex melts above 50°C, it does not prevent the probe from binding to the template under dPCR conditions. When the drop-off probe is cleaved by polymerase, the 5'-FAM fragment is too short to re-bind to the poly(A) quencher oligo at room temperature (or detection temperature), and the signal is maintained.

[0080] As shown in Figure 4, the quencher oligo worked well in the BAT26 microsatellite, reducing background noise, but not in BAT25. Shortening the length of the poly(A) quencher oligo to 16 nucleotides resulted in effective quenching and reduced background noise in both microsatellites.

[0081] Example 3: Alternative method for improving quenching As shown in Figure 5, we designed an alternative approach to improve drop-off probe quenching. In this approach, the reference probe partially overlaps with the drop-off probe, so the reference probe hybridizes to the drop-off probe at a lower temperature, quenching the phosphor. Figure 5 shows that these approaches, with 16 or 12 nucleotides overlapping, reduce background noise as well as the independent 16-mer quencher probe approach.

[0082] Example 4: Hairpin Probe We also attempted to use a hairpin-structured probe as a drop-off probe. In this case, the complete probe forms a hairpin structure through intramolecular base pairing, allowing the quencher to approach the phosphor more closely (see, for example, Figure 6 and Sequence ID No. 3). The nucleotides shown in shaded area in Sequence ID No. 3 form a hairpin and hybridize to the template. Since the hairpin melts above 50°C, the probe binds to the target and is hydrolyzed by polymerase.

[0083] Example 5: Internal Quencher Another approach to reduce background / improve drop-off probe quenching is to bring the quencher closer to the phosphor on the probe. Figure 7 shows drop-off probes for the BAT25 marker (SEQ ID NO: 4) and the BAT26 marker (SEQ ID NO: 5), with the quencher located at the 3' end or internally. The plot on the left shows the results of MSI assays for BAT25 (top) and BAT26 (bottom) with a 3' quencher, and the plot on the right shows the results of MSI assays for BAT25 (top) and BAT26 (bottom) with an internal quencher. In both assays, the internal quencher was more effective in reducing background fluorescence from the drop-off probe that was not hydrolyzed.

[0084] Example 6: Detection of small insertions / deletions Although most tumors exhibit several nucleotide shifts within microsatellite repeat sequences, a large proportion have only one or two nucleotide shifts. For example, CRC tumors average six nucleotide shifts, but 12% of the tumors examined showed only one nucleotide shift. In EMC, the average shift is three nucleotides, but 76% of the samples examined showed only one nucleotide shift.

[0085] We attempted to investigate whether the method of the present invention could detect shifts of a small number of nucleotides. Figure 8 shows that this method was effective in distinguishing signals from several different MSI cell lines with deletions of varying sizes.

[0086] Example 7: Detection of MSI in FFPET samples DNA from formalin-fixed paraffin-embedded tissue (FFPET) samples is often damaged and fragmented. Since tumor tissue is frequently preserved as FFPET, our method is considered ideal even for damaged DNA.

[0087] BAT25 and BAT26 MSI assays were performed on DNA from wild-type (MSS) FFPET samples and MSI FFPET samples. The results demonstrate that the MSI assays described herein function even with FFPET DNA.

[0088] Example 8: Multiple MSI detection To further streamline our assay compared to current capillary electrophoresis methods, we investigated whether the MSI assay described herein could be performed in the same dPCR reaction. A drop-off probe, a reference probe, and amplification primers for BAT25 and BAT26 microsatellites were combined. Multiplex assays were performed in RAJI and Jurkat cell lines, both of which have MSI at both sites.

[0089] Figure 10 shows that the probe and primer did not interfere with each other, and the results were clear. The left and center figures show the single reactions, respectively, revealing the presence of two alleles in each cell line. The results of the double reaction are shown on the right, revealing the same result.

[0090] Example 9: Detection of five types of MSI markers using an internal quencher probe Digital PCR assays were designed for five commonly detected mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24, Mono-27). All five markers exhibited the expected phenotypes in 2D scattering plots when tested against genomic DNA from MSS cell lines (BT-549, top figure; positive clusters showed high fluorescence in both the drop-off probe FAM channel and the reference probe HEX channel) and MSI-H cell lines (RKO cell lines, bottom figure; positive clusters showed high fluorescence only in the reference probe HEX channel) (Figure 11).

[0091] The drop-off probe sequences for BAT-25 and BAT-26 are the same internal quencher probes shown in Figure 7 (sequences 4 and 5, respectively). Sequence number 6 shows the internal quencher drop-off probe sequence for NR-21.<FAM_Thr> CCTTTTTTT<BHQ_2> TTTTTTTTTTTTTTTAGCAAC <phos>Sequence ID 7 shows the sequence of the internal quencher drop-off probe for NR-24.<FAM_Thr> TCCTATTTTTT<BHQ_2> TTTTTTTTTTTTTTTTTTTTGTGAG <phos>Sequence ID 8 shows the sequence of the internal quencher dropoff probe for Mono-27.<FAM_Thr> ACTCTTTTTT<BHQ_2> TTTTTTTTTTTTTTTTTTTTTGAG <phos>).

[0092] Example 10: NR-21 assay for detecting subtle differences in nucleotide deletions Daudi cells exhibit a two-nucleotide shortening of the NR-21 MSI marker compared to BT549 cells, either heterozygous or homozygous. Drop-off probes sometimes fail to distinguish such subtle differences in repeat length. The first drop-off probe designed to detect NR-21 did not adequately distinguish between the MSI marker from Daudi cells and that from BT549 cells. Therefore, we designed a drop-off probe with sufficient sensitivity to distinguish between the two-nucleotide difference in the NR-21 MSI marker.

[0093] Figure 12 shows the results of comparing DNA from BT549 cells (top two figures) and Daudi cells (bottom two figures) using probe 1 and probe 5. Probe 1 is sequence<FAM_Thr> TGGCCTTTTTTT<BHQ_2> TTTTTTTTTTTTTTTAGCA <phos> This represents the first drop-off probe with (SEQ ID NO: 9). Probe 5 is already shown above as SEQ ID NO: 6. As shown in Figure 12, probe 5 distinguishes the higher-order FAM signal from the wild-type NR-21 copy from the lower-order FAM signal from the NR-21 copy with the deletion.< / phos> < / phos> < / phos> < / phos>

Claims

1. a) A drop-off probe having a sequence complementary to a wild-type microsatellite repeat sequence in genomic DNA, and having at least one complementary additional nucleotide at the 5' end and at least one complementary additional nucleotide at the 3' end of the wild-type microsatellite repeat sequence, and attached to a first label and a first quencher, wherein the complementary additional nucleotides at the 3' and 5' ends are complementary to a sequence adjacent to the wild-type microsatellite repeat sequence in genomic DNA, and the first quencher of the drop-off probe is attached to a site within 10 nucleotides of the first label; b) A reference probe having a sequence complementary to a reference sequence that is not a microsatellite repeat sequence in the genomic DNA, and attached to a second label and a second quencher; c) An amplification primer set for annealing 5' upstream and 3' downstream of the binding site of the drop-off probe and the reference probe, wherein the amplification primer set is designed to amplify both the binding site of the drop-off probe and the reference probe, and A kit that includes this.

2. The kit according to claim 1, wherein the drop-off probe has at least three nucleotides at the 5' end and at least three nucleotides at the 3' end of the wild-type microsatellite repeat sequence.

3. The kit according to claim 1 or 2, wherein the first quencher of the drop-off probe is attached within a sequence of the drop-off probe that is complementary to a wild-type microsatellite repeat sequence.

4. The kit according to any one of claims 1 to 3, wherein the drop-off probe has at least four nucleotides at the 5' end of the microsatellite repeat sequence.

5. c) A quencher oligonucleotide complementary to the drop-off probe, wherein the quencher oligonucleotide comprises a first quencher and quenches the first label of the drop-off probe. A kit according to any one of claims 1 to 4, further comprising a quencher oligonucleotide.

6. The kit according to any one of claims 1 to 5, wherein the drop-off probe forms a hairpin structure at a temperature below 50°C.

7. The kit according to any one of claims 1 to 6, wherein the reference probe comprises a sequence complementary to at least six nucleotides of the drop-off probe, and further comprises a first quencher at its 3' end, which quenches the first label of the drop-off probe.

8. The kit according to claim 7, wherein the reference probe comprises a sequence complementary to at least 10 nucleotides of the drop-off probe, and further comprises a first quencher at its 3' end, which quenches the first label of the drop-off probe.

9. The kit according to any one of claims 1 to 8, wherein the kit comprises at least one drop-off probe having a sequence complementary to different wild-type microsatellite repeat sequences in genomic DNA, and having at least one nucleotide at the 5' end and at least one nucleotide at the 3' end of the different wild-type microsatellite repeat sequences, wherein a third label and a third quencher are attached to the at least one drop-off probe.

10. The kit according to any one of claims 1 to 8, wherein the kit comprises at least four additional drop-off probes having sequences complementary to different wild-type microsatellite repeat sequences in genomic DNA, and each having at least one nucleotide at the 5' end and at least one nucleotide at the 3' end of the different wild-type microsatellite repeat sequences, wherein the at least four additional drop-off probes are attached to different labels that are different from the first label, the second label, and the third label, and to corresponding quenchers.

11. The kit according to claim 9, wherein the kit comprises at least one additional reference probe having a sequence complementary to a different reference sequence in genomic DNA that is not a microsatellite repeat sequence, the at least one additional reference probe being attached to a fourth quencher with a different label that is different from the first label, the second label, and the third label.

12. The kit according to claim 10, wherein the kit comprises at least four additional reference probes having sequences complementary to different reference sequences in genomic DNA that are not microsatellite repeat sequences, the at least four additional reference probes being attached to quenchers corresponding to different labels that are different from the first label, the second label, and the third label.

13. The kit according to any one of claims 1 to 12, further comprising a heat-stable DNA polymerase.

14. A method for detecting microsatellite instability, a) Genomic DNA, i) A drop-off probe having a sequence complementary to a wild-type microsatellite repeat sequence in genomic DNA, and having at least one nucleotide at the 5' end and at least one nucleotide at the 3' end of the wild-type microsatellite repeat sequence, wherein the drop-off probe is attached to a first label and a first quencher, and the first quencher of the drop-off probe is attached within 10 nucleotides of the first label; ii) A reference probe having a sequence complementary to a reference sequence that is not a microsatellite repeat sequence in the genomic DNA, and attached to a second label and a second quencher; iii) A set of amplification primers for annealing 5' upstream and 3' downstream of the binding sites of the drop-off probe and the reference probe, wherein the set of amplification primers is designed to amplify the binding sites of both the drop-off probe and the reference probe. Make contact with it; b) By performing dPCR, the binding of the drop-off probe and the reference probe to genomic DNA is amplified and detected; c) Detecting microsatellite instability when the binding of the drop-off probe to genomic DNA is less than the control value or threshold value. A method that includes this.

15. The method according to claim 14, which is performed in multiple steps using two or more sets of drop-off probes, a reference probe, and a set of amplification primers, arranged to detect instability at the locations of two or more microsatellite repeat sequences.

16. The method according to claim 14, which is performed multiple times using a set of five or more sets of drop-off probes positioned to detect instability at the locations of five or more microsatellite repeat sequences, a reference probe, and a set of amplification primers.

17. a) A drop-off probe having a sequence complementary to a wild-type microsatellite repeat sequence in genomic DNA, and having at least one nucleotide at the 5' end and at least one nucleotide at the 3' end of the wild-type microsatellite repeat sequence, and attached to a first label and a first quencher, wherein the first quencher of the drop-off probe is attached to a site within 10 nucleotides of the first label; b) A reference probe that has a sequence complementary to a reference sequence that is not a microsatellite repeat sequence in the genomic DNA, and is attached to a second label and a second quencher; c) A set of amplification primers for annealing 5' upstream and 3' downstream of the binding sites of the drop-off probe and the reference probe, wherein the set of amplification primers is designed to amplify the binding sites of both the drop-off probe and the reference probe; d) With heat-stable DNA polymerase; e) Genome DNA A reaction mixture containing the following:

18. The at least one additional drop-off probe having a sequence complementary to a different wild-type microsatellite repeat sequence in genomic DNA, and having at least one nucleotide at the 5' end and at least one nucleotide at the 3' end of the different wild-type microsatellite repeat sequence, wherein the at least one additional drop-off probe is attached to a third label and a third quencher; At least one additional reference probe having a sequence complementary to a different reference sequence that is not a microsatellite repeat sequence in the genomic DNA, and to which a fourth label and a fourth quencher are attached, which are different from the first, second, and third labels. The reaction mixture according to claim 17, further comprising:

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