Patent Description:
A commonly encountered situation in genetic analysis entails the need to identify a low percent of variant DNA sequences (`minority alleles') in the presence of a large excess of non-variant sequences (`majority alleles'). Methods to determine the original abundance of mutant alleles of one or more barcoded target sequences following mutation enrichment and sequencing are described in <CIT>.

The following are examples of situations in which minority alleles need to be identified. (a) identification and sequencing of a few mutated alleles in the presence of a large excess of normal (wild-type) alleles, a commonly encountered situation in cancer. (b) identification of a few methylated alleles in the presence of a large excess of unmethylated alleles (or: vice versa) in epigenetic analysis. (c) identification and genotyping of a few fetal DNA sequences circulating in the maternal blood where a large excess of maternal DNA sequences are also present. (d) identification of tumor-circulating DNA in blood or in urine of cancer patients (or abnormal DNA in people suspected of having cancer) in the presence of a large excess of wild type alleles. Detection of low-prevalence somatic mutations in tumors with heterogeneity, stromal contamination or in bodily fluids is difficult. However, the clinical significance of identifying these mutations is huge in many situations. For example, (a) in lung adenocarcinoma, low-level EGFR mutations that cannot be identified by regular sequencing can predict positive response to tyrosine kinase inhibitors or drug resistance; (b) mutations in plasma useful as biomarkers for early detection or tumor response to treatment cannot be sequenced using conventional methods; and (c) mutations in tumors with frequent stromal contamination, such as pancreatic or prostate cancer, can be 'masked' by presence of wild type alleles, thus requiring laborious micro-dissection or missing mutations altogether.

In some cases, minority alleles are microsatellites in which there are one or more deletions compared to microsatellites in majority alleles.

Microsatellites (e.g., homopolymer repeats) abide in the human genome. These comprise multiple repeats of a single or more nucleotides, such as poly-adenines (e.g., 15A's). A special type of mutation found in certain cancers involves insertions or deletions ('indels') in microsatellites, usually anywhere between <NUM>-<NUM> bp long. During cell division, polymerases performing DNA duplication often produce indels when they synthesize over microsatellites. These indels are often repaired by DNA mismatch repair enzymes. However, in many diseases (e.g., cancers), the indels found in microsatellites are increased due to mismatch repair deficiency. This leads to microsatellite instability (MSI). High levels of MSI are predictive for outcome during chemotherapy for various types of cancers (e.g., colorectal cancer (CRC)) and have been associated with distinct characteristics and favorable results including better prognosis, a higher <NUM>-year survival, and lesser metastasis. Tumors with MSI are also more amenable to treatment via immunotherapy. Hence it is of great interest to screen tumors for MSI. At the same time, monitoring of MSI in plasma by screening tumor circulating DNA instead of testing the tumor can be clinically valuable for prognostic or predictive applications or as a marker for assessment of residual tumor load, to detect minimal residual disease (MRD).

However, detection of MSI and microsatellite indels is difficult. For example, while MSI is more frequent in colon cancers than other cancers, MSI detection in colonoscopy-obtained polyps, as well as in cfDNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. Improvements in sensitivity of MSI detection include utilization of long nucleotide repeats that display increased instability as compared to shorter repeats (<NPL>). Further, COLD-PCR technology (<NPL>;<NPL>; and <NPL>) has recently been adapted to enrich altered microsatellites and suppress wild-type (WT) alleles for sensitive detection of single microsatellite sequences in the HSP110 microsatellite (<NPL>). This approach utilizes a `blocker' DNA oligonucleotide that blocks polymerase amplification by hybridizing to wild-type HSP110 microsatellites. In contrast, the blocker oligonucleotide prevents amplification of deletion-containing microsatellites since the blocker only hybridizes to the wild-type microsatellite alleles. The DNA blocker approach for enrichment of deletions at microsatellites provides an improvement in the sensitivity of detection for given microsatellite targets.

Despite these improvements, the enrichment of mutations via PCR is ultimately limited by polymerase-introduced errors (`stutter bands') (<NPL>; and <NPL>) that introduce wild-type allele changes indistinguishable from genuine indels. Thus, small indels comprising few nucleotide changes unavoidably fall within stutter bands that confound interpretation when capillary electrophoresis is employed for endpoint detection. High resolution melting-based MSI detection enables convenient assessment of MSI, but is also PCR-based and liable to stutter artifacts. Similarly, sequencing (e.g., Next generation sequencing (NGS)) is error-prone when it comes to identifying changes in microsatellites. While bioinformatic approaches provide opportunities to correct or over-look polymerase and sequencing errors, detection of small indels within large homopolymers remains a problem that limits the otherwise highly promising NGS-based detection of MSI.

This disclosure provides compositions and methods for using a modified version of Nuclease-assisted Mutation Enrichment (NaME) to enrich microsatellites containing deletions relative to wild-type microsatellites that do not contain deletions. The methods disclosed herein enable simultaneous enrichment of deletion-containing microsatellites at multiple targets or throughout the entire genome (g- NaME) as opposed to previous approaches that enrich indels in one or few DNA sequences or microsatellites at a time. This high throughput approach is anticipated to enable an unprecedented increase in sensitivity and the ability to detect presence of microsatellite deletions with a limited amount of whole-genome sequencing. This approach has unique applications such as detection of minimal residual disease by screening circulating DNA in blood samples obtained from patients that underwent therapy. Alternatively, methods disclosed herein can be used for early cancer detection in patients that do not have signs of disease.

Methods provided herein allow for multiplexed enrichment of microsatellites having deletions in a way that does not require knowledge of sequence of either microsatellites or sequences flanking microsatellites.

Contemplated herein is the use of NaME or NaME-with probe overlap (NaME-PrO) (<NPL>) with generic probes. Generic probes, as described herein, comprise of a region that is complementary to a microsatellite sequence and which may be flanked by nucleotides that base-pair without specificity (e.g., inosines). The nucleotides of the probes that flank the region that is complementary to a microsatellite sequence do not form a sequence that is specifically complementary to sequences flanking microsatellites in genomic DNA. Herein, "flanking" is used to mean that there is no nucleotide between the sequence being flanking and the sequence/nucleotides that is flanking.

Specifically, provided is a method for enriching deletion-containing microsatellite targets in a sample of genomic DNA, the method comprising:.

subjecting the nucleic acid sample to a third temperature that allows the DSN to preferentially cleave the complementary wild-type nucleic acid-probe duplexes relative to partially complementary target nucleic acid-probe duplexes, wherein the DSN is added:.

In some embodiments, the third temperature is higher than the melting temperature of the target nucleic acid-probe duplexes.

In some embodiments, a DSN is thermostable.

In some embodiments, a first and a second oligonucleotide probes each comprise at least one modified nucleotides (e.g., locked nucleotide (LNA), peptide nucleic acid (PNA), or xeno nucleic acid (XNA)) that increases the difference in melting temperatures of the fully-matched wild-type nucleic acid-probe sequence and target nucleic acid-probe mismatched sequences.

In some embodiments, a first and a second oligonucleotide probes each comprise <NUM> to <NUM> modified nucleotides (e.g., LNAs, PNAs, or XNAs), wherein the distance between the innermost LNAs, PNAs, or XNAs is greater than <NUM> nucleotides. In some embodiments, the region of the first and second probes that is complementary to the microsatellite sequence in the top and bottom strands of the wild-type nucleic acid is <NUM>-<NUM> nucleotides long. In some embodiments, a first and second probes comprise <NUM>-<NUM> inosines that flank one or both sides of the region that is complementary to the microsatellite sequence. In some embodiments, the number of inosines in the <NUM>' end of the first probe is not equal to the number of inosines in the <NUM>' end of the second probe, and the number of inosines in the <NUM>' end of the first probe is not equal to the number of inosines in the <NUM>' end of the second probe. In some embodiments, a first and second probes are <NUM> to <NUM> nucleotides long. In some embodiments, a first and second probes are <NUM> to <NUM> nucleotides long.

In some embodiments, a method for enriching deletion-containing microsatellite targets using probes further comprises adding Mg<NUM>+ to the nucleic acid sample so that the concentration of the Mg<NUM>+ in the nucleic acid sample is between <NUM>-<NUM> when the nucleic acid sample is subjected to the second temperature and/or third temperature.

In some embodiments, a microsatellite is a mono-nucleotide repeat, di-nucleotide repeat, or tri-nucleotide repeat.

In some embodiments, a first and second probes are in molar excess of <NUM>-fold to <NUM> billion-fold compared to wild-type and target nucleic acids.

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

Nuclease-assisted Mutation Enrichment (NaME) that results in selective degradation of target wild type DNA or RNA, thereby providing enrichment of mutated target sequences has been previously disclosed in <CIT> and child applications thereof. NaME utilizes a double-strand specific nuclease (DSN, e.g., from crab or shrimp) that selectively degrades double stranded nucleic acid (e.g., DNA, or DNA/RNA hybrids), while it has minimal action on single stranded nucleic acid (e.g., ssDNA or ssRNA).

NaME can be used before, during or after PCR or other amplification methods. Subsequently, mutation-enriched sequences can be screened via any currently available method to identify the mutations (e.g., Sanger Sequencing, SSCP, next generation sequencing, MALDI-TOF for known mutations, High Resolution Melting HRM for pre-screening unknown mutations, and Single Molecule Sequencing, or third generation sequencing).

An adaptation of NaME to detect microsatellites on specific DNA targets is described in <NPL>. By using NaME before PCR, wild-type microsatellites are eliminated before generation of the `stutter bands', hence bypassing the false positives generated by such polymerase errors. Thus, NaME was shown to be an excellent way to eliminate wild type microsatellites, thereby enriching microsatellites with deletions and thus enhancing the detection of indel-containing microsatellites without being affected by stutter bands. This NaME approach for enriching specific micro-satellite containing targets having deletions uses probes that have a region that is complementary to a microsatellite and which also has sequences flanking the region that is complementary to the microsatellite, wherein the flanking sequence is complementary to the sequence in a wild-type nucleic acid that flanks the microsatellite.

Herein, novel methods of enriching microsatellites with deletions (herein referred to as "target microsatellites") are provided in which either generic probes (that do not comprise sequences that flank a region that is complementary to microsatellites that are complementary to sequence on wild-type nucleic acids that flank the microsatellites) or no probes are used in NaME so that multiple target microsatellites are enriched in a sample of genomic nucleic acid simultaneously.

The applications and field of use of the presently-disclosed method include any enrichment of any target sequence in a manner that achieves very high multiplexity in a single tube reaction, i.e. it is effectively a genome-wide enrichment for microsatellite repeat sequences with deletions. Thus g-NaME can be used for a very high sensitivity detection of deletions in microsatellites over the whole genome, thereby increasing sensitivity and reducing the amount of sequencing and sequencing reagents that need to be consumed to identify deletions present in samples that have a low level of target sequence to be detected, e.g., liquid biopsies in cancer patients. Highly sensitive detection of minimal residual disease in cancer patients can be achieved by use of the method disclosed herein. By applying g-NaME to tumors, it is possible to identify tumor-specific deletions present at microsatellites (e.g., homopolymers) and then trace these to the corresponding circulating DNA, thereby identifying minimal residual disease with high sensitivity and low-pass sequencing, using minimal amount of circulating DNA.

If genome-wide sequencing is performed without any enrichment, some deletions might still be detected albeit at lower sensitivity and with the requirement of deeper and more expensive sequencing. Therefore, provided herein is a method of improving the sensitivity of detecting deletion-containing microsatellite targets in a sample of genomic DNA.

The invention as claimed is directed to a method for enriching deletion-containing microsatellite targets in a sample of genomic DNA, the method comprising:.

The oligonucleotides probes are designed to have a region that is complementary to at least part of a microsatellite in wild-type nucleic acid and this region. These generic-design probes, targeting both the sense and the antisense DNA strands can be added to the reaction prior to denaturation and addition of the DSN enzyme (see e.g., <FIG>). Generic-design NaME probes are designed for both the sense and the anti-sense DNA strands, (e.g., for a homopolymer comprising adenosines and thymine, polyA probes plus polyT probes).

The following provides an example of the method. By using probes in high excess relative to the target nucleic acid (e.g., <NUM>-fold or <NUM>-fold or more molar excess to the poly-A sequences present in the sample), the deletion enrichment using this embodiment is anticipated to be more efficient than without using probes. for polyA15 microsatellites comprising <NUM> sequential adenines the perfect match is poly-15T; and so on. Following the denaturation step, the temperature is lowered to a temperature at or below the Tm of the probe added and DSN enzyme is added to the solution. If there is a deletion in a polyA15, the probe will not bind well, since the Tm of the target-probe duplex will be lower than the Tm of the full complement, 15A binding to 15T. Accordingly, the wild type poly-A15 microsatellites will form duplexes with poly-T15 and be preferentially digested by DSN relative to microsatellites having a deletion that duplex with the poly-T15 on partially. Following PCR amplification of the intact sequences the deletion-containing polyA will be preferentially enriched over the wild type alleles. <FIG> provides an example of an overall process from the starting genomic DNA to sequencing.

The nucleic acid sample is of genomic DNA. In some embodiments, genomic DNA is isolated from a biological sample (e.g., blood, plasma, serum, urine, CSF, buccal-swab, or solid tissue (e.g., a tumor tissue)). In some embodiments, genomic DNA is cell-free circulating DNA. In some embodiments, genomic DNA as provided in a sample of nucleic acid is fragmented. In some embodiments, DNA fragments are <NUM>-<NUM> bp long (e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> bp long).

In some embodiments, a sample of DNA is suspected of containing at least one microsatellite. In some embodiments, a sample of DNA is suspected of containing at least one microsatellite with one or more insertions or deletions. A microsatellite is a tract of repetitive DNA in which certain DNA motifs (ranging in length from one to six or more base pairs) are repeated, typically <NUM>-<NUM> times. Microsatellites occur at thousands of locations within an organism's genome. They have a higher mutation rate than other areas of DNA. Microsatellites are also called short tandem repeats (STRs) or simple sequence repeats (SSRs). Microsatellites as referred to as herein also include mini satellites that are of larger length (e.g., up to <NUM> bp). Microsatellites in a sample of nucleic acid can be of multiple types and of varying length. Non-limiting examples of microsatellites are mono-nucleotide repeats (e.g., AAAAAAAAA), di-nucleotide repeats (e.g., ACACACACACACACA (SEQ ID NO: <NUM>)), or tri-nucleotide repeats (e.g.,CAGCAGCAGCAGCAGCAG (SEQ ID NO: <NUM>)). In some embodiments, a microsatellite has a repeat of more than three nucleotides. A single sample of DNA can have mono-nucleotide repeats, di-nucleotide repeats, and/or trinucleotide repeats, each of varying length. For example, a sample of nucleic acid may comprise a poly-A repeat that is of <NUM> bp, 18bp, 25bp, and <NUM> bp. It may also comprise CAG repeats of multiple lengths.

In some embodiments, a microsatellite on wild-type nucleic acids is <NUM>-<NUM> (e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>,<NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM>) bp or nucleotides long. In some embodiments, a microsatellite is at least <NUM> (e.g., at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM>) bp or nucleotides long. In some embodiments, a microsatellite on wild-type nucleic acids is <NUM>-<NUM> (e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>,<NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM>) repeats long. For mono-nucleotide repeats, a repeat is one nucleotide long. For di-nucleotide repeats, a repeat is two nucleotides long. For tri-nucleotide repeats, a repeat is three nucleotides long. Therefore, a microsatellite with di-nucleotide repeats having the same number of repeats as a microsatellite with mono-nuclear repeats will be twice as long as the microsatellite with mono-nuclear repeats.

In some embodiments, a microsatellite is at least <NUM> (e.g., at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM>) repeats long.

A deletion in a microsatellite may be up to <NUM> bp long (e.g., up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, up to <NUM>, or up to <NUM> bp long). In some embodiments, a deletion is one or more than <NUM> bp (e.g., <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, or more than <NUM> bp).

Oligonucleotides probes as used in the method are also referred to as generic oligonucleotide probes. Generic oligonucleotide probes do not comprise any sequence that is complimentary to a sequence flanking the microsatellite. They are complementary to and bind all microsatellites that are as long as or longer than the probe. They are generic because they bind to multiple microsatellites, each at a different location in the genome. They are not specific to a single sequence in the genome. As described below, generic probes can include inosines flanking the portion complementary to the microsatellite. The inosine may then bind portions of the microsatellite not bound by the microsatellite specific sequences or bind to regions flanking the microsatellite but not part of the repeating sequence of the satellite. Oligonucleotide probes as used here are described in more detail below. In some embodiments, a pair of oligonucleotide probes comprise of a first probe and a second probe, wherein the first probe comprises a region that is complementary to a microsatellite sequence in a first wild-type nucleic acid top strand and the second probe comprises a region that is complementary to a first microsatellite sequence in the wild-type nucleic acid bottom strand. In some embodiments, the first and second probes are complementary to each other. In some embodiments, the length of the region that is complementary to the microsatellite sequences is at least n nucleotides, wherein n is <NUM>-<NUM> (e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>,<NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM>) nucleotides long. In some embodiments, oligonucleotides probes are added so that they are in molar excess compared to wild-type and target nucleic acids. In some embodiments, probes are in molar excess of <NUM>-fold to <NUM> billion-fold (e.g., <NUM>-fold, <NUM>-fold, <NUM>,<NUM>-fold, <NUM>,<NUM>-fold, <NUM>,<NUM>,<NUM>-fold, <NUM>,<NUM>,<NUM>-fold, <NUM>,<NUM>,<NUM>, or 1billion-fold) compared to wild-type and target nucleic acids.

The method of enriching deletion-containing microsatellite targets comprises adding to the nucleic acid sample a double strand specific nuclease (DSN), also referred to as duplex-specific nuclease. In some embodiments, a DSN is sourced from crabs. In some embodiments, a DSN is sourced from shrimp. DSNs are enzymes that preferentially digest or cleave nucleotides that are duplexed, e.g., dsDNA, or dsDNA-RNA hybrid duplexes. In some embodiments, DSNs are thermostable so that they do not get deactivated at higher temperatures (e.g., above <NUM> or <NUM>) and/or under certain conditions. Activity of DSN can be measured using a Kunitz assay (<NPL>). One unit of DSN activity can be defined as the amount of DSN added to <NUM>µg/ml calf thymus DNA that causes an increase of <NUM> absorbance units per minute; Activity assay was performed at <NUM>, in <NUM> Tris-HCl buffer, pH <NUM>, containing <NUM> MgCl<NUM>.

In some embodiments, the sample of nucleic acid is denatured to form ssDNA from dsDNA.

In some embodiments, the sample of nucleic acid is only destabilized. Destabilization of wild-type and target nucleic acids permits hybridization of the probes to their corresponding sequences on the wild type and mutant nucleic acids thereby forming complementary wild-type- probe duplexes on top and bottom strands, and partially complementary mutant-probe duplexes. By "destabilizing" it is meant that the double stranded wild type and target mutant nucleic acids denature to such an extent so as to allow the probes to hybridize to their corresponding sequences, but the wild type and target mutant nucleic acids do not denature completely. A condition that destabilizes is an increased temperature (e.g., <NUM> - <NUM> including <NUM>, <NUM>, <NUM>, <NUM>). This destabilizing temperature is typically about <NUM>-<NUM> below the melting temperature (Tm) of the nucleic acid sequence. At this temperature, the oligonucleotide probes invade and bind to their corresponding sequences on the wild type and mutant nucleic acids. The probes fully match the sequences on the wild type nucleic acid and can, thus, form complimentary wild type probe duplexes (i.e., with no mis-matches). Therefore, the first temperature is one that either destabilizes the wild-type and target nucleic acids, or denatures them. The second temperature is a temperature at which probes hybridize to wild-type and target nucleic acids to form complementary wild-type nucleic acid-probe duplexes or partially complementary target nucleic acid-probe duplexes. In some embodiments, the second temperature is the melting temperature or slightly below (up to <NUM>) the melting temperature of wild-type nucleic acid-probe duplexes. In some embodiments, the second temperature is above the melting temperature or slightly above (up to <NUM>) the melting temperature of target nucleic acid-probe duplexes.

The third temperature is a temperature at which DSN is active and digests duplexed sequence.

The solvent lowers the Tm of the nucleic acids, without inhibiting or diminishing the activity of DSN. In some embodiments, addition of solvent lowers the Tm of the nucleic acids allowing the first temperature for denaturation/destabilization of the nucleic acids to be lower, such that there is lower degree of deactivation of the DSN. Examples of such solvents include, but are not limited to DMSO, betaine or formamide. In some embodiments, <NUM>-<NUM>% of DMSO is included in the reaction mixture or nucleic acid sample. In some embodiments, DSN is added after subjecting the nucleic acid sample to the second temperature. In some embodiments, a thermostable DSN is used that can withstand a higher temperature without its activity being diminished either partially or completely.

In some embodiments, once a nucleic acid sample is subjected to a third temperature to allow the DSN to digest dsDNA, the DSN is then inactivated either partially or completely, for example, by heating the sample to <NUM> for <NUM>-<NUM>.

In some embodiments, DSN is added so that its concentration in the reaction is <NUM>-<NUM> units (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> units).

In some embodiments, the Mg<NUM>+ concentration in the reaction mixture is adjusted so that it is between <NUM>-<NUM> (e.g., <NUM>-<NUM>). In some embodiments, Mg<NUM>+ concentration is adjusted before subjecting the sample to the first temperature. In some embodiments, Mg<NUM>+ concentration is adjusted before subjecting the sample to the second temperature.

In some embodiments, a single reaction is performed using more than one pair of probes (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM> or more pairs of probes), wherein each pair of probe has a sequence that is different from that of the other pairs of probes. For example, a first pair of probes may have a region that is complementary to a poly-A repeat, while a second pair of probes may have a region that is complementary to a poly-G repeat, while a third pair of probes may have a region that is complementary to a poly-AT repeat.

In some embodiments, a sample if nucleic acid suspected of comprising a target nucleic acid is subjected to multiple NaME reactions (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more than <NUM> reactions), each reaction conducted with a different pair of probes. In some embodiments, the pairs of probes in a first reaction differ from a pair of probes in a second reaction only by the length of the region that is complementary to wild-type nucleic acids. For example, a pair of probes in a first reaction and the second reaction may comprise first and second probes of poly-A and poly-T sequence, example that the probes in the first reaction have a region that is complementary to the wild-type strands that is <NUM> nucleotides long, whereas the probes in the second reaction have a region that is complementary to the wild-type strands that is <NUM> nucleotides long. In some embodiments, the sequence of probes in a first and second reaction are different. For example, the probes of a first reaction may comprise a region that is complementary to a mono-nucleotide repeat and the probes of a second reaction may comprise a region that is complementary to a di-nucleotide repeat or a mono-nucleotide repeat that is different from the mono-nucleotide repeat of the probes of the first reaction.

Provided herein are generic oligonucleotide probes. In some embodiments, generic probes are provided in a pair comprising a first and second probe, wherein the first probe and second probe each have a region that is complementary to the top and bottom strands of wild-type microsatellite sequences. The regions of the first and second probes that are complementary to microsatellite sequences may be flanked by nucleotides, however these nucleotides do not form a sequence that is specifically complementary to sequences that flank microsatellite sequences on wild-type nucleic acids. Herein, "flanking" is used to mean that there is no nucleotide between the sequence being flanking and the sequence/nucleotides that is flanking.

As describes above, generic oligonucleotide probes do not comprise any sequence that is complimentary to a sequence flanking the microsatellite. They are complementary to and bind all microsatellites that are as long as or longer than the probe. They are generic because they bind to multiple microsatellites, each at a different locations in the genome. They are not specific to a single sequence in the genome. As described below, generic probes can include inosines flanking the portion complementary to the microsatellite. The inosine may then bind portions of the microsatellite not bound by the microsatellite specific sequences or bind to regions flanking the microsatellite but not part of the repeating sequence of the satellite.

In some embodiments, generic probes, as described herein, comprise of a region that is complementary to a microsatellite sequence and which may be flanked by nucleotides that base-pair without specificity (e.g., inosines). The nucleotides of the probes that flank the region that is complementary to a microsatellite sequence do not form a sequence that is specifically complementary to sequences flanking microsatellites in genomic DNA. Herein, "flanking" is used to mean that there is no nucleotide between the sequence being flanking and the sequence/nucleotides that is flanking.

In some embodiments, generic probes, as described herein, consist of a region that is complementary to a microsatellite sequence and which may be flanked by nucleotides that base-pair without specificity (e.g., inosines). The nucleotides of the probes that flank the region that is complementary to a microsatellite sequence do not form a sequence that is specifically complementary to sequences flanking microsatellites in genomic DNA. Herein, "flanking" is used to mean that there is no nucleotide between the sequence being flanking and the sequence/nucleotides that is flanking.

Probes contain a region (S) that is complementary to the satellite, optionally flanked by inosines (I)<NUM>-<NUM> and/or an adaptor (A). The probe is of the formula I*S*I, A*I*S*I*A, or A*S*A, where I is <NUM>-<NUM> inosines and * is a bond.

In some embodiments, a probe consists of a mono-nucleotide repeat, di-nucleotide repeat, or tri-nucleotide repeat, in each case optionally flanked at each end by <NUM>-<NUM> inosines.

In some embodiments, the region that is complementary to the top strand of a first probe is complementary to the region that is complementary to the bottom strand of a second probe. <FIG> provides an example of a first probe that has a region that is complementary to the microsatellite sequence is the top strand of a wild-type nucleic acid (as shown in <FIG>).

In some embodiments, the region of a probe that is complementary to wild-type sequences is <NUM>-<NUM> (e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>,<NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM>) nucleotides long. In some embodiments, the region of a probe that is complementary to wild-type sequences is <NUM>, <NUM>, <NUM>,<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> nucleotides long.

Oligonucleotide probes for use in any one of the methods disclosed herein may comprise natural deoxynucleotides (DNA), or natural nucleotides (RNA), or modified deoxynucleotides or nucleotides. The modification may comprise one or more artificial nucleotides such as: locked nucleic acid (LNA), peptide nucleic acid (PNA), and xeno nucleic acid (XNA); or any other modified nucleotide that increases the difference in melting temperatures of the fully-matched wild-type nucleic acid-probe sequence and target nucleic acid-probe mismatched sequences. In a preferred embodiment, the modified nucleotides are placed at the two ends of the probe (see e.g., <FIG>). As an example, in the same setting where poly-T probes are directed against poly-A microsatellites, if the LNA-containing poly-T15 binds to the corresponding wild type poly-A15 microsatellite the binding will be stronger than the equivalent natural nucleotide bonding and will have a higher Tm in view of the LNA-modified ends. In some embodiments, an LNA-containing T is placed one nucleotide from each end of the probe (or, in some embodiments, each end of the probe that is complementary to a microsatellite). In contrast, if there is a small deletion in a microsatellite, then the probes will not bind well, since only one of the two LNA-containing nucleotides will be binding, but not both. Hence the Tm of the hybrid with a deletion-containing microsatellite will be much lower and the duplex will not form. Accordingly, addition of the DSN enzyme will not digest such deletion containing sequences, which will then be enriched following PCR.

In some embodiments, each of the first and second probes of a pair of probes comprises at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, or at least <NUM>) modified nucleotide (e.g., LNA, PNA, or XNA) that increases the difference in melting temperatures of the fully-matched wild-type nucleic acid-probe sequence and target nucleic acid-probe mismatched sequences. In some embodiments, there is at least one (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) modified nucleotide on each of the <NUM>' and <NUM>' ends of the regions that are complementary to wild-type strand. For example, there may be <NUM> modified nucleotide on the <NUM>' end of the region that is complementary to a wild-type strand and two modified nucleotides in the <NUM>' end of the region that is complementary to a wild-type strand. In some embodiments, a modified nucleotides (e.g., LNA, PNA, or XNA) is located on the first nucleotide of the <NUM>'-end portion of the probe that is complementary to the microsatellite, and/or on the first nucleotide of the <NUM>'-end portion of the probe that is complementary to the microsatellite. In some embodiments, a modified nucleotides (e.g., LNA, PNA, or XNA) is located on the second nucleotide of the <NUM>'-end portion of the probe that is complementary to the microsatellite, and/or on the first and or second nucleotide of the <NUM>'-end portion of the probe that is complementary to the microsatellite.

In some embodiments, a probe comprises only LNAs as modified nucleotides. In some embodiments, a probe may have more than one type of modified nucleotide, e.g., one LNA and two XNAs.

In some embodiments, the modified nucleotides are placed at the two ends of the probe so that both bind to wild-type sequence but only one or none modified nucleotides bind to target sequence. In some embodiments, the inner-most modified nucleotides on a probe is at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> nucleotides apart.

In some embodiments, inosines are added on either side of the oligonucleotide probe (see e.g., <FIG>). These inosines can base-pair with any DNA base, thereby enabling stronger binding to all microsatellites and their flanking sequences. This is advantageous since the optimal digestion by DSN is about <NUM>-<NUM>, thereby requiring binding of the probe to flanking sequences as well to achieve optimal performance of the present protocol.

In some embodiments, probes targeting microsatellites on both the sense and antisense DNA strands are always added. To avoid probes for the sense strand binding to probes for the antisense strand, a different number of inosines are designed into the probe that is complementary to the top strand and the probe that is complementary to the bottom strand. Thus, the probes for the top and bottom strand will be non-overlapping. At the temperature close to the probe Tm, the sense and antisense strand probes would not be expected to bind substantially to each other using this design. In some embodiments, each of a first and second probe comprises <NUM>-<NUM> (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) inosines that flank either or both sides of the region that is complementary to the microsatellite sequence in a wild-type nucleic acid. In some embodiments, the number of inosines in the <NUM>' end of the first probe is not equal to the number of inosines in the <NUM>' end of the second probe, and the number of inosines in the <NUM>' end of the first probe is not equal to the number of inosines in the <NUM>' end of the second probe. For example, if the <NUM>' end of a first probe has <NUM> inosines, then the <NUM>' end of the second probe should not have <NUM> inosines; it may have <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> inosines. This unequal pairing of number of inosines in the <NUM>' and <NUM>' ends of the first and second probes, or <NUM>' and <NUM>' ends of the first and second probes, respectively, ensures that the probes do not hybridize to each other.

In some embodiments, a probe is <NUM>-<NUM> (e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM>) nucleotides long with a region that is complementary to a wild-type microsatellite that is up to <NUM> nucleotides long and having up to <NUM> inosines on either or both ends flanking the region that is complementary to a wild-type microsatellite. A microsatellite in a wild-type sequence may be a mono-nucleotide repeat, dinucleotide repeat, or tri-nucleotide repeat. In some embodiments, a microsatellite has a repeat of more than three nucleotides.

The following sections entitled "Self-g NaME", "Non-enzymatic elimination of wild-type microsatellites at multiple genomic targets using blocking probes", "Non-enzymatic enrichment of A:T microsatellites at multiple genomic targets using COLD-PCR", and "Universal microsatellite target-enrichment using restriction endonucleases" disclose further methods for enriching deletion-containing microsatellite targets in a sample of genomic DNA. These methods are not encompassed by the invention as set out in the appended claims.

Self- NaME method for enriching and detecting microsatellite deletions takes advantage of DSN properties to preferentially degrade larger microsatellites and preserve short microsatellites such as those resulting from deletions occurring on wild-type (WT) microsatellites. It is similar to the method described herein which uses generic probes, however, does not use generic probes, and instead relies on hybridization of wild-type and target duplexes. See for example <FIG>. Genomic DNA is first denatured (e.g., using a temperature of <NUM> or higher), after which the temperature is reduced to an appropriate temperature just below the melting temperature (Tm) of the microsatellites being interrogated. For example, for 15A homopolymers, a hybridization temperature of <NUM> is used. At this temperature the wild-type 15A homopolymers hybridize with other poly-adenine homopolymers in the genome and are digested by DSN. On the other hand, homopolymers containing deletions (e.g., a 10A homopolymer resulting from a 5A deletion on a wild-type 15A homopolymer) do not hybridize substantially at this temperature; being single-stranded, they are not digested by the DSN. Thereby, deletion-containing DNA is preferentially left un-degraded relative to corresponding wildtype microsatellites having no deletions. Hence a subsequent amplification (e.g., by PCR reaction) after DSN digestion is expected to amplify preferentially the deletion-containing alleles that remain substantially single stranded and unaffected by DSN. Because the self-hybridization takes place on all genomic homopolymer regions at once, thousands of deletions on homopolymers over the genome can be enriched at the same time, thus providing a highly multiplexed approach. Following enrichment, the deletion homopolymers can be detected via genome-wide sequencing.

Accordingly, also contemplated herein is a method for enriching deletion-containing microsatellite targets in a sample of genomic DNA, the method comprises:.

When NaME is performed on samples having or suspected of having microsatellite targets without the use of generic probes, then the nucleic acid has to be denatured; destabilization will not work, because unless the nucleic acids are first denatured before allowed them to hybridize again, then the DSN will digest most of the nucleic acids.

Also contemplated herein is a multi-site enrichment approach that does not require action of an DSN enzyme to suppress the wild-type microsatellites. <FIG> provides an illustration of this approach, in which oligonucleotide probes as described herein are used during amplification of nucleic acid strands using ligated adaptors, wherein the probes act as 'polymerase blockers' that preferentially bind to wild-type microsatellites and inhibit amplification. In contrast, when a microsatellite contains a deletion, the binding of the blocker probes to this microsatellite has a lower melting temperature (Tm), hence it does not bind well and allows polymerase extension and amplification. The amplification can be done using either standard PCR conditions, or COLD-PCR conditions. Any variation of COLD-PCR (e.g., temperature independent/tolerant COLD-PCR) can also be performed using the oligonucleotide probes disclosed herein. COLD-PCR and its derivatives, e.g., Temperature-Tolerant COLD-PCR are disclosed in the following patent application: <CIT>, <CIT>, <CIT>, <CIT>, <CIT>.

Enrichment of microsatellite indels via COLD-PCR was described previously by <NPL>). However, in How-Kit et al. , the oligonucleotide blocker probes were designed to match a specific, unique sequence. In contrast the blocker probes employed in methods disclosed herein have a generic design that bind to multiple genomic sites ('genome-wide') as opposed to binding to a single unique sequence, thereby achieving genome-wide enrichment of indel-containing microsatellites.

Accordingly, further contemplated herein is a method for enriching deletion-containing microsatellite targets in a sample of genomic DNA, the method comprising:.

Oligonucleotide probes can be any pair of generic oligonucleotides probes as disclosed herein. Adaptors are single-stranded or double-stranded and are at least <NUM> bp long (e.g., at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> bp long). In some embodiments, adaptors are ligated to one or more tag via which the adaptor can be immobilized. In some embodiments, a tag is biotin. In some embodiments, biotinylated adaptors, which are ligated to nucleic acid fragments are bound to streptavidin (e.g., on a bead).

The oligonucleotide probes as disclosed herein may also be used in temperature-independent COLD-PCR. Accordingly, a method of COLD-PCR using a first critical denaturation temperature is repeated using a second critical denaturation temperature that is higher than the first critical denaturation temperature. The method may further comprise repeating steps (e) to (g) at least once, wherein step (e) is repeated at a second Tc of a second double-stranded wild-type nucleic acid containing a microsatellite and a second double-stranded target nucleic acid suspected of containing a microsatellite corresponding to the wild-type microsatellite with at least one deletion target, the second Tc being above the first Tc and below the melting temperature of second wild-type nucleic acid-probe duplex to permit preferential denaturation of second target nucleic acid-probe duplexes relative to second wild-type nucleic acid-probe duplexes.

Instead of relying on critical denaturation temperature at which there is preferential denaturation of first target nucleic acid-probe duplexes relative to first wild-type nucleic acid-probe duplexes, a critical hybridization temperature may be relied upon to preferentially permit preferential hybridization of first wild-type nucleic acid-probe duplexes relative to first target nucleic acid-probe duplexes. Accordingly, also contemplated is a method comprising: (a) providing a nucleic acid sample comprising at least a first double-stranded wild-type nucleic acid containing a microsatellite and at least a first double-stranded target nucleic acid suspected of containing a microsatellite corresponding to the wild-type microsatellite with at least one deletion, and wherein each end of each strand of the double-stranded nucleic acids are ligated to an adaptor;.

A method relying on a first critical hybridization temperature may further comprise repeating steps (d) to (f) at least once, wherein step (d) is repeated at a second Th of a second double-stranded wild-type nucleic acid containing a microsatellite and a second double-stranded target nucleic acid suspected of containing a microsatellite corresponding to the wild-type microsatellite with at least one deletion target, the second Th being below the first Th and above the melting temperature of second target nucleic acid-probe duplex to permit preferential hybridization of second wild-type nucleic acid-probe duplexes relative to second target nucleic acid-probe duplexes.

A method for enriching deletion-containing microsatellite targets in a sample of genomic DNA may involve simple PCR. Accordingly also contemplated here is a method comprising (a)providing a nucleic acid sample comprising at least a first double-stranded wild-type nucleic acid containing a microsatellite and at least a first double-stranded target nucleic acid suspected of containing a microsatellite corresponding to the wild-type microsatellite with at least one deletion relative to the wild-type microsatellite, and wherein each end of each strand of the double-stranded nucleic acids are ligated to an adaptor;.

In some embodiments, a method using simple PCR further comprises repeating at least once steps (c)-(e).

Any of the PCR or COLD-PCR techniques that utilize the generic probes as disclosed herein to enrich target nucleic acid comprising a microsatellite having a deletion relative to wild-type nucleic acid comprising a corresponding microsatellite with no deletion can be preceded by any of the NaME methods disclosed herein. When any of the NaME methods disclosed herein precedes any of the PCR methods disclosed herein, the DSN is inactivated prior to the PCR method.

Any of the PCR or COLD-PCR techniques that utilize the generic probes as disclosed herein to enrich target nucleic acid comprising a microsatellite having a deletion relative to wild-type nucleic acid comprising a corresponding microsatellite with no deletion can be follow by any of the NaME methods disclosed herein.

Genomic DNA may be fragmented before ligating to adaptors.

Also contemplated herein is a simple approach to enrich the A:T-rich genomic fragments or sequences over G:C rich fragments or sequences (including polyA/T with deletions and WT microsatellites without deletions), so that during sequencing, most of the sequencing reads concentrate or focus on the targets that contain the clinically useful information, i.e. the AT-rich sequences. In some embodiments, this approach is called 'target enrichment', as opposed to `deletion enrichment' which refers to enrichment of altered microsatellites relative to their WT alleles.

<FIG> demonstrates and example of target enrichment of the A:T-rich portion of the genome, which involves PCR amplification at lower denaturation temperature, COLD-PCR. COLD-PCR and its derivatives, e.g., Temperature-Tolerant COLD-PCR are disclosed in the following patent application: <CIT>, <CIT>, <CIT>, <CIT>, <CIT>.

Following ligation of adaptors to the genomic fragments to be interrogated (e.g., circulating DNA from a blood sample; or genomic DNA from a tumor biopsy), a series of COLD-PCR cycles may be applied, where the denaturation temperature at each cycle is not high enough to denature fragments of high melting temperature Tm (e.g., high GC fragments), but it is adequate for denaturation of low Tm fragments like polynucleotide repeats that include polyA and polyT. For example, the denaturation temperature can be set to <NUM>, or <NUM> instead of say <NUM> for denaturation during PCR.

Accordingly, also contemplated herein is a method of enriching A:T containing microsatellites in a sample of genomic DNA, the method comprising:.

Any of the PCR or COLD-PCR techniques that utilize the generic probes as disclosed herein to enrich A:T-rich sequencing containing target nucleic acid can be preceded by any of the NaME methods disclosed herein. When any of the NaME methods disclosed herein precedes any of the PCR methods disclosed herein, the DSN is inactivated prior to the PCR method.

Any of the PCR or COLD-PCR techniques that utilize the generic probes as disclosed herein to enrich A:T-rich sequencing containing target nucleic acid can be follow by any of the NaME methods disclosed herein.

It is further contemplated that microsatellite target enrichment may be accomplished by using one or more restriction endonucleases that are specific for GC-rich sequences. These would be anticipated to leave A:T-rich regions intact, such as polyA/T repeat mononucleotides, or AT dinucleotide repeats. Additionally, AC, AG dinucleotide repeats, or CAG trinucleotide repeats would also remain intact. On the other hand, other sequences as well as most of the flanking sequences around microsatellites would be digested heavily.

For example, by using CviPII enzyme (<NPL>) which cuts at CCA and CCG (and to a lesser extent at CCT) the microsatellite BAT <NUM> shown on Figure 12A remains intact. In contrast, the remaining flanking sequences as well as all GC-rich part of the genome are digested in small fragments and can be removed by size-selection filtration, or by additional digestion with another -CG-targeting enzyme.

Next, by ligating adaptors to the undigested microsatellite sequences, a ligation-mediated PCR using standard PCR conditions (or COLD-PCR with reduced denaturation temp) can form a DNA library that can be sequenced. Accordingly, sequencing can be focused almost exclusively at microsatellites throughout the genome, thus saving cost, time and effort.

CviPII can be made to cut only at CCA sequences, and not at CCG. Because the enzyme does not cut CG-methylated DNA (CpG sites), the DNA that is to be screened can first be fully methylated by treatment with DNA methyltransferases or other enzymes that methylate the G within CpG sequences. Then subsequent treatment with CviPII can be regulated to cut only CCA. In this way the generation of fragments from enzymatic treatment can be adapted as needed, to perform either very frequent cutting or modestly frequent cutting.

While both approaches to enrich A:T-rich regions as disclosed herein (i.e., using COLD-PCR and using GC-enzyme cutter) are able to generate target enrichment at polyA or polyT mononucleotide repeats, the COLD-PCR methods have the advantage of simplicity (no enzymatic cutting), while enzymatic cutting methods have the advantage that they can enrich more complex microsatellites, like dinucleotides (CA)n or trinucleotides (GAC)n more easily, or also higher order microsatellites, since the approach does not depend on the Tm of the microsatellite. Thus even a high Tm microsatellite like (GAC)n will also be preferentially enriched easily.

The method disclosed herein can be used to enhance identification of microsatellite deletions in tumors, circulating DNA or in other liquid biopsies obtained from a subject (e.g., a human subject suffering from or is suspected to suffer from cancer), for detection of minimal residual disease, tumor mutational burden, or to monitor tumor dynamics. Alternatively, the method disclosed herein can be used to screen apparently healthy individuals for signs of early disease (e.g., cancer) by periodic testing of biological samples (e.g., blood, urine, buccal swap, or tissue biopsy).

In some embodiments, genomic DNA is cell-free circulating DNA. In some embodiments, genomic DNA is obtained from a biological sample. In some embodiments, a biological sample is serum, plasma, blood, urine, solid tissue (e.g., a tumor), feces, skin, hair, a buccal swab, or a pulmonary brushing.

Small deletions or insertions (commonly <NUM>-30bp in size) at microsatellites take place even in the absence of mismatch repair deficiency and microsatellite instability, but at a lower level than in repair-deficient cells. Such indels are clonally expanded in tumors due to tumor cell proliferation. The specific indel distribution in a tumor genome can serve as a signature for that tumor. The signature can then be traced in the blood as a tumor biomarker, thereby serving to identify minimal residual disease during or after cancer therapy.

<FIG> and <FIG> demonstrate experimental validation for the self- NaME embodiment as shown in <FIG>. Two randomly selected poly-A15 microsatellites (#<NUM> and #<NUM>) were tested using mixtures of DNA from deletion-containing cell lines and wild type DNA, and then tested following application of the no-probe self- NaME protocol. PCR was then applied and the presence of deletion was tested via high resolution melting (HRM). The deletion can be detected after application of self- NaME, while when NO- NaME was run in parallel as a control the deletion cannot be detected. <FIG> demonstrates similar results by using capillary electrophoresis as the endpoint detection. Since the microsatellites tested were randomly chosen, it is likely that the enrichment of deletions is taking place over the entire population of poly-A15 microsatellites in the genome.

<FIG> demonstrates similar conclusions as <FIG>, but by using generic probes with inosines at the two ends, directed against both the sense and the antisense strands containing the randomly-chosen microsatellite #<NUM>.

<FIG> demonstrates that the compositions and materials shown in <FIG> show enhanced enrichment of the same randomly-chosen microsatellite #<NUM> when the Mg++ concentration during NaME-PrO using probes as shown in <FIG> is increased to <NUM> or <NUM> instead of the standard <NUM>.

<FIG> shows a method of using the frequent cutting enzyme CviPII enzyme which cuts at CCA and CCG (and to a lesser extent at CCT) the microsatellite BAT <NUM>. In contrast, the remaining flanking sequences as well as all GC-rich part of the genome are digested in small fragments and can be removed by size-selection filtration, or by additional digestion with another -CG-targeting enzyme.

Next, by ligating adaptors to the undigested microsatellite sequences, a ligation-mediated PCR using standard PCR conditions (not necessarily COLD-PCR with reduced denaturation temp) can form a DNA library that can be sequenced. Accordingly, sequencing can be focused almost exclusively at microsatellites throughout the genome, thus saving cost, time and effort.

<FIG> shows use of the CviPII to cut only at CCA sequences, and not at CCG. Because the enzyme does not cut CG-methylated DNA (CpG sites), the DNA that is to be screened can first be fully methylated by treatment with DNA methyltransferases or other enzymes that methylate the G within CpG sequences. Then the subsequent treatment with CviPII can be regulated to cut only CCA. In this way the generation of fragments from enzymatic treatment can be adapted as needed, to perform either very frequent cutting or modestly frequent cutting.

It is to be understood that the foregoing embodiments are presented by way of example only.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents referenced herein, and/or ordinary meanings of the defined terms.

The phrase "and/or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases.

The phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.

Claim 1:
A method for enriching deletion-containing microsatellite targets in a sample of genomic DNA, the method comprising:
providing a nucleic acid sample comprising at least a first double-stranded wild-type nucleic acid containing a microsatellite and at least a first double-stranded target nucleic acid suspected of containing a microsatellite corresponding to the wild-type microsatellite with at least one deletion relative to the wild-type microsatellite;
adding to the nucleic acid sample a pair of oligonucleotide probes comprising a first probe and a second probe, wherein the first probe comprises a region that is complementary to the microsatellite sequence in the wild-type nucleic acid top strand and the second probe comprises a region that is complementary to the microsatellite sequence in the wild-type nucleic acid bottom strand; wherein the length of the region that is complementary to the microsatellite sequences is at least <NUM> nucleotides;
adding to the nucleic acid sample a double strand specific nuclease (DSN);
subjecting the nucleic acid sample to a first temperature that destabilizes or denatures the at least first double-stranded wildtype and the at least first target mutant nucleic acids;
subjecting the nucleic acid sample to a second temperature that allows preferential formation of complementary wild-type nucleic acid-probe duplexes relative to partially complementary target nucleic acid-probe duplexes; and
subjecting the nucleic acid sample to a third temperature that allows the DSN to preferentially cleave the complementary wild-type nucleic acid-probe duplexes relative to partially complementary target nucleic acid-probe duplexes;
wherein the DSN is added:
(i) after subjecting the nucleic acid sample to the second temperature, or
(ii) before subjecting the nucleic acid sample to the first temperature, and wherein the method further comprises adding to the nucleic acid sample an organic solvent that lowers the melting temperature of the wild-type and target nucleic acids to inhibit inactivation of the DSN when the nucleic acid sample is subjected to the first temperature.