Selective degradation of wild-type dna and enrichment of mutant alleles using nuclease

Abstract

The present disclosure provides methods for preparing a target mutant nucleic acid for subsequent enrichment relative to a wild-type nucleic acid using nucleases that have a substantially higher activity on double stranded DNA versus single stranded DNA or RNA. The present disclosure also includes methods for enriching a target mutant nucleic acid and for preparing unmethylated/methylated nucleic acids of interest for subsequent enrichment.

What is claimed is: 
     
         1 . A method for enriching a target mutant nucleic acid, comprising:
 (a) preparing an amplification reaction mixture comprising:   a double-stranded wild-type nucleic acid, a double-stranded target nucleic acid suspected of containing a mutation, a thermostable double strand-specific nuclease (DSN), a pair of oligonucleotide probes, one of which is complementary to the wild-type nucleic acid top strand and the other is complementary to the wild-type nucleic acid bottom strand, wherein at least one of the probes overlaps a sequence on the target nucleic acid containing the suspected mutation and PCR amplification components and the two probes only partially overlap each other;   (b) subjecting the reaction mixture to a denaturing temperature to permit denaturation of the wild-type nucleic acid and the target mutant nucleic acid;   (c) reducing the temperature to permit hybridization of the probes to their corresponding sequences on the wild-type and target mutant nucleic acids thereby forming complementary wild-type-probe duplexes on top and bottom strands, and partially complementary target mutant-probe duplexes, wherein the DSN cleaves the complementary wild-type-probe duplexes but not the partially complementary target mutant-probe duplexes; and   (d) subjecting the reaction mixture to an amplification condition thereby enriching the uncleaved target mutant nucleic acid relative to the cleaved wild-type nucleic acid.   
     
     
         2 . The method of  claim 1 , further comprising repeating steps (b) and (c) for two or more cycles before executing step (d). 
     
     
         3 . The method of  claim 2 , further comprising repeating steps (b), (c) and (d) for two or more cycles. 
     
     
         4 . The method of  claim 1 , wherein the reaction mixture further comprises an organic solvent. 
     
     
         5 . The method of  claim 1 , wherein the denaturing temperature is between 65-85° C. 
     
     
         6 . The method of  claim 1 , wherein both probes overlap the suspected mutation. 
     
     
         7 . The method of  claim 1 , wherein each probe is modified at the 3′ end to prevent polymerase extension. 
     
     
         8 . The method of  claim 1 , wherein primers used for PCR amplification have a melting temperature that is below the temperature applied in step (c). 
     
     
         9 . The method of  claim 1 , wherein the method is used to enrich two or more different target mutant nucleic acids relative to wild-type nucleic acids and the method further comprises one or more additional pairs of probes directed to the different wild-type nucleic acids, wherein for each pair of probes, one of the probes is complementary to the wild-type nucleic acid top strand and the other is complementary to the wild-type nucleic acid bottom strand. 
     
     
         10 . The method of  claim 1 , wherein the amplification condition is PCR; full COLD-PCR, fast COLD-PCR; ice-COLD-PCR, temperature-tolerant COLD-PCR and limited denaturation time COLD-PCR. 
     
     
         11 . A method for preparing unmethylated nucleic acids of interest for subsequent enrichment relative to corresponding methylated nucleic acids comprising:
 (a) ligating bisulfite-resistant adaptors to double stranded nucleic acids of interest;   (b) subjecting the adaptor-linked nucleic acids to sodium bisulfite treatment and a nucleic acid amplification reaction to form double-stranded bisulfite-treated nucleic acids;   (c) subjecting the bisulfite-treated nucleic acids to a temperature that permits preferential denaturation of unmethylated nucleic acids while methylated nucleic acids remain double-stranded or denaturation of both unmethylated and methylated nucleic acids to form unmethylated and methylated single stranded nucleic acids;   (d) exposing the unmethylated and methylated nucleic acids to double strand-specific nuclease (DSN) or an exonuclease.   
     
     
         12 . The method of  claim 11 , wherein the temperature permits preferential denaturation of unmethylated nucleic acids while methylated nucleic acids remain double-stranded. 
     
     
         13 . The method of  claim 12 , wherein the unmethylated and methylated nucleic acids are exposed to double strand-specific nuclease (DSN) and conditions for optimal DSN activity, wherein the DSN cleaves the methylated double-stranded nucleic acids but not the unmethylated single-stranded nucleic acids. 
     
     
         14 . The method of  claim 12 , wherein the unmethylated and methylated nucleic acids are exposed to an exonuclease and conditions for optimal exonuclease activity, wherein the exonuclease cleaves the unmethylated single-stranded nucleic acids but not the methylated double-stranded nucleic acids. 
     
     
         15 . The method of  claim 11 , wherein the temperature permits denaturation of both unmethylated and methylated nucleic acids to form unmethylated and methylated single stranded nucleic acids. 
     
     
         16 . The method of  claim 15 , further comprising reducing the temperature to permit preferential formation of methylated duplexes, but not unmethylated duplexes. 
     
     
         17 . The method of  claim 16 , wherein the unmethylated and methylated nucleic acids are exposed to double strand-specific nuclease (DSN) and conditions for optimal DSN activity, wherein the DSN preferentially cleaves the methylated duplexes but not the unmethylated single-stranded nucleic acids. 
     
     
         18 . The method of  claim 16 , wherein the unmethylated and methylated nucleic acids are exposed to an exonuclease and conditions for optimal exonuclease activity, wherein the exonuclease cleaves the unmethylated single-stranded nucleic acids but not the methylated double-stranded nucleic acids. 
     
     
         19 . The method of  claim 11 , wherein the nucleic acid amplification reaction of step (b) is selected from the group consisting of: PCR; full COLD-PCR, fast COLD-PCR; ice-COLD-PCR, temperature-tolerant COLD-PCR and limited denaturation time COLD-PCR. 
     
     
         20 . The method of  claim 14 , wherein the cleaved unmethylated single stranded nucleic acids and the uncleaved methylated duplexes are subjected to an amplification condition using the bisulfite resistant adaptors ligated in step (a). 
     
     
         21 . The method of  claim 18 , wherein the cleaved unmethylated single stranded nucleic acids and the uncleaved methylated duplexes are subjected to an amplification condition using the bisulfite resistant adaptors ligated in step (a). 
     
     
         22 . The method of  claim 11 , wherein naturally AT-rich sequences are removed prior to the sodium bisulfite treatment.