Methods for variant detection

Abstract

The invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as SNPs and indels. The invention also provides a method for performing inexpensive multiplex assays. The invention also provides methods for detection of DNA sequences altered after cleavage by a targetable endonuclease, such as the CRISPR Cas9 protein from the bacterium Streptococcus pyogenes.

What is claimed is: 
     
         1 . A blocked-cleavable primer for rhPCR, the primer comprising:
   5′-A-B-Z-E-3′
   
       wherein
 A is optional and is a tail extension that is not complementary to a target; 
 B is a sequence domain that is complementary to a target; 
 Z comprises:
 C, a discrimination domain, and 
 D, a cleavage domain that, when hybridized to the target, is cleavable by RNase H2; and 
 
 E is a blocking domain that prevents extension of the primer. 
 
     
     
         2 . The primer of  claim 1 , wherein the discrimination domain C is located 5′ of the cleavage domain D. 
     
     
         3 . The primer of  claim 1 , wherein the discrimination domain C is located 3′ of the cleavage domain D. 
     
     
         4 . The primer of  claim 1 , wherein the discrimination domain C overlaps with the cleavage domain D. 
     
     
         5 . The primer of  claim 1 , wherein C comprises 1-3 RNA bases. 
     
     
         6 . The primer of  claim 1 , wherein C comprises 1 RNA base. 
     
     
         7 . The primer of  claim 6 , wherein the discrimination domain C consists of the 1 RNA base. 
     
     
         8 . The primer of  claim 1 , wherein the cleavage domain comprises one or more of the following moieties: a DNA residue, an abasic residue, a modified nucleoside, or a modified phosphate internucleotide linkage. 
     
     
         9 . The primer of  claim 8 , wherein a sequence flanking the cleavage site contains one or more internucleoside linkages resistant to nuclease cleavage. 
     
     
         10 . The primer of  claim 9 , wherein the nuclease resistant linkage is a phosphorothioate. 
     
     
         11 . The primer of  claim 5 , wherein the 3′ oxygen atom of at least one of the RNA residues is substituted with an amino group, thiol group, or a methylene group. 
     
     
         12 . The primer of  claim 1 , wherein the blocking group is attached to the 3′-terminal nucleotide of the primer. 
     
     
         13 . The primer of  claim 1 , wherein A comprises a region that is identical to a universal forward primer and optionally a probe binding domain. 
     
     
         14 . A method of detecting a variation in a target DNA sequence, the method comprising:
 (a) providing a reaction mixture comprising
 (i) an oligonucleotide primer having a cleavage domain positioned 5′ of a blocking group, the blocking group linked at or near the end of the 3′-end of the oligonucleotide primer wherein the blocking group prevents primer extension and/or inhibits the primer from serving as a template for DNA synthesis, 
 (ii) a sample nucleic acid that may or may not have the target sequence, and where the target sequence may or may not have the variation 
 (iii) a cleaving enzyme and 
 (iv) a polymerase; 
   (b) hybridizing the primer to the target DNA sequence to form a double-stranded substrate;   (c) cleaving the hybridized primer, if the primer is complementary at the variation, with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the primer; and   (d) extending the primer with the polymerase.   
     
     
         15 . The method of  claim 14 , wherein the cleaving enzyme is a hot start cleaving enzyme comprising at least one amino acid substitution as compared to its wild type, which is thermostable and has reduced activity at lower temperatures, and which exhibits enhanced mismatch discrimination during rhPCR as compared to its wild type. 
     
     
         16 . The method of  claim 15 , wherein the hot start cleaving enzyme is  Pyrococcus abyssi  RNase H2 comprising (a) a G12A amino acid substitution; (b) a P13T amino acid substitution; (c) a G169A amino acid substitution; or (d) a combination thereof. 
     
     
         17 . The method of  claim 15 , wherein the cleaving enzyme is chemically modified. 
     
     
         18 . The method of  claim 15 , wherein the cleaving enzyme is reversibly inactivated through interaction with an antibody at lower temperatures. 
     
     
         19 . The method of  claim 14 , wherein the cleavage domain comprises at least one RNA base. 
     
     
         20 . The method of  claim 19 , wherein the cleavage domain is located 3′ of the position of variation. 
     
     
         21 . The method of  claim 19 , wherein the cleavage domain is located 5′ of the position of variation. 
     
     
         22 . The method of  claim 19 , wherein the cleavage domain overlaps with the position of variation. 
     
     
         23 . The method of  claim 14 , wherein the cleavage domain comprises one or more 2′-modified nucleosides, and the cleaving enzyme cleaves between the position complementary to the variation and the one or more modified nucleosides. 
     
     
         24 . The method of  claim 23 , wherein the one or more modified nucleosides are 2′-fluoronucleosides. 
     
     
         25 . The method of  claim 14 , wherein the polymerase is a high-discrimination polymerase. 
     
     
         26 . The method of  claim 14 , wherein the polymerase is a mutant H784Q Taq polymerase. 
     
     
         27 . The method of  claim 26 , wherein the mutant H784Q Taq polymerase is reversibly inactivated via chemical, aptamer or antibody modification. 
     
     
         28 . The method of  claim 14 , wherein the primer contains a 5′ tail sequence that comprises a universal primer sequence and optionally a universal probe sequence, wherein the tail is non-complementary to the target DNA sequence. 
     
     
         29 . The method of  claim 14 , wherein the target DNA sequence is a sample that has been treated with a gene editing enzyme. 
     
     
         30 . The method of  claim 14 , wherein the target DNA sequence is a sample that has been treated with a CRISPR enzyme. 
     
     
         31 . The method of  claim 14 , wherein the target DNA sequence is a sample that has been treated with a Cas9 or Cpf1 enzyme. 
     
     
         32 . A method of target enrichment comprising:
 (a) providing a reaction mixture comprising
 (i) a first oligonucleotide primer having a tail domain that is not complementary to a target sequence, the tail domain comprising a first universal primer sequence; a cleavage domain positioned 5′ of a blocking group, the blocking group linked at or near the end of the 3′-end of the first oligonucleotide primer wherein the blocking group prevents primer extension and/or inhibits the first primer from serving as a template for DNA synthesis, 
 (ii) a sample nucleic acid that may or may not have the target sequence, 
 (iii) a cleaving enzyme and 
 (iv) a polymerase; 
   (b) hybridizing the first primer to the target DNA sequence to form a double-stranded substrate;   (c) cleaving the hybridized first primer, if the first primer is complementary to the target, with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the first primer; and   (d) extending the first primer with the polymerase.   
     
     
         33 . The method of  claim 32 , the method further comprising a second primer in reverse orientation to support priming and extension of the first primer extension product. 
     
     
         34 . The method of  claim 33 , wherein the second primer further comprises a tail domain comprising a second universal primer sequence. 
     
     
         35 . The method of  claim 34 , wherein steps (b)-(d) are performed 1-10 times. 
     
     
         36 . The method of  claim 35 , further comprising removing unextended primers from the reaction and hybridizing universal primers to the extension product to form a second extension product. 
     
     
         37 . The method of  claim 36 , wherein the universal primers further comprise tailed sequences for addition of adapter sequences to the second extension product. 
     
     
         38 . The method of  claim 37 , wherein sequencing is performed on the second extension product to determine the sequence of the target. 
     
     
         39 . The method of  claim 32 , wherein the target DNA sequence is a sample that has been treated with a gene editing enzyme. 
     
     
         40 . The method of  claim 32 , wherein the target DNA sequence is a sample that has been treated with a CRISPR enzyme. 
     
     
         41 . The method of  claim 32 , wherein the target DNA sequence is a sample that has been treated with a Cas9 or Cpf1 enzyme. 
     
     
         42 . The method of  claim 32 , wherein the cleaving enzyme is a hot start cleaving enzyme comprising at least one amino acid substitution as compared to its wild type, which is thermostable and has reduced activity at lower temperatures, and which exhibits enhanced mismatch discrimination during rhPCR as compared to its wild type. 
     
     
         43 . The method of  claim 42 , wherein the hot start cleaving enzyme is  Pyrococcus abyssi  RNase H2 comprising (a) a G12A amino acid substitution; (b) a P13T amino acid substitution; (c) a G169A amino acid substitution; or (d) a combination thereof. 
     
     
         44 . The method of  claim 42 , wherein the cleaving enzyme is chemically modified. 
     
     
         45 . The method of  claim 32 , wherein the cleaving enzyme is a hot start cleaving enzyme that is reversibly inactivated through interaction with an antibody at lower temperatures. 
     
     
         46 . The method of  claim 32 , wherein the cleaving domain comprises at least one RNA base. 
     
     
         47 . The method of  claim 46 , wherein the cleavage domain is located 3′ of the position of variation. 
     
     
         48 . The method of  claim 46 , wherein the cleavage domain is located 5′ of the position of variation. 
     
     
         49 . The method of  claim 46 , wherein the cleavage domain overlaps with the position of variation. 
     
     
         50 . The method of  claim 32 , wherein the cleaving domain comprises one or more 2′-modified nucleosides, and the cleaving enzyme cleaves between the position complementary to the variation and the one or more modified nucleosides. 
     
     
         51 . The method of  claim 50 , wherein the one or more modified nucleosides are 2′-fluoronucleosides. 
     
     
         52 . The method of  claim 32 , wherein the polymerase is a high-discrimination polymerase. 
     
     
         53 . The method of  claim 32 , wherein the polymerase is a mutant H784Q Taq polymerase. 
     
     
         54 . The method of  claim 53 , wherein the mutant H784Q Taq polymerase is reversibly inactivated via chemical, aptamer or antibody modification.