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.

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 - 54 . (canceled)