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.

1 - 9 . (canceled) 
     
     
         10 : 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 and 3′ of a position of variation, 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.   
     
     
         11 : The method of  claim 10 , wherein the cleaving enzyme is a hot start cleaving enzyme that is thermostable and has reduced activity at lower temperatures. 
     
     
         12 : The method of  claim 10 , wherein the cleaving enzyme is a chemically modified hot start cleaving enzyme that is thermostable and has reduced activity at lower temperatures. 
     
     
         13 : The method of  claim 12 , wherein the hot start cleaving enzyme is a chemically modified  Pyrococcus abyssi  RNase H2. 
     
     
         14 : The method of  claim 10 , wherein the cleaving enzyme is a hot start cleaving enzyme that is reversibly inactivated through interaction with an antibody at lower temperatures. 
     
     
         15 : The method of  claim 10 , wherein the cleaving domain is comprised of at least one RNA base, and the cleaving enzyme cleaves between the position complementary to the variation and the RNA base. 
     
     
         16 : The method of  claim 10 , wherein the cleaving domain is comprised of 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. 
     
     
         17 : The method of  claim 16 , wherein the one or more modified nucleosides are 2′-fluoronucleosides. 
     
     
         18 : The method of  claim 10 , wherein the polymerase is a high-discrimination polymerase. 
     
     
         19 : The method of  claim 10 , wherein the polymerase is a mutant H784Q Taq polymerase. 
     
     
         20 : The method of  claim 19 , wherein the mutant H784Q Taq polymerase is reversibly inactivated via chemical, aptamer, or antibody modification. 
     
     
         21 : The method of  claim 10 , 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. 
     
     
         22 - 53 . (canceled)