Methods and compositions for pcr using blocked and universal primers

Abstract

Provided herein are methods and compositions for performing PCR with primers with blocked 3′-ends that are unblocked when these primers anneal to the template. The multiplexed PCR can be used as real-time qPCR, for end-point detection or as enrichment method for next generation sequencing (NGS). Also described herein are methods and compositions to improve sensitivity of mutation-specific PCR when targeting closely-spaced mutations.

1 - 10 . (canceled) 
     
     
         11 . A blocked PCR primer and an enhancer 3′-blocked oligo that anneals downstream from the blocked primer, forming no gap or a single base gap between the primer and the oligo when both are annealed to the target in a PCR or isothermal amplification mixture comprising an endonuclease that cleaves off a cleavable group at the single base gap and enables primer extension. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . A multiplex allele (mutation) specific PCR reaction mixture used to detect two or more closely spaced mutations, comprising:
 (a) mutation (allele) specific primers for each mutation to be detected, wherein the primers comprise the same target-specific 3′-regions, except for different 3′-ends; and   (b) the primers of (a) further comprising different non-target-specific 5′ tags; such that after the first two PCR cycles the first allele-specific primer that was extended in the first PCR cycle continues PCR by annealing to the amplicons that are 100% complementary to the whole first primer, but other allele-specific primers with 5′-tags not matching the amplicons generated by the first primer do not anneal at the annealing temperature used in PCR.   
     
     
         15 . The PCR reaction mixture of  claim 14 , wherein
 the allele (mutation) and target-specific primers of (a) and (b) further comprise universal 5′-tags and the different non-target specific tags of (b) are situated between the 3′-target-specific region and the universal 5′ tags of the allele specific primers; and   wherein the reaction mixture further comprises (c) at least one pair of universal primers that can prime on the universal 5′ tags.   
     
     
         16 . The reaction mixture of  claim 14 , where at least one of the allele or locus specific primers is blocked. 
     
     
         17 . A method of using PCR to detect two or more closely-spaced mutations with increased sensitivity, comprising the use of mutation-specific primers for each mutation with 5′-non target tags different for each primer, such that after first two PCR cycles, an increased annealing temperature is used to decrease completion between different mutation-specific primers, thus increasing assay sensitivity. 
     
     
         18 . A PCR amplification method comprising the use of at least one blocked PCR primer with a 3′-end complementary to the template, such that unblocking is performed by a 3′-exonuclease activity and extension by a polymerase activity, respectively, and wherein the two enzymatic activities are performed by a single enzyme or two separate enzymes. 
     
     
         19 . A PCR reaction mixture comprising at least one 3′-blocked primer comprising a 3′-end having a nucleotide sequence 100% complementary to a template; and either a polymerase with 3′-exonuclease activity, or a separate 3′-exonuclease and a polymerase; such that after the 3′-blocked primer anneals to the template the exonuclease unblocks the primer(s) and the polymerase extends the unblocked primer(s) during PCR.