Methods and compositions for PCR using blocked and universal primers

摘要

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.

主权项

1. A PCR reaction mixture comprising:
(a) at least one pair of target-specific primers comprising universal 5′ tags, where at least one target-specific primer cannot be extended by polymerase (3′-blocked primer); (b) at least one pair of universal primers adapted to prime on a complement of the universal 5′ tags, and at least one of the universal primers comprises a bar-code identifying a sample; (c) an enzyme that unblocks the 3′-blocked primer after it anneals to the DNA target generating a 3′-OH end; and (d) a polymerase that extends unblocked primers during one or more initial PCR cycles; and subsequently extends universal primers driving amplification in a closed tube reaction.