Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer degeneracy.

ABSTRACT: Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR ("PEX PCR") to separate linear copying and exponential amplification stages of PCR to reduce PCR bias. In this manuscript, we describe an improved deconstructed PCR ("DePCR") protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with anneal

SUBMITTER: Naqib A 

PROVIDER: S-EPMC6573857 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature