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

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.

Making and Sequencing Heavily Multiplexed, High-Throughput 16S Ribosomal RNA Gene Amplicon Libraries Using a Flexible, Two-Stage PCR Protocol.

Deep sequencing of polymerase chain reaction (PCR)-amplified small subunit (16S or 18S) ribosomal RNA (rRNA) genes fragments is commonly employed to characterize the composition and structure of microbial communities. Preparing genomic DNA for sequencing of such gene fragments on Illumina sequencers can be performed in a straightforward, two-stage PCR method, described herein. The protocol described allows for up to 384 samples to be sequenced simultaneously, and provides great flexibility in choice of primers.

Genomics Analysis of Replicative Helicase DnaB Sequences in Proteobacteria.

Replicative Helicase DnaB interacts with DnaA, DnaC, DnaG, and DNA polymerase III to commence replication, increase the movement rate of the replication fork, and to assemble part of the primosome. The formation of the replication fork is limited by the ability to load DnaB to the DNA, thus DnaB has shown to be vital to a large extent. In the absence of DnaB, the replication fork is not maintained and in a state of inactivity the replication fork degrades and collapses.