Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler.

ABSTRACT: Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucl

SUBMITTER: TerMaat JR 

PROVIDER: S-EPMC3691701 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature