Improved PCR Amplification of Broad Spectrum GC DNA Templates.
Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10-90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.
Research & Development, Gen9 Inc, Cambridge, Massachusetts, United States of America.
Nicholas
N
Guido
Research & Development, Gen9 Inc, Cambridge, Massachusetts, United States of America.
Elena
E
Starostina
Research & Development, Gen9 Inc, Cambridge, Massachusetts, United States of America.
Devin
D
Leake
Research & Development, Gen9 Inc, Cambridge, Massachusetts, United States of America.
Ishtiaq
I
Saaem
D001482:Base Composition; D018899:CpG Islands; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*
D006147:Guanine; D004247:DNA; C066856:7-deazaguanine