Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers.
Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.
Hill
Janet E
JE
National Research Council Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada. Janet.Hill@nrc-cnrc.gc.ca
Town
Jennifer R
JR
Hemmingsen
Sean M
SM
D001419:Bacteria / Q000145:classification / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics*; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D018834:Chaperonin 60; D017931:DNA Primers; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S