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.
National Research Council Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada. Janet.Hill@nrc-cnrc.gc.ca
Janet E
JE
Hill
Jennifer R
JR
Town
Sean M
SM
Hemmingsen
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