The invention disclosed relates to a PCR primer pair for amplication of
chaperonin-60 (cpn60) targets having high G+C content and to a PCR primer
"cocktail" to improve the representation of diverse sequences in
chaperonin-60 based PCR product libraries derived from complex templates.
In previous cpn60-based and 16S rDNA-based studies of mammalian
intestinal microbiota, it has been observed that some classes of
organisms such as the Actinobacteria, which are known through
culture-based studies to be present in large numbers in these
environments, are underrepresented or even absent from PCR product
libraries. Using library sequence data and reference cpn60 sequence data
from cpnDB, the chaperonin sequence database, we designed a pair of PCR
primers which can be used alone for higher G+C content targets and, when
used in combination with a previously developed degenerate, universal
cpn60 primer pair, improve the representation of complex templates with
high G+C content. We have validated these primers using a combination of
traditional and quantitative real-time PCR on both artificially
constructed complex templates and biological samples. The development and
optimization of this primer cocktail represents a significant advance in
our ability to generate cpn60 PCR product libraries which more closely
represent the biodiversity in complex microbial communities.