| 摘要 |
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 o f 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 artificial ly contracted complex templates and biological samples. The development and optimization o f 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.
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