Monday, October 04, 2010

New manuscript about reproducibility of GC clamps

One more thing to worry about!

A recent article of interest in FEMS Microbiology Letters:

Rettedal et al. 2010. GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles. DOI: 10.1111/j.1574-6968.2010.02097.x

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GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles
Elizabeth A. Rettedal, Sharon Clay, Volker S. Brözel.

Abstract
Fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene pools have become a popular tool for comparisons between microbial communities. The GC-clamp portion of primers for DGGE amplicon preparation provides a key component in resolving fragments of similar size but different sequence. We hypothesized that repeat syntheses of identical 40-base GC-clamp primers lead to different DGGE profiles. Three repeat syntheses of the same GC-clamp primer and two different GC-clamp primers directed at the V3–5 region of the 16S rRNA gene were compared. Genomic DNA of two separate soil bacterial communities and three bacterial species was amplified and resolved by DGGE. The DGGE profiles obtained with repeat-synthesized primers differed among each other as much as with alternate primers, for both soil DNA and pure single species. The GC-clamp portion of members of amplicon pools varied among each other, deviating from the design sequence, and was the likely cause for multiple bands derived from a single 16S rRNA gene sequence. We recommend procuring an oligonucleotide batch large enough to conduct an entire project. This should help to avoid any DGGE profile variations due to performance differences between repeat syntheses of GC-clamp oligonucleotide primers.

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