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GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles.
FEMS Microbiol Lett. 2010 Nov; 312(1):55-62.FM

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.

Authors+Show Affiliations

Department of Biology, South Dakota State University, Brookings, SD 57007, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

20831594

Citation

Rettedal, Elizabeth A., et al. "GC-clamp Primer Batches Yield 16S rRNA Gene Amplicon Pools With Variable GC Clamps, Affecting Denaturing Gradient Gel Electrophoresis Profiles." FEMS Microbiology Letters, vol. 312, no. 1, 2010, pp. 55-62.
Rettedal EA, Clay S, Brözel VS. GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles. FEMS Microbiol Lett. 2010;312(1):55-62.
Rettedal, E. A., Clay, S., & Brözel, V. S. (2010). GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles. FEMS Microbiology Letters, 312(1), 55-62. https://doi.org/10.1111/j.1574-6968.2010.02097.x
Rettedal EA, Clay S, Brözel VS. GC-clamp Primer Batches Yield 16S rRNA Gene Amplicon Pools With Variable GC Clamps, Affecting Denaturing Gradient Gel Electrophoresis Profiles. FEMS Microbiol Lett. 2010;312(1):55-62. PubMed PMID: 20831594.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles. AU - Rettedal,Elizabeth A, AU - Clay,Sharon, AU - Brözel,Volker S, Y1 - 2010/09/10/ PY - 2010/9/14/entrez PY - 2010/9/14/pubmed PY - 2011/1/28/medline SP - 55 EP - 62 JF - FEMS microbiology letters JO - FEMS Microbiol Lett VL - 312 IS - 1 N2 - 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. SN - 1574-6968 UR - https://www.unboundmedicine.com/medline/citation/20831594/GC_clamp_primer_batches_yield_16S_rRNA_gene_amplicon_pools_with_variable_GC_clamps_affecting_denaturing_gradient_gel_electrophoresis_profiles_ L2 - https://academic.oup.com/femsle/article-lookup/doi/10.1111/j.1574-6968.2010.02097.x DB - PRIME DP - Unbound Medicine ER -