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Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA... Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes. G C Wang and Y Wang Institute of Molecular and Cell Biology, National University of Singapore, Singapore. ABSTRACT PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention. CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? « Previous | Next Article » Table of Contents This Article Appl. Environ. Microbiol. December 1997 vol. 63 no. 12 4645-4650 » Abstract PDF Services Email this article to a colleague Similar articles in ASM journals Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of AEM Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Wang, G. C. Articles by Wang, Y. Search for related content PubMed PubMed citation Articles by Wang, G. C. Articles by Wang, Y. Related Content Load related web page information Social Bookmarking CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? current issue December 2011, volume 77, issue 23 Alert me to new issues of AEM About AEM Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy AEM RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0099-2240 Online ISSN: 1098-5336 Copyright © 2011 by the American Society for Microbiology. For an alternate route to AEM .asm.org, visit: http://intl- AEM .asm.org | More Info» var gaJsHost = (("https:" == document.location.protocol) ? "https://ssl." : "http://www."); document.write(unescape("%3Cscript src='" + gaJsHost + "google-analytics.com/ga.js' type='text/javascript'%3E%3C/script%3E")); var pageTracker = _gat._getTracker("UA-5821458-4"); pageTracker._trackPageview(); http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied and Environmental Microbiology American Society For Microbiology

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

Applied and Environmental Microbiology , Volume 63 (12): 4645 – Dec 1, 1997

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

Applied and Environmental Microbiology , Volume 63 (12): 4645 – Dec 1, 1997

Abstract

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes. G C Wang and Y Wang Institute of Molecular and Cell Biology, National University of Singapore, Singapore. ABSTRACT PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention. CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? « Previous | Next Article » Table of Contents This Article Appl. Environ. Microbiol. December 1997 vol. 63 no. 12 4645-4650 » Abstract PDF Services Email this article to a colleague Similar articles in ASM journals Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of AEM Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Wang, G. C. Articles by Wang, Y. Search for related content PubMed PubMed citation Articles by Wang, G. C. Articles by Wang, Y. Related Content Load related web page information Social Bookmarking CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? current issue December 2011, volume 77, issue 23 Alert me to new issues of AEM About AEM Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy AEM RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0099-2240 Online ISSN: 1098-5336 Copyright © 2011 by the American Society for Microbiology. For an alternate route to AEM .asm.org, visit: http://intl- AEM .asm.org | More Info» var gaJsHost = (("https:" == document.location.protocol) ? "https://ssl." : "http://www."); document.write(unescape("%3Cscript src='" + gaJsHost + "google-analytics.com/ga.js' type='text/javascript'%3E%3C/script%3E")); var pageTracker = _gat._getTracker("UA-5821458-4"); pageTracker._trackPageview();

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Publisher
American Society For Microbiology
Copyright
Copyright © 1997 by the American society for Microbiology.
ISSN
0099-2240
eISSN
1098-5336
Publisher site
See Article on Publisher Site

Abstract

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes. G C Wang and Y Wang Institute of Molecular and Cell Biology, National University of Singapore, Singapore. ABSTRACT PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention. CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? « Previous | Next Article » Table of Contents This Article Appl. Environ. Microbiol. December 1997 vol. 63 no. 12 4645-4650 » Abstract PDF Services Email this article to a colleague Similar articles in ASM journals Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of AEM Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Wang, G. C. Articles by Wang, Y. Search for related content PubMed PubMed citation Articles by Wang, G. C. Articles by Wang, Y. Related Content Load related web page information Social Bookmarking CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? current issue December 2011, volume 77, issue 23 Alert me to new issues of AEM About AEM Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy AEM RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0099-2240 Online ISSN: 1098-5336 Copyright © 2011 by the American Society for Microbiology. For an alternate route to AEM .asm.org, visit: http://intl- AEM .asm.org | More Info» var gaJsHost = (("https:" == document.location.protocol) ? "https://ssl." : "http://www."); document.write(unescape("%3Cscript src='" + gaJsHost + "google-analytics.com/ga.js' type='text/javascript'%3E%3C/script%3E")); var pageTracker = _gat._getTracker("UA-5821458-4"); pageTracker._trackPageview();

Journal

Applied and Environmental MicrobiologyAmerican Society For Microbiology

Published: Dec 1, 1997

There are no references for this article.