ExCyto PCR amplification

Vinay Dhodda, Ronald Godiska, Jeffrey D. vanWye, David Mead, Rebecca Hochstein, Lynne Sheets, Sarah vande Zande, Chris Niebauer, Douglas L. Crawford, Marjorie F. Oleksiak

Research output: Contribution to journalArticle

Abstract

Background: ExCyto PCR cells provide a novel and cost effective means to amplify DNA transformed into competent bacterial cells. ExCyto PCR uses host E. coli with a chromosomally integrated gene encoding a thermostable DNA polymerase to accomplish robust, hot-start PCR amplification of cloned sequences without addition of exogenous enzyme. Results: Because the thermostable DNA polymerase is stably integrated into the bacterial chromosome, ExCyto cells can be transformed with a single plasmid or complex library, and then the expressed thermostable DNA polymerase can be used for PCR amplification. We demonstrate that ExCyto cells can be used to amplify DNA from different templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours. Further, PCR amplification with ExCyto cells is comparable to amplification using commercial DNA polymerases. The ability to transform a bacterial strain and use the endogenously expressed protein for PCR has not previously been demonstrated. Conclusions: ExCyto PCR reduces pipetting and greatly increases throughput for screening EST, genomic, BAC, cDNA, or SNP libraries. This technique is also more economical than traditional PCR and thus broadly useful to scientists who utilize analysis of cloned DNAs in their research.

Original languageEnglish (US)
Article numbere12629
Pages (from-to)1-5
Number of pages5
JournalPloS one
Volume5
Issue number9
DOIs
StatePublished - 2010

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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    Dhodda, V., Godiska, R., vanWye, J. D., Mead, D., Hochstein, R., Sheets, L., vande Zande, S., Niebauer, C., Crawford, D. L., & Oleksiak, M. F. (2010). ExCyto PCR amplification. PloS one, 5(9), 1-5. [e12629]. https://doi.org/10.1371/journal.pone.0012629