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
Article numbere12629
Pages (from-to)1-5
Number of pages5
JournalPLoS One
Volume5
Issue number9
DOIs
StatePublished - Nov 1 2010

Fingerprint

DNA-Directed DNA Polymerase
Amplification
DNA-directed DNA polymerase
Polymerase Chain Reaction
DNA
Plasmids
cells
plasmids
Gene encoding
Expressed Sequence Tags
Ice
Chromosomes
Escherichia coli
Libraries
Screening
Complementary DNA
Throughput
Bacterial Chromosomes
ice
screening

ASJC Scopus subject areas

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

Cite this

Dhodda, V., Godiska, R., vanWye, J. D., Mead, D., Hochstein, R., Sheets, L., ... Oleksiak, M. F. (2010). ExCyto PCR amplification. PLoS One, 5(9), 1-5. [e12629]. https://doi.org/10.1371/journal.pone.0012629

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

In: PLoS One, Vol. 5, No. 9, e12629, 01.11.2010, p. 1-5.

Research output: Contribution to journalArticle

Dhodda, V, Godiska, R, vanWye, JD, Mead, D, Hochstein, R, Sheets, L, vande Zande, S, Niebauer, C, Crawford, DL & Oleksiak, MF 2010, 'ExCyto PCR amplification', PLoS One, vol. 5, no. 9, e12629, pp. 1-5. https://doi.org/10.1371/journal.pone.0012629
Dhodda V, Godiska R, vanWye JD, Mead D, Hochstein R, Sheets L et al. ExCyto PCR amplification. PLoS One. 2010 Nov 1;5(9):1-5. e12629. https://doi.org/10.1371/journal.pone.0012629
Dhodda, Vinay ; Godiska, Ronald ; vanWye, Jeffrey D. ; Mead, David ; Hochstein, Rebecca ; Sheets, Lynne ; vande Zande, Sarah ; Niebauer, Chris ; Crawford, Douglas L ; Oleksiak, Marjorie F. / ExCyto PCR amplification. In: PLoS One. 2010 ; Vol. 5, No. 9. pp. 1-5.
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