ExCyto PCR Amplification

Vinay Dhodda,Sarah Vande Zande,Lynne Sheets,Marjorie F Oleksiak,Rebecca Hochstein,Jeffrey D Vanwye,Chris Niebauer,David Mead,Ronald Godiska,Douglas L Crawford,Ching-Hong Yang

doi:10.1371/journal.pone.0012629

Abstract

BackgroundExCyto 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.ResultsBecause 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.ConclusionsExCyto 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.

Highlights

DNA amplification plays a critical role in many molecular biology procedures [1]
This method requires adding the purified DNA polymerase to a master mix and preventing non-specific amplification by hot-start or other approaches. In this manuscript we describe novel methods, which utilize E. coli cells (‘‘ExCyto polymerase chain reaction (PCR)’’ cells) that express a chromosomally integrated gene for a thermostable DNA polymerase, facilitating robust PCR amplification without the addition of exogenous enzyme
Because the tsDNA polymerase is integrated into the chromosome of the ExCyto PCR cells, one can transform these cells with plasmids or BACs and maintain the expression of the tsDNA polymerase

Summary

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 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. 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

Introduction

Results and Discussion

Materials and Methods