ExCyto PCR amplification.
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.
Dhodda
Vinay
V
Lucigen Corporation, Middleton, Wisconsin, United States of America.
Godiska
Ronald
R
Vanwye
Jeffrey D
JD
Mead
David
D
Hochstein
Rebecca
R
Sheets
Lynne
L
Vande Zande
Sarah
S
Niebauer
Chris
C
Crawford
Douglas L
DL
Oleksiak
Marjorie F
MF
D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014169:Transformation, Bacterial
D004269:DNA, Bacterial