ExCyto PCR amplification

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.

Figure 1. ExCyto PCR of chromosomal integrants.

Two µl of overnight cultures from chromosomal integrants were used for PCR with primers flanking the melAmelB bicistron integration site. Integrants show a band at 4.5 kb. Wild type (WT, last lane) has a band at 2 kb. One kb molecular weight markers are shown in the first lane.

Figure 2. ExCyto PCR amplification.

A cDNA library was transformed into ExCyto cells. Twenty-four different cDNAs were amplified without addition of exogenous polymerase using (A) single colonies, (B) 2 µl of frozen glycerol stocks, or (C) 2 µl of overnight cultures. One kb molecular weight markers are shown in the last lane.

Figure 3. ExCyto PCR and plasmid copy numbers.

Clones from a shotgun library of genomic DNA transformed into ExCyto cells were grown overnight, and 2 ul of the overnight cultures were amplified with ExCyto PCR from the (A) pEZSEQ high copy vector (∼300 copies/cell), (B) pSMART GC low copy vector (approx. 20 copies/cell), and (C) pSMART VC single-copy vector (1 copy per cell). In total, 54 different genomic DNA clones were amplified. One kb molecular weight markers are shown in the first and last lanes.

Figure 4. Dilution series of ExCyto PCR cells.

Eight different clones from a cDNA library transformed into ExCyto cells were grown overnight, and a serial dilution was used for amplification (undiluted, 100-, and 1000-fold dilutions). In (A), bacteria were diluted, reducing both the target DNA and the tsDNA polymerase. In (B), to compensate for the serial dilution of the tsDNA polymerase, 2 ul of ExCyto cells (OD 1.1) without plasmids was added. In (C), to compensate for the serial dilution of plasmid, 2 ul of bacteria with the same plasmid, but without the tsDNA polymerase was added. A 10-fold dilution is not shown, but gives similar amplification to that seen with undiluted cells. One kb molecular weight markers are shown in the last lane.

Figure 5. Robustness of ExCyto PCR.

Eight different clones from a cDNA library transformed into ExCyto cells were grown overnight, and 2 ul of the overnight cultures were added to reaction mix and left on ice for 0, 30, 60 and 120 minutes prior to ExCyto PCR amplification. One kb molecular weight markers are shown in the last lane.

Figure 6. ExCyto versus standard PCR.

(A) Twenty-four different clones from a cDNA library transformed in ExCyto cells were grown overnight, and 2 µl of overnight cultures were used for ExCyto PCR amplification. (B, C, & D) Standard PCR on 2 µl of overnight cultures from the same 24 clones as in (A) above, except clones were transformed into parent cells without the integrated thermostable polymerase. The cDNA inserts were amplified using Invitrogen's Taq DNA Polymerase (B), Promega's GoTaq DNA Polymerase (C), or Stratagene's Taq DNA Polymerase (D). One kb molecular weight markers are shown in the last lane.