ExCyto PCR amplification.

Area of Science:

• Molecular Biology
• Genetics

Background:

• ExCyto PCR cells utilize host E. coli with a chromosomally integrated gene for thermostable DNA polymerase.
• This enables robust, hot-start PCR amplification of cloned DNA sequences without exogenous enzyme addition.

Purpose of the Study:

• To introduce and validate ExCyto PCR as a novel, cost-effective method for DNA amplification.
• To demonstrate the utility of endogenously expressed DNA polymerase for PCR in bacterial cells.

Main Methods:

• Competent bacterial cells (E. coli) were engineered with a chromosomally integrated gene encoding a thermostable DNA polymerase.
• These ExCyto PCR cells were transformed with various DNA templates, including plasmids and libraries.

Main Results:

• ExCyto cells successfully amplified DNA from diverse templates, plasmids of varying copy numbers, and master mixes stored for extended periods.
• PCR amplification efficiency using ExCyto cells was comparable to commercial DNA polymerases.
• This study demonstrates for the first time the transformation of a bacterial strain for endogenous protein-based PCR amplification.

Conclusions:

• ExCyto PCR significantly reduces pipetting steps and increases throughput for screening EST, genomic, BAC, cDNA, and SNP libraries.
• The technique is more economical than traditional PCR, offering broad utility for researchers analyzing cloned DNAs.