Extension temperature of 60 degree C required for PCR amplification of large DNA fragments (>5kb) from a low GC bacterium Clostridium acetobutylicum

摘要

PCR amplification of DNA fragments has been routinely used in gene cloning and engineering of microbial strains for biotechnological purposes such as production of biofuels and green chemicals. However, it is often a challenge to amplify large DNA fragments (>5kb) from low GC microorganisms using the standard PCR protocols. In this brief communication, we report a modified PCR method with an extension temperature of 60 degree C, which efficiently amplified a 5.3 and a 5.5kb DNA fragment (an extension time of 6min) from a low GC bacterium Clostridium acetobutylicum (~30% GC). A lower than normal extension temperature (72 degree C) approach may also facilitate PCR amplification of large DNA fragments (>5kb) from other low GC microorganisms.