Analysis on Causes of Multi-bands in Researching on Microbe Populations by PCR-DGGE

Abstract:

Microbe populations from chicken intestinal were analyzed by PCR-DGGE. DNA fragment represented by a band from DGGE gel was retrieved. DNA fragment was two times repeated analyzed with PCR-DGGE, furthermore PCR reamplification and using high-fidelity DNA polymerase amplification also be applied to study on causes of multi-bands in PCR-DGGE analysis. The results showed that the causes of multi-bands in PCR-DGGE analysis may be that DNA templates for PCR mixed with other DNA fragment, and it was difficult to eliminate phenomenon of multi-bands in PCR-DGGE analysis. While the DNA fragments represented by bands from DGGE gel was sequenced, cloned this DNA fragments and extracted plasmid DNA of positive bacteria, and the plasmid DNA was amplified and analyzed again on DGGE gel to verify its position. Colonies whose positions were the same with the original DNA were selected for DNA sequencing to improve the veracity of DNA sequencing.