PCR-DGGE研究微生物种群中多条带产生原因分析

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

摘要:

鸡肠道微生物菌群经PCR-DGGE分析, 回收PCR-DGGE分析胶上的一条DNA片段, 回收的DNA片段再重复进行2次PCR-DGGE分析, 以及分别用PCR反复循环扩增和PCR高保真酶扩增后再进行DGGE分析等方法研究PCR-DGGE分析中多条带产生原因。结果显示PCR-DGGE分析中多条带产生原因可能是作PCR扩增模板的DNA混杂有少量其他DNA片段, 多条带现象不易被消除。DGGE分析胶上的DNA片段测序时, 将该DNA片段回收、PCR扩增后克隆, 提取多个阳性克隆菌的质粒DNA片段, 分别与其原目的DNA片段进行DGGE分析, 在DGGE分析胶上选取与原目的DNA片段处于同一电泳位置的质粒DNA测序, 提高测序的准确性。

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.