Comparison of Total DNA Extraction Methods for Studying Endophyte Diversity in Mulberry / 广州中医药大学学报

ABSTRACT

Objective To compare the advantages and disadvantages of the four DNA extraction methods according to the endophytic diversity in the roots, stems, and leaves of mulberry analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) , and by taking the yield, purity and PCR amplification as indexes. Methods Four common methods, i.e., cetyltrimethyl ammonium bromide ( CTAB) , sterile phosphate buffered saline (SPBS) vibration, liquid nitrogen grinding (LNG) , and KIT methods, were used to extract the total DNA from different tissues of mulberry, and then were compared based on the diversity analysis results for endophyte by PCR-DGGE. Results From the roots and stems of mulberry, we got the highest concentration of DNA by LNG extraction method, and got the lowest concentration by SPBS extraction method. But for the leaves of mulberry, the results of the four extraction methods were completely opposite to those for the roots and stems. For different tissues of mulberry, the purity of DNA extracted by KIT method was the best. According to the endophytic bacteria diversity analyzed by 16S rDNA PCR-DGGE, the appropriate method for extraction of DNA was LNG or CTAB, but was not KIT. And according to the results of endophytic fungi diversity analyzed by ITS PCR-DGGE, the best extraction method was KIT, and the unsuited methods varied from the tissues of mulberry. Conclusion The optimum DNA extraction method for mulberry varies from the tissues of mulberry and endophtic bacteria.