第三代测序细菌基因组DNA提取方法的比较

Comparative Study of Bacterial DNA Extraction Methods for the Third Generation Sequencing Technology

为筛选有效提取细菌菌株总DNA的方法,分别采用SDS法、CTAB-SDS法、试剂盒法和改良试剂盒法对苏云金芽胞杆菌、粘细菌和放线菌进行总DNA的提取、电泳检测、NanoDrop 2000测定、PCR扩增.对放线菌和苏云金芽胞杆菌,用改良试剂盒法所提取的总DNA纯度较高,电泳条带清晰,DNA主带位于23 kb,单位体积菌液所提取到的总DNA较SDS法和CTAB-SDS法高,能满足下游的PCR扩增等分子操作.对待测样品所做的质检报告也证实样品合格,可用于后续建库、测序.而粘细菌提取到的总DNA由于它们的OD260/230在2.0以下,没有达到第三代测序样品的要求.改良试剂盒法所提取到的总DNA大多数可以满足第三代测序技术样品制备的要求.

To screen the methods for effectivly extracting bacterial genomic DNA to the genome sequencing sample preparation for the third generation sequencing. The sodium dodecyl sulphate（ SDS） extraction method,hexadecyltrimethylammonium bromide-sodium dodecyl sulphate（ CTAB-SDS） extraction method,genomic DNA kit extraction method and improved genomic DNA kit extraction method were compared to extract the total DNA from the Bacillus thuringiensis,Myxobacteria and Actinomycetes. Then,the DNA samples were evaluated through the agarose gelelectrophoresis,Nano Drop 2000 analysis,and PCR amplification,respectively. On Actinomycetes and Bacillus thuringiensis,the method based on improved genomic DNA kit extraction could obtain the higher purity DNA. The electrophoretic bands were clear,and main strip was located in the 23 kb. The total DNA content per unit volume extracted is much higher than SDS method and CTAB-SDS method,which could meet the need of downstream molecular application. The quality inspection report also confirmed the samples qualified,might be used for subsequent library construction and sequencing. However,the myxobateria total DNA extracted can not meet the requirements for the third sequencing sample because of their OD260 /360 far below 2. 0. The total DNA improved by genomic DNA kit extraction method could fulfill the sequencing technology requirements.