穰杰+李莉+唐琼+杨琦+何恋+丁学知+夏立秋

基金项目：国家自然科学基金资助项目（31370116）;国家“973”基础研究计划资助项目（2012CB722301）;国家“863”高技术研究发展计划资助项目（2011AA10A203）;湖南省教育厅重点资助项目（13CY002， 10CY013，12K033）;湖南省2011协同创新中心资助项目（20134486）

*通讯作者，Email：xialq@hunnu.edu.cn（湖南师范大学生命科学学院，微生物分子生物学国家重点实验室培育基地，中国 长沙410081）

摘要为筛选有效提取细菌菌株总DNA的方法，分别采用SDS法、CTABSDS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CTABSDS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.

关键词第三代测序技术;DNA提取方法;CTABSDS法;改良溶液型试剂盒法

中图分类号Q939.9文献标识码A文章编号10002537（2015）06001407

Comparative Study of Bacterial DNA Extraction Methods

for the Third Generation Sequencing Technology

RANG Jie， LI Li， TANG Qiong， YANG Qi， HE Lian， DING Xuezhi， XIA Liqiu*

（College of Life Sciences， Hunan Normal University，

State Key Laboratory Breeding Base of Microbial Molecular Biology， Changsha 410081， China）

AbstractTo 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 bromidesodium dodecyl sulphate（CTABSDS） 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， NanoDrop 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 CTABSDS 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.

Key wordsThe third sequencing technology; DNA extraction method; CTABSDS method; improved solution genomic DNA kit extraction method

细菌全基因组测序在当今生命科学领域研究中具有非常重要的作用，其测序方法也由早先的Sanger双脱氧法经过第二代高通量测序技术，更新换代为单分子荧光实时测序技术（SelfMonitoring Analysis and Reporting Technology，SMART技术）.该测序方法与第二代测序方法相比展示了无与伦比的优势.例如，第二代测序技术读长较短，对后续的序……