含发夹结构的Gateway质粒DNA测序方法改进  

Improvement of DNA Sequencing Method for Gateway Plasmids Containing Hairpin Structure

作　　者：刘耘[1] 熊家军[1] 杨利国[1] 

机构地区：[1]华中农业大学动物遗传育种与繁殖教育部重点实验室,武汉430070

出　　处：《华中农业大学学报》2008年第4期441-444,共4页Journal of Huazhong Agricultural University

基　　金：国家“863”计划项目(2007AA10Z152);国家“十一五”支撑项目(2006BAD04A02-11)资助

摘　　要：Gateway克隆技术现已广泛用于cDNA文库构建。然而,用常规DNA测序程序对Gateway cDNA进行测序时,反向重复的att序列易形成发夹结构,导致测序质量较差,且测得的序列较短,成为Gateway克隆技术应用的限制因素。本研究报道了1种Gateway cDNA克隆高温变性测序方法,即在Gateway cDNA测序PCR反应之前,先将DNA在98℃预变性5 min,可有效防止测序峰值的快速下降,极显著地增加Gateway cDNA克隆测序长度(质量Q≥20的序列平均长度从528 bp提高到816 bp)。该方法在常规DNA测序程序的基础上仅新增了5 min的高温预变性,不增加任何实验成本和操作步骤,非常适合Gateway质粒DNA的大规模测序。The Gateway cloning technique is extensively used to construct cDNA library. The resultant cDNA clones contain inverted repeats of attB or attL at both ends of the inserts. When sequenced using the standard plasmid DNA cycle sequencing protocol, the inverted repeats form hairpin structure which reduces the sequence quality and length of Gateway clones. We preheated the Gateway cDNA at 98℃ for 5 min,which could efficiently prevent the formation of hairpin structure and significantly improve the quality and length of sequences. A total of 155 Gateway cDNA clones were sequenced using this technique and the average length of sequence quality Q≥20 is 816 nucleotides,which is significantly longer than that of Gateway cDNAs sequenced using the standard protocol. This high-temperature predenaturation sequencing method is simple, and did not add any cost and manipulations, and is suitable for large-scale sequencing of Gateway plasmid DNAs prone to forming hairpin structure.

关 键 词：Gateway克隆系统 DNA测序 发夹 高温预变性 

分 类 号：Q785[生物学—分子生物学]