利用高效的大片段DNA回收方法改良Fosmid文库的构建

An Improved Method for Large Size DNA Recovery Facilitates Fosmid Library Preparation

外源DNA插入片段为40 kb左右的Fosmid文库在基因组学研究中有广泛的应用,但长期以来,40 kb外源片段的分离与纯化依赖于传统的切胶并电洗脱至透析袋的方法,难以得到足够量的DNA片段,极大降低Fosmid文库构建的成功率。通过改进全自动核酸/蛋白质回收系统SageELF的操作流程,建立一种简单、便捷、高效地回收40 kb左右DNA片段的方法,并用其成功地构建高质量的Fosmid文库。从文库中随机挑选的25个单克隆,经过测序及酶切分析,发现该文库中插入的DNA片段大小为37.9±5.2 kb。以上结果表明,利用改良的操作方法回收40 kb基因组DNA,操作简捷、高效,片段大小精准;另外,用该DNA片段构建的Fosmid文库,插入片段比较集中,有利于后续的基因组学分析。

The Fosmid library with inserted DNA of around 40 kb has been widely used in genomics research.However,the traditional method of agarose gel separation and electroelution into dialysis bag has long been used for large DNA fragment isolation,which makes it difficult to obtain sufficient amount of DNA fragments for ligation,thus dramatically reduces the success rate in Fosmid library preparation.In this study,a simple,convenient and efficient method for 40 kb DNA fragments recovery was established exploiting a modified program on SageELF,a fully automated nucleic acid/protein recovery system.The method was used to prepare Fosmid libraries with improved quality successfully.Through Sanger sequencing and restriction fragment analysis of 25 random selected Fosmid clones,we found the size of the inserted DNA was 37.9±5.2 kb.These results showed that,our improved method could recover 40 kb DNA accurately,simply and efficiently;In addition,the inserts of the Fosmid library constructed with the recovered 40 kb DNA were more concentrated,which will facilitate the following genomics analysis.