常规PCR与RACE结合法快速从cDNA文库中克隆基因

Rapidly Cloning Gene from cDNA Liprary Using Common PCR and RACE Combined Method

首先根据Fe(Ⅱ ) 转运蛋白基因家族的保守区设计同源的常规PCR引物 (F1,R1) ,扩增出 4 90bp的特异片段 .然后根据RACE法原理 ,巧妙地设计 3′RACE(F2 )和 5′RACE(R3)特异性引物 ,使两者既能分别与文库载体上的筛选扩增引物配对扩增cDNA 3′和 5′末端序列 ,又能使扩增出的 3′RACE和 5′RACE产物序列有重叠部分 ;另外设计引物时还兼顾到使F2和R3也能配对 ,从而能对 3′RACE和 5′RACE扩增产物进行双特异性引物常规PCR鉴定 .该方案不但能够直接根据序列鉴定 3′RACE和 5′RACE产物是否为同一基因序列 ,还能快捷地用常规PCR鉴定 3′RACE和5′RACE扩增产物是否为特异性扩增 ,避免了对非特异性扩增产物的克隆测序等繁琐的鉴定过程 ,优化了基因克隆策略 .最后根据拼读出的全长序列设计引物 (F4 ,R4 )扩增出完整编码区 .应用该方法成功地从小金海棠缺铁处理的根系cDNA文库中克隆了Fe(Ⅱ ) 转运蛋白基因的cDNA .

We have got 490 bp of Fe(Ⅱ)-transporter gene first by planning primers for common PCR according to the conserved domains of Fe(Ⅱ)-transporter gene family.Then,we plan 3′RACE (F2) and 5′RACE(R3) primers which not only can anneal with screening amplimer of cDNA library vector respectively,but also can make the sequence of 3′RACE and 5′RACE have overlapped part.On the other hand,these two primers can anneal with each other which can rapidly identify the products of 3′RACE and 5′RACE according to their sequences.This plan not only ensures that the products of 3′RACE and 5′RACE are the parts of one same gene,but also can identify if the products are special product rapidly,which avoids cloning and sequencing each product for identifying and makes the clone process easily.At last,we amplified the full ORF by planning primers(F4,R4) according to the full cDNA sequence.We have cloned the cDNA of Fe(Ⅱ)-transporter gene successfully from the cDNA library of Malus xiaojinensis Cheng et Jiang,which proves that this plan is a rapid method to clone gene from cDNA library.