单引物预掺入PCR与抗体可变区基因的体外放大

Single Primer Pre-incorporation PCR and Ampliflcation of Antlhody Variable RegionGene.

用针对引导序列的５′－引物和针对恒定区的３′－引物，常规ＰＣＲ程序只使所选５株单抗１０个可变区ｃＤＮＡ中的４个得到放大。新设计的程序增设了一个反应时相：９４℃ｌｍｉｎ，３７℃６－８ｍｉｎ，循环１－３次，只加入５′－引物，补充３′－引物后，转入常规ＰＣＲ循环，１０个可变区ｃＤＮＡ均获放大。此程序被命名为＂单引物预掺入ＰＣＲ＂。

Using 5′- primers corresponding toleader sequence,3′- primers to constant re-gion,and conventional PCR protocol,only 4out of 10 variable region cDNAs of the 5 mo-noclonal antibodies were amplified.A novelprotocol was designed and tested which in-cluded one more time phase of reaction:94℃1min,37℃ 6-8min,1-3 cycles,with 5′-primer but no 3′-primer.Conventional PCRcvcles started after 3′- primer was supple-mented.As a result,all 10 variable regioncDNAs were successfully amplified.Thisnovel protocol was named 'single primer pre-incorporation PCR'.