一个更加通用的抗体可变区cDNA体外扩增法

More applicable protocol for in vitro amplification of antibody variable region cDNAs

在对一组单克隆抗体可变区ｃＤＮＡ进行体外扩增时，为了克服通用可变区引物的局限性，我们在ｏｌｉｇｏ（ｄＴ）－ｃｅｌｌｕｌｏｓｅ上合成固相ｃＤＮＡ，并采用一种称为“单引物预掺入ＰＣＲ”的新ＰＣＲ程序，即：在只有一侧引物存在的情况下，先让ＰＣＲ反应在低退火延伸温度下运转３个循环；追加另一侧引物后，转入常规ＰＣＲ循环。新方法扩展了“通用引物”的适用范围，并为引物设计和一些其它基因的ＰＣＲ放大提供了思路。

In an attempt to amplify the variable region cDNAs of a group of monoclonal antibodies while overcoming the limited utility of universal V region primers,solid-phase cDNA was synthesized onto oligo(dT)-cellulose,and a novel PCR protocol called single primer pre-incorporation PCR was adopted,in which PCR reactions were initiated with 3 cylces of low-temperature annealing and chain exension at the presence of merely one primer,followed by convertional PCR cycles upon the supplement of another primer.Our method extended the utility of universal primers and provided implications for primer design and PCR amplification of some other genes.