鸡Visfatin基因9 bp indel杂合突变异源双链DNA的鉴定

内脂素(Visfatin)是脂肪细胞因子家族的新成员,主要由内脏脂肪组织产生.研究表明内脂素具有类胰岛素样作用.在检测固始鸡-安卡鸡资源群体3代(亲本,F1,F2)964只鸡Visfatin基因9bp插入/缺失(9 bp 'TAACCTGTG' insertion-deletion)多态的过程中,发现其杂合子的变性和非变性聚丙烯酰胺胶上除2条同源双链DNA(282bp和273bp)外有2条未知条带(命名为A和B).A,B条带经回收、二次PCR、再次聚丙烯酰胺凝胶电泳及DNA测序表明:Visfatin基因第10内含子中9bp insertion-deletion突变杂合子的PCR产物中,本身包含2种同源双链DNA片段和2种异源双链DNA片段,不需要经过额外的变性、退火处理,其PCR产物可以直接进行突变检测,在229个杂合突变中异源双链DNA的检出率为100%.因此,通过异源双链DNA这一标示物作为基因分型时的依照或者参考,建立适当的异源双链DNA分析法可进行基因中几个核苷酸插入/缺失多态的检测.

Identification of Heteroduplex DNA in Heterozygous Mutations of 9 bp Insertion-deletion in Chicken Visfatin Gene

Visfatin is a new member of adipokines secreted by visceral fat and mimics the effects of insulin. Detecting a 9 bp insertion-deletion of Visfatin gene among 964 chickens derived from Gushi-Anka resource population (parents,F1,F2), we found that except two homoduplex DNAs in length of 282 and 273 bp, another two unknown bands (named A and B) were unexpectedly detected by denaturing and non-denaturing polyacrylamide gel electrophoresis. Via recovering, reamplifying and DNA sequencing of two unknown bands were used to confirm that A and B bands were heteroduplex DNA fragments. Namely, heterozygous mutations of 9 bp insertion-deletion in Visfatin gene contained two homoduplex and two heteroduplex DNA fragments simultaneously. The PCR products can be directly used for mutation detecting, without additional denaturation and annealing. The detection rates of the heteroduplex DNAs among 229 heterozygous mutations were 100%. Therefore, these results showed that heteroduplex DNA can be as a reference in genotyping of insertion-deletion mutation of several nucleotides.