CAJ | 학술논문

Y形接头延伸法(Y-shaped adaptor dependent extension,YADE)是一种扩增已知DNA片段相邻序列的方法,但其效率常受到已知序列周围酶切位点数目的限制.在Y形接头延伸中采用由多种酶切和连接产生的DNA作模板,大大提高了该方法扩增相邻序列的效率,实现了相邻序列的连续扩增.利用该方法通过2轮连续的扩增从7种酶切连接产物中成功地获得了1个棉花小GTP酶基因(GhRacB)的2228 bp上游序列.结果表明,多模板Y形接头延伸法是一种从复杂基因组中扩增相邻序列的有用方法.
Y형접두연신법(Y-shaped adaptor dependent extension,YADE)시일충확증이지DNA편단상린서렬적방법,단기효솔상수도이지서렬주위매절위점수목적한제.재Y형접두연신중채용유다충매절화련접산생적DNA작모판,대대제고료해방법확증상린서렬적효솔,실현료상린서렬적련속확증.이용해방법통과2륜련속적확증종7충매절련접산물중성공지획득료1개면화소GTP매기인(GhRacB)적2228 bp상유서렬.결과표명,다모판Y형접두연신법시일충종복잡기인조중확증상린서렬적유용방법.
Y-shaped adaptor dependent extension(YADE)method is a useful tool to amplify the flanking sequence of a known DNA sequence,but its efficiency is frequently limited by the restriction sites around the known sequence.In this paper,we demonstrated that using multiple templates derived from several restrictions and ligations could dramatically increase the efficiency of YADE method and render it suitable for sequential amplification of flanking sequences.With templates originating from 7 digestions,a 2228-bp 5'-upstream sequence of a cotton small GTPase gene was obtained by two rounds of sequential YADE amplifications.The results demonstrated that the YADE method with multiple templates may be a useful tool for sequential PCR walking in complex genomes.