筛查基因突变的新技术──循环双脱氧指纹图谱法及其应用  

A New Technique of Screening of Gene Mutations Cycle-dideoxy Fingerprinting and Its Applications

作　　者：刘敬忠[1] 向华[1] 张纪平 毛燕玲[1] 赵离钟[1] 张鹏[1] 朱章菱[1] 翁心植[1] 卢圣栋 

机构地区：[1]首都医科大学附属北京红十字朝阳医院,中国医学科学院基础医学研究所,武汉市儿童医院

出　　处：《高技术通讯》1996年第1期37-41,共5页Chinese High Technology Letters

摘　　要：建立了适用于大批量标本基因突变筛查的双脱氧指纹图谱法即ｄｄＦ。该技术集测定ＤＮＡ序列的％脱氧末端终止法及ＳＳＣＰ二者长处于一身，即只作一个（例如用ｄｄＡ）双脱氧末端终止反应，然后将反应产物在与ＳＳＣＰ相同的电泳条件下进行聚丙烯酰胺凝胶电泳，这样有突变的样品不会漏检，阳性检出率高达１００％。简化了原方法中ＲＮＡ转录及逆转录等步骤，直接以ＰＣＲ产物为模板，将双链ＤＮＡ循环测序的反应用作ｄｄＦ反应，称之为Ｃｙｃｌｅ－ｄｄＦ，省去了Ｔ７ＲＮＡ聚合酶及ＭＭＶ逆转录酶二种酶。这样，Ｃｙｃｌｅ－ｄｄＦ既保持了阳性检出率高的优点，又降低了成本，简化了步骤，使之便于在大多数实验室推广开展。运用Ｃｙｅｌｅ－ｄｄＦ技术进行了乙型血友病（ＨＢ）患者九因子基因（Ｆ　Ⅸ）及β地中海贫血患者β珠蛋白基因突变的筛查。对筛查出的有异常指纹图谱的片段，进而用ＡＢＤ３７３Ａ－０１型ＤＮＡ自动测序仪有针对性地测出了具体突变点。The ddF was simplified by replacing T7 RBA transcription and MMV reverse transcriptation with a dsDNA cycle sequencing that was named cycle ddF (Cycle-dideoxy Fingerprinting). The cost of the screening test was decreased. Ninth DNA fragments of F Ⅸ gene from Hemophilia B patients and 30 DNA fragments of β globin gene from β-thalassemia were screened by using the cycle ddF technique. The DNA fragments with point mutations, were found out obviously and confirmed by DNA sequencing with ABD 373A-01 type autosequencer.

关 键 词：指纹图谱法 基因突变筛查 DNA测序 

分 类 号：Q343[生物学—遗传学]