摘要
目的竞争ＰＣＲ可用于ｍＲＮＡ的定量测定，为了得到白血病中ｂｃｒ－ａｂｌｍＲＮＡ定量ＰＣＲ的竞争内标物。方法利用重组ＰＣＲ技术，实施对ｂ３ａ２型ｂｃｒ－ａｂｌｃＤＮＡ中一２７６ｂｐ片段的缺失法定点突变。结果经ＤＮＡ序列分析证实，重组后的ｃＤＮＡ片段与重组前相比，５′和３′端大部分序列相同，仅中间５５ｂｐ的部分序列缺失，同时引入１９ｂｐ外源ＤＮＡ片段，即净缺失３６ｂｐ，得到２４０ｂｐ的重组ＰＣＲ产物。较ｂ２ａ２型ｂｃｒ－ａｂｌｃＤＮＡ相应的２０１ｂｐ扩增片段长３９ｂｐ，使之适于作为ｂ３ａ２和ｂ２ａ２型两类ｂｃｒ－ａｂｌｍＲＮＡ定量ＰＣＲ的通用内标物。
Objective Competitive PCR is a powerful method for quantification of mRNA. This study sought to construct an internal standard which can be used as a competitor for bcr abl fusion cDNA. Methods Using recombinant PCR for site directed mutagenesis. Results A 240bp mimic of a 276bp fragment in b3a2 type of bcr abl cDNA was obtained. The difference in mimic, which was verified by DNA sequencing, was a 55bp deletion of the target sequence and a 19bp insertion of exogenous unrelated sequence with a Xba I restriction site. In it, except for the inner 36bp, the mimic contained the same sequence and shared the same primer recognition sites as the target b3a2 cDNA(276bp) or as the target b2a2 cDNA(201bp).The obtained recombinant vector, named pGEM mimic, can be used as a common competitor for either b3a2 or b2a2 type of bcr abl cDNA. Conclusion This provided a simple and reliable method for constructing an internal standard used in competitive PCR.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
北大核心
1998年第3期143-146,共4页
Chinese Journal of Medical Genetics
