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重组PCR构建bcr |
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李巍△ 杜传书
【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-abl mRNA定量PCR的竞争内标物。方法 利用重组PCR技术，实施对b3a2型bcr-abl cDNA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-abl cDNA相应的201bp扩增片段长39bp，使之适于作为b3a2和b2a2型两类bcr-abl mRNA定量PCR的通用内标物。结论 表明重组PCR是一种获得靶基因的竞争性内标物的简便而可靠的方法。
【关键词】 bcr-abl融合基因 重组PCR 竞争PCR mRNA定量
CONSTRUCTION OF A COMPETITOR FOR bcr-abl cDNA BY RECOMBINANT PCR Li Wei, Du Chuanshu*.* Department of Medical Genetics, Sun Yat-sen University of Medical Sciences. Guangzhou 510098 P.R.China
【Abstract】 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.
【Key words】 bcr-abl fusion gene Recombinant PCR Competitive PCR Quantitation of mRNA
断裂点簇集区基因bcr与abl原癌基因形成的bcr-abl融合基因，是慢性粒细胞白血病(CML)和急性淋巴细胞白血病(ALL)等白血病中存在的费城染色体(Ph)的分子标志。临床上，动态检测白血病患者外周血或骨髓中bcr-abl mRNA的含量变化比单纯定性检测bcr-abl基因或Ph染色体，对于疗效和预后的正确评价
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