实时聚合酶链反应熔解曲线分析检测APC基因突变  

Detection of APC mutation by real-time polymerase chain reaction and melting curve analysis

作　　者：郑薇薇[1] 刘忠臣[2] 李庆阁[1] 

机构地区：[1]厦门大学生命科学学院生物医学科学系 [2]厦门市中山医院

出　　处：《中华病理学杂志》2005年第10期669-671,共3页Chinese Journal of Pathology

基　　金：国家自然科学基金资助项目(30170834);福建省自然科学基金重点项目(C0220001)

摘　　要：目的建立一种准确、方便、价廉、适合临床的APC基因突变检测方法。方法采用SYBRGreenⅠ为实时聚合酶链反应(PCR)指示剂,首先从基因组DNA中扩增包含突变位点的靶基因(270bp),并通过熔解曲线分析确定产物;再以上述片段为模板扩增特定长度的小片段(40/35bp),以0.5℃/step的速率,获得从65℃到99℃的熔解曲线以检测APC_1309位5bp缺失突变。结果临床标本检测结果表明:18例结直肠肿瘤患者石蜡组织标本中检出APC_1309位5bp缺失突变7例,20例正常人外周血标本检测结果均为阴性。结论实时PCR熔解曲线法设计简单,操作简便,结果可靠,可望应用于临床APC基因突变检测,并可推荐用于其他基因突变的检测。Objective To establish a simple, reliable and low cost approach for clinical detection of APC mutation. Methods Using SYBR Green I as the real-time polymerase chain reaction （PCR） product indicator, a DNA fragment of 270 bp targeting APC_1309 mutation （5 bp deletion） was amplified from the sample DNA. A short fragment （40/35 bp） was then amplified from the 270 bp PCR product, followed by melting curve analysis from 65℃ to 99℃at 0.5℃/step. Results A total of 18 paraffin-embedded .tumor samples were analyzed, of which 7 were tested positive for the mutation and 11 were negative. No mutation was detected in any of the 20 normal peripheral blood samples. Conclusions Real-time PCR melting curve analysis can be used for routine APC mutation detection. The simple design, low cost and high reliability should allow similar applications to the analysis of a variety of other gene mutations.

关 键 词：聚合酶链反应 基因 APC 结肠直肠肿瘤 

分 类 号：R735.3[医药卫生—肿瘤]