BRCA2基因突变荧光偏振检测方法的建立及优化
A fluorescence polarization-based method to detect BRCA2 mutation
薛丽 1杨芳 2张文红 2赵锦荣 2白玉杰1
作者信息
- 1. 海南医学院科学实验中心,海南,海口,510035;第四军医大学药学系基因诊断研究所,陕西,西安,710033
- 2. 第四军医大学药学系基因诊断研究所,陕西,西安,710033
- 折叠
摘要
目的:建立一种适合于大量标本的基因突变筛查方法.方法:PCR扩增包含BRCA2基因突变位点的片段,经模板指导的荧光素标记碱基掺入反应(Template Directed Dye-terminator Incorporation,TDI),荧光素标记终止碱基(R110-acyGTp或TAMRA-acyTTP)特异性掺入到检测探针的3'末端,通过检测荧光偏振值(Flourscence Polarization,FP)确定BRCA2基因突变.结果:建立了基于TDI-FP原理的突变检测方法,并对引物、dNTPs浓度、PCR反应循环数等进行了优化,对健康人及乳腺癌患者外周血标本的检测结果与PCR产物直接测序法结果完全吻合.结论:此方法可快速、高通量检测BRCA2基因突变,适用于临床诊断和大量人群筛查.
Abstract
AIM: To develop a rapid to detect BRCA2 gene mutation for large-scale screening. METHODS: The DNA fragment covering the mutation site was amplified with PCR method. The fluorescents labeled terminator (R110-acyGTP or TAMRA-acy TTP) was incorporated to the 3'-end of detecting probe via template-directed dye-terminator incorporation ( TDI) reaction. The mutation was detected by fluorescence polarization (FP) measurement. RESULTS: The new method has been established based on the TDI-FP platform. The concentration of primers and dNTPs and the number of PCR cycle have been optimized. To evaluate this method, genomic DNA samples from breast cancer patients and healthy controls were analyzed. The results were perfectly consistent with those determined by direct sequencing. CONCLUSION: This new method enables the high throughput, accurate and rapid detection of BRCA2 mutation. It can be used to large-scale screening in clinic laboratories.
关键词
实验诊断学/BRCA2/荧光偏振/乳腺癌/模板指导的荧光素标记碱基掺入反应Key words
laboratory diagnosis/BRCA2, fluorescence polarization/breast cancer/template-directed dye-terminator incorporation引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据