摘要
背景与目的 BIM基因编码的蛋白属于BCL2蛋白家族的成员,作为凋亡调控因子参与多种细胞活动。BIM基因上2,903bp的插入缺失片段与非小细胞肺癌EGFR-TKI靶向用药的获得性耐药相关。建立并优化HRM方法有助于快速、准确地检测BIM基因2,903 bp片段的缺失,为临床工作提供指导性的意见。本研究建立与稳定了HRM的检测方法,并在30例肺癌样本以及30例正常对照样本中检测BIM基因缺失情况。方法设计、合成针对BIM基因片段缺失的引物,优化高分辨率熔解曲线(high resolution melting,HRM)分析方法。并选取部分样本通过普通PCR方法和直接测序法检测BIM基因缺失情况。野生型模板扩增产物其解链温度要高于缺失型产物的解链温度。野生型和缺失型产物的熔解曲线可见明显差异,相应的Tm值相差约2.5 oC。结果经过HRM基因突变分析方法,在30例肺癌样本中检测到1例为BIM基因纯合缺失型,7例为杂合缺失型,22例为野生型。在30例正常对照样本中检测,2例为杂合缺失型,28例为野生型。结论本研究建立的对BIM基因片段缺失的高分辨率熔解曲线法是一种,灵敏、准确、快速、高通量的方法。
Background and objective The aim of this study is to establish a HRM （high resolution melting curve） method for detection of deletion in human BIM gene and to detect this site deletion with the above method in 30 lung cancer samples and 30 normal samples. Methods The primers for detection of BIM deletion were designed and synthesized. The HRM method for geng deletion was established. And select the part of samples to detect BIM delection by normal PCR and sequencing assay. The Tm value of wild type PCR products was higher than that of the deletion PCK products. The difference of the corresponding Tm value is 2.5 ℃. Results By detection with HRM methods, 1 samples were confirmed to be mutant, 7 samples were confirmed to be heterozygous and the other 22 samples were all wild type in the lung cancer samples. 2 samples were confirmed to be heterozygous and the other 28 samples were all wild type in the normal samples. Conclusion The HRM method for detection of BIM deletion established in this study is a sensitive, accurate, simple and high throughput method.
出处
《中国肺癌杂志》
CAS
北大核心
2014年第3期238-242,共5页
Chinese Journal of Lung Cancer
