构建检测心肌肌钙蛋白Ⅰ基因Arg145Trp突变的寡核苷酸探针

Developing oligonucletides probes to detect Arg145Trp mutation of cardiac troponin I gene

目的:等位基因特异性寡核苷酸杂交是基于分子杂交原理而建立的经典的突变检测方法,也是DNA芯片技术的重要组成部分,常用于突变筛查。实验拟建立一种适合快速大规模检测心肌肌钙蛋白Ⅰ基因Arg145Trp突变(C4693T)的方法。方法:实验于2002-07/2004-06在南京医科大学第一附属医院心血管病研究所临床生物学诊断与治疗实验室完成。直接测序研究证实的具有cTnⅠ基因Arg145Trp突变的肥厚型心肌病患者2例,正常对照1名,受试者对实验均知情同意。自外周血白细胞抽提DNA,聚合酶链反应扩增心肌肌钙蛋白Ⅰ基因含有突变位点的外显子,点于硝纤膜上,分别在不同的温度条件(48,52,56℃)下再与生物素标记的突变及野生探针杂交,摸索最佳杂交条件。根据最佳杂交条件,分别在100倍浓度无关探针条件下行非特异性竞争杂交,在100倍浓度未标记的特异寡核苷酸探针条件下行特异性竞争杂交。结果:采用碱性磷酸酶标记的链霉亲和素和NBT/BCIP观察杂交结果:①突变及野生探针在48℃杂交时,除阴性对照外,2个患者及正常对照样本均见明显阳性信号。②在52℃及56℃条件下突变探针正确检出两个突变样本,但以56℃结果最佳。③100倍浓度无关探针杂交存在时,对生物素标记的突变寡核苷酸探针杂交结果没有影响,2个患者样本均见明显阳性信号。100倍浓度未标记的突变寡核苷酸探针存在时杂交,完全抑制生物素标记的突变寡核苷酸探针杂交结果。结论:所设计的寡核苷酸探针能够检测出肥厚型心肌病患者心肌肌钙蛋白Ⅰ基因Arg145Trp突变(C4693T),适合该突变的快速大规模筛选。

AIM： Allele specific oligonucleotide hybridization is a mutation detection method based on molecular hybridization principles, and a key component of DNA array technique to screen mutation. In this study, we developed a method based on the principles of allele-specific oligonucleotide hybridization, which is suitable for rapid large-scale screening of cardiac troponin Ⅰ gene Arg145Trp mutation （C4693T） in patients with hypertrophic cardiomyopathy. METHODS： From July 2002 to June 2004, the experiment was performed at the Clinical Bio Diagnostic ＆ Bio Therapeutic Laboratory, Research Institute of Cardiovascular Diseases, First Affiliated Hospital of Nanjing Medical University. Two patients （with Arg145Trp mutation） with hypertrophic cardiomyopathy and l normal control were enrolled. Informed consents were obtained from all subjects. Genomic DNA was extracted from their peripheral blood leukocytes. The mutation-bearing exon of cardiac troponin I gene was amplified by PCR. The amplification products were dropped on nitrocellulose membrane, and then hybridized with two biotin-labeled oligonucletides probes, complementary to normal or mutant sequence, at different temperature conditions, respectively to identify optimal hybridization condition. RESULTS： The streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium detection results showed that ①the designed oligonuclefides probe complementary to mutant sequence successfully detected Arg145Trp mutation in the patients at 48 ℃. ②Two mutation samples were detected both at 52℃ and 56℃, but the latter was better. ③Hybridization of biotin-labeled oligonucletides probes was not influenced when the concentration of unspecific probe was 100 times higher than that of the biotin-labeled mutant probe. Positive signals were found in samples of two patients. Hybridization was inhibited completely when the concentration of unlabeled mutant probe was 100 times higher than that of the biotin-labeled mutant probe. CONCLUSION： This synthesized oligonucletides probe is suitable for rapid large-scale screening of Arg145Trp mutation （C4693T） in patients with hypertrophic cardiomyopathy.