摘要
目的 建立一种简便快速筛查非缺失型α-地中海贫血点突变的反向点杂交 (reverse dotblot,RDB)技术。方法 将生物素直接标记在引物上 ,选择性扩增人α2珠蛋白基因 ,将 PCR产物与固化在膜条上的寡核苷酸探针杂交 ,洗膜、显色后分析结果。结果 采用生物素标记的引物 Bio- C1和 Bio- C3可选择性扩增α2珠蛋白基因 ,PCR产物长度为 10 85 bp,该标记片段与固相探针构成的反向点杂交检测体系 ,可以分辨出中国人群中已知的 6种非缺失型α-地中海贫血点突变。结论 该反向点杂交检测体系无需巢式 PCR扩增 ,一步选择性扩增的α2珠蛋白基因产物即可用于杂交 ;无需进行检测标记物 (如生物素 )反应 ,而将生物素直接标记在引物上 ;无需不同的洗膜条件 ,故较已报道的同类方法更为简单易行。RDB技术可用于快速诊断中国人非缺失型α-地中海贫血点突变。
Objective: To establish a rapid and convenient method of reverse dot blot (RDB) analysis for detecting the point mutations of non-deletion α-thalassemia in Chinese. Methods: Label biotin to primers and amplify human α2 globin gene selectively, then hybridize PCR products with a set of oligonucleotide probes immobilized on strips, and develop colour to detect non-deletion α-thalassemia defects. Results: The PCR system using biotin-labeled primers could specifically amplify a 1085 bp fragment of human α2 globin gene which encompasses all six α-thalassemia mutations. After being hybridized with sequence-specific oligonucleotide probes and colour development, it could simultaneously identify all six types of non-deletion α-thalassemias encountered in Chinese. Conclusion This method does not need semi-nested PCR, and the products amplified by biotinylated primers can be used directly to hybridize with the probes on strips under the identical conditions of hybridization. So, it is a specific and multiplex detection assay for screening non-deletion α-thalassemia defects in Chinese.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
2003年第4期345-347,共3页
Chinese Journal of Medical Genetics
基金
国家重点基础研究发展规划" 973"项目(2 0 0 1 CB51 0 30 8)
广东省科委和广东省卫生厅联合攻关重点项目(99B0 670 4 G)~~
