State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Biomedical Engineering Center, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, PR China.
Talanta. 2010 Mar 15;80(5):1725-9. doi: 10.1016/j.talanta.2009.10.013. Epub 2009 Oct 14.
This report has described a convenient genotyping method capable of detecting point mutations directly in human genomic DNA based on the combination of ligase chain reaction (LCR) and microbead-enrichment technique. LCR primers, including a biotin-labeled common primer and two fluorescence-labeled allele-specific primers, are designed for two alleles of a mutated site. When genomic DNA carries the mutated site, the common primer and allele-specific primer are ligated to form exponential amplified biotin-labeled fluorescence ligation products. These ligated products are enriched by streptavidin-coated microbeads, and genotypes are identified conveniently according to the fluorescence color of microbeads using fluorescent microscopy. Due to amplification of LCR process and enrichment of microbeads, the detection limit of the proposed method is as low as 10(-15)mol/L templates. The method provides a convenient and simple strategy to detect point mutation directly in human genome. We have confirmed the efficiency of this approach with the identification of beta-globin gene point mutation, which results in the reduced production of globin in an inherited hemoglobin disorder thalassemia disease.
本报告描述了一种简便的基因分型方法，该方法基于连接酶链反应（LCR）和微珠富集技术，能够直接检测人基因组 DNA 中的点突变。LCR 引物包括一个生物素标记的通用引物和两个荧光标记的等位基因特异性引物，用于突变位点的两个等位基因。当基因组 DNA 携带突变位点时，通用引物和等位基因特异性引物连接形成指数扩增的生物素标记荧光连接产物。这些连接产物通过链霉亲和素包被的微珠富集，并使用荧光显微镜根据微珠的荧光颜色方便地鉴定基因型。由于 LCR 过程的扩增和微珠的富集，该方法的检测限低至 10(-15)mol/L 模板。该方法为直接检测人类基因组中的点突变提供了一种简便的策略。我们已经通过鉴定导致遗传性血红蛋白病地中海贫血中珠蛋白产量减少的β-珠蛋白基因突变证实了该方法的有效性。
