Direct fluorescence detection of point mutations in human genomic DNA using microbead-based ligase chain reaction.
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.
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.
Xiangxian
X
Meng
Xiaohai
X
Yang
Kemin
K
Wang
Qiuping
Q
Guo
Yongjun
Y
Tan
Qiuhua
Q
Mo
Xiangmin
X
Xu
D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods*; D017354:Point Mutation; D012680:Sensitivity and Specificity; D055544:beta-Globins / Q000235:genetics*; D017086:beta-Thalassemia / Q000235:genetics
D055544:beta-Globins; D004247:DNA