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Specific Gene Capture Combined with Restriction-Fragment Release for Directly Fluorescent Genotyping of Single-Nucleotide Polymorphisms in Diagnosing Spinal Muscular Atrophy
- Wang, Chun-Chi, Chen, Chung-An, Jong, Yuh-Jyh, Kou, Hwang-Shang
- Analytical chemistry 2018 v.90 no.19 pp. 11599-11606
- DNA, diagnostic techniques, fluorescence, fluorescent labeling, genes, genotype, genotyping, magnetism, medicine, muscular atrophy, nucleotides, patients, polymerase chain reaction, restriction endonucleases, single nucleotide polymorphism, streptavidin
- In this study, a fast and simple fluorescent genotyping strategy, streptavidin magnetic beads combined with biotin-coupled PCR and restriction-fragment release, was developed for determination of nucleotide variants. This method was further applied for analyzing SMN1 gene in diagnosis of spinal muscular atrophy (SMA). After biotin-coupled PCR, the streptavidin magnetic beads would capture the biotin-labeled SMN genetic fragments, and then the restriction enzyme of HPY188I could only digest and release the fluorescent end of SMN1 genetic fragment into the supernatant. Therefore, the SMN1 gene could be easily fluorescently quantified, and SMN2 would not, for diagnosis of SMA. The copy number of the SMN1 gene could be regressed using the relative fluorescent unit versus the known copy number, and the coefficient of correlation is equal to 0.9617 (r = 0.9617). In this research, a total of 16 blind DNA samples were analyzed, including 6 wild types, 5 carriers, and 5 SMA patients. Importantly, this fast, simple, and highly efficient method is universal for detection of all nucleotides variants by replacing the specific restriction enzyme. This technique has the potency to be served as a tool for fast and accurate diagnosis of genotypes in clinical medicine.