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Universal fluorescent tri-probe ligation equipped with capillary electrophoresis for targeting SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy
- Wang, Chun-Chi, Shih, Chi-Jen, Jong, Yuh-Jyh, Wu, Shou-Mei
- Analytica chimica acta 2014 v.833 pp. 40-47
- DNA, analytical chemistry, capillary electrophoresis, fluorescence, fluorescent dyes, genes, high performance liquid chromatography, motor neurons, muscular atrophy, single nucleotide polymorphism
- This is the first ligase chain reaction used for diagnosis of spinal muscular atrophy (SMA). Universal fluorescent tri-probe ligation (UFTPL), a novel strategy used for distinguishing the multi-nucleotide alternations at single base, is developed to quantitatively analyze the SMN1/SMN2 genes in diagnosis of SMA. Ligase chain reaction was performed by adding three probes including universal fluorescent probe, connecting probe and recognizing probe to differentiate single nucleotide polymorphisms in UFTPL. Our approach was based on the two UFTPL products of survival motor neuron 1 (SMN1) and SMN2 genes (the difference of 9 mer) and analyzed by capillary electrophoresis (CE). We successfully determined various gene dosages of SMN1 and SMN2 genes in homologous or heterologous subjects. By using the UFTPL-CE method, the SMN1 and SMN2 genes were fully resolved with the resolution of 2.16±0.37 (n=3). The r values of SMN1 and SMN2 regression curves over a range of 1–4 copies were above 0.9944. Of the 48 DNA samples, the data of gene dosages were corresponding to that analyzed by conformation sensitive CE and denatured high-performance liquid chromatography (DHPLC). This technique was found to be a good methodology for quantification or determination of the relative genes having multi-nucleotide variants at single base.