Dumbbell-Shaped DNA Analytes Amplified by Polymerase Chain Reaction for Robust Single-Nucleotide Polymorphism Genotyping by Affinity Capillary Electrophoresis

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• 作者：Hideaki Shibata ; Atsushi Ogawa ; Naoki Kanayama ; Tohru Takarada ; Mizuo Maeda
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：June 4, 2013
• 年：2013
• 卷：85
• 期：11
• 页码：5347-5352
• 全文大小：319K
• 年卷期：v.85,no.11(June 4, 2013)
• ISSN：1520-6882

文摘

A sample preparation method was developed for single-nucleotide polymorphism (SNP) genotyping based on hybridization between a single-stranded DNA (ssDNA) analyte and an allele-specific oligonucleotide (ASO) probe. When the SNP site is located in the stable secondary structure, the folding of this analyte imposes kinetic penalties on the hybridization with the ASO probe. To address this issue, the sequence of the ssDNA analyte was converted from the original one so that the analyte exhibited a clear dumbbell-shaped structure composed of two stem鈥搇oop moieties and an unfolded probe-binding site. The as-prepared analyte was structurally favorable for hybridization with the ASO probe, irrespective of the original sequence and secondary structure of the analyte. The sequence conversion was easily achieved by polymerase chain reaction using forward and reverse primers having an additional sequence at the 5鈥?terminus. These ssDNA analytes were subjected to affinity capillary electrophoresis using a diblock copolymer probe composed of an ASO segment and a poly(ethylene glycol) segment. The 70-base dumbbell-shaped analytes with a single-base difference were clearly separated within 12 min, although the original ones exhibited almost no separation due to the undesired folding of the probe-binding site. This sample preparation method should open up a wide range of applications for the ASO probes in genetic analysis.