A multiplex single nucleotide polymorphism genotyping method using ligase-based mismatch discrimination and CE-SSCP.
Accuracy, simplicity, and cost-effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase-based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray-based detection, the design process and multiplex assay procedure are complicated because of the DNA size-dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition-optimized ligation and high-resolution CE-based SSCP. With this high-resolution CE-SSCP system, we are able to use similar-sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single-base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
Woong
W
Choi
Gi Won
GW
Shin
Hee Sung
HS
Hwang
Seung Pil
SP
Pack
Gyu Yong
GY
Jung
Gyoo Yeol
GY
Jung
D020137:Base Pair Mismatch; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D018807:Polymorphism, Single-Stranded Conformational; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D016159:Tumor Suppressor Protein p53; D011088:DNA Ligases
CE-SSCP; Diagnostic assay; Ligase-based genotyping assay; Multiplex analysis; SNP