고해상도 CE-SSCP 및 ligation 반응 기술을 이용한 다중 단일염기다형성 분석 시스템 개발

Abstract

Single nucleotide polymorphisms (SNPs) are defined as DNA sequence variation containing single base substitution, small deletions or insertions that are present in at least 1% of population. SNPs have been found to be associated with disease susceptibility. Notably, SNPs account for about 90% of all human genetic variations. Numerous researchers have devoted years to developing SNP genotyping methods as validation tool for disease-related SNP marker candidates and diagnostic method using them. For the development of clinically useful genotyping method for SNP markers, accuracy, simplicity, sensitivity, and cost-effectiveness are the most important criteria. Among the developed many detection methods to date, Sequencing-based methods and real-time PCR based methods are widely used for SNP genotyping. However, both have low multiplexing power. As alternatives, single-base extension based methods have been developed for multiplex analysis, but they suffer from complicated procedures or error-prone detection methods. Although ligation dependent methods are considered the simplest multiplex genotyping method for SNPs, multiplex assays using this method are limited by the detection method and mismatched hybridization cannot be often discriminated at the SNP sites. In addition, sensitivity is not guaranteed by the ligation reaction alone. Although capillary electrophoresis (CE) is an attractive alternative to error-prone hybridization-based detection, the multiplex assay process is complicated because of the size-based DNA separation principle.

In this dissertation, a novel SNP detection method by using improved ligation-based reaction coupled with high resolution CE-based single strand conformation polymorphism (SSCP) are developed as an accurate, sensitive and simple multiplex genotyping technology. To improve ligation reaction method, here we introduced various strategies to enhance accuracy, multiplex capacity, sensitivity, and precision of the analysis for a number of SNPs spread in the specific genes.

First, to improve the specificity of the ligation reaction for discrimination of mismatched hybridization at SNP sites, strategies using optimization of ligation reaction condition and thermostable ligase which have activity near the melting temperature of probes. As a result, mismatched hybridizations were efficiently discriminated and specific SNP allele targets were successfully detected by optimizing ligation time and ligase concentration.

Second, as strategies to improve the sensitivity and multiplexity of SNP detection, ligase detection reaction (LDR), ligase-dependent amplification, using three-different dyes labeled probes was introduced. The approaches are demonstrated that is sufficiently sensitive SNP detection without exponential amplification after ligation reaction and is a feasible validation tool for quantitative and multiplex population studies of SNPs.

Lastly, the precisely detection of ligation-based reaction for a number of SNPs spread in the specific genes defined as highly polymorphic regions was improved to be good enough for discrimination of multiple SNPs on a single codon. To accomplish this, we employed gap filling ligation reaction and gap-forming probes to target highly polymorphic regions. By this approaches, closely multiple highly polymorphic regions could be accurately and simply analyzed without non-specific hybridization.