Establishment of a high throughput single-nucleotide-polymorphisms (SNPs) screening system based on the re-annealing and digestion reaction
| Project/Area Number |
15590592
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Legal medicine
|
|---|
| Research Institution | Kansai Medical University |
|---|
Principal Investigator |
AKANE Atsushi Kansai Medical University, Faculty of Medicine, Professor, 医学部, 教授 (70202520)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YOSHIDA Manabu Kansai Medical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (20122004)
OKII Yutaka Kansai Medical University, Faculty of Medicine, Assistant, 医学部, 助手 (20121915)
YOSHIMURA Sumitaka Kansai Medical University, Faculty of Medicine, Assistant, 医学部, 助手 (70167005)
|
|---|
| Project Period (FY) |
2003 – 2006
|
| Project Status |
Completed (Fiscal Year 2006)
|
| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2006: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2005: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 2004: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
|
|---|
| Keywords | Se blood group / ABO blood group / mitochondrial DNA / temperature-gradient gel / heteroduplex / homoduplex / electrophoresis / allele / single nucleotide polymorphism (SNP) / 再会合 |
|---|
| Research Abstract |
Se blood group is important in the forensic field because it is associated with the secretion of ABO blood group antigens in body fluids. In this study, first, Se1, Se2, sej and se alleles of the Se blood group which are common in Japanese population were genotyped using the temperature-gradient gel electrophoresis method. Heteroduplex bands occurred in the heterozygotic samples showed genotype-specific electrophoresis patterns. When one mismatched nucleotide existed, one or two heteroduplex bands were found in the gel. In the samples with two mismatched bases, four heteroduplex bands were observed. These findings suggested that numbers of the mismatched nucleotides could be estimated even when an unknown allele was analyzed by the method.
This method was applied to the genotyping of ABO blood group. When exons 6 and 7 of the four typical alleles, A, B, O^<A-> and 0G were analyzed, the homoduplex bands showed allele-specific electrophoresis patterns due to difference in the GC contents. Different heteroduplex bands could be detected after mixture of different alleles, so that ABO blood group could be genotyped by this method.
This method was also applied to identify the htpervariable region 1 of mitochondrial DNA (mtDNA HV1). Homoduplex bands of mtDNA HV1 showed different electrophoresis patterns, and mixture of different samples revealed specific heteroduplex bands, so that mtDNA HV1 variants could be identified by this method.
Mismatched nucleotides in heteroduplex bands could not be cleaved by means of the reported techniques. So, the temperature-gradient gel electrophoresis was the most suitable, high throughput screening method to detect single nucleotide polymorphisms (SNPs) in alleles and variants.
|
Report
(5 results)
Research Products
(23 results)
