|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 2003 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 2002 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Real-time PCR methods using AS-TaqMan PCR, TaqMan PCR with MBG probe, melting curve analyses and mismatch amplification assay were developed.
1.I have developed AS-TaqMan PCR assay and SYBR Green PCR assay for detecting of glycophorin A (GYPA), low density lipoprotein receptor (LDLR), hemoglobin G (HBGG), D7S8 and group specific component (GC) alleles which are typed by AmpliType PM + OQA1 PCR Amplification and Typing. The differences of threshold cycles (Ct) values between different genotypes on each of the loci were statistically differed significantly. Further analysis was carried out about GYPA, because it shows a strong correlation between glycophorin A and MN blood group. Then, AS-TaqMan PCR assay and SYBR Green PCR assay for genotyping of MN blood group have developed.
2.TaqMan PCR with MBG hybridization probe for ABO genotyping has developed. The base deletion at 261^<st> base substitutions at 703^<rd>, 796^<th> and 930^<th> of cDNA of ABO glycosyltransferase were detected by TaqMan PCR with MBG probe.
3.I have developed the hybridization probe assay to rapidly detect the base deletion at 261^<st> and base substitutions at 793^<rd> and 930^<th> of cDNA of ABO glycosyltransferase. Two fluorescent-labeled hybridization probe were designed and detection of deletion and substitutions were performed by melting curve analyses.
4.MN genotyping by mismatch amplification assay and melting curve analyses have developed. Mismatch amplification assay involves mismatched PCR primers that have different binding efficiencies within a real time PCR. MN genotyping by melting curve analyses has also developed.
Developed real-time PCR assays are simple, rapid, and accurate, as well as suitable for high-throughput applications.