1996 Fiscal Year Final Research Report Summary
The development of DNA diagnosis by PCR and Capillary electrophoresis
| Project/Area Number |
07672326
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Showa University |
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Principal Investigator |
ARAKAWA Hidetoshi Showa University, School of Pharmaceutical Sciences Associate Professor, 薬学部, 助教授 (70129807)
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| Co-Investigator(Kenkyū-buntansha) |
MAEDA Masako Showa University, School of Pharmaceutical Sciences Professr, 薬学部, 教授 (00053869)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Capillary electrophoresis / DNA diagnosis / PCR / Double strand DNA / SSCP |
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| Research Abstract |
Capillary electrophoresis (CE) was studied for the analysis of PCR amplified sample. Alowcross linked polyacrylamide gel and entangled polymer solution (polymer solution) were used for CE.Laser induced fluorescence (LIF) detection was performed using Thiazole orange as the fluorescent intercalating dye. The highly sensitive LIF-CE was applied to detection of allele specific PCR for medium-chain acyl-CoA dehydrogenase deficiency and phenylketonuria mutation, and of PCR-restriction fragment length polymorphism for mutant dive E gene. Analysis of single strand conformation polymorphism (SSCP) by CE was also developed. The conformational change of the single strand DNA is caused by a mutation in DNA fragment. The change is detected as mobility shift on CE.The effects of acrylamide gel concentration, running-temperature and fragment size amplified by PCR were studied to develop the separation of SSCP.The results obtained in this report showed that CE is well suited for clinical DNA analysis using PCR.Analysis of SSCP by laserinduced fluorescence capillary electrophoresis in entangled polymer solution (CE-LIF) has benn developed also in this study. K-ras genes including seven mutations were amplified with primer labeled with Texas Red at its 5' end. The labeled PCR products were separated with capillary gel electrophoresis and He-Ne laser-excited fluorescence detection. All fragments having normal (Gly) and mutated (Ala, Arg, Cys, Ser, Val, Asp) sequences at codon 12 can be distinguished by this method. Further more, SSCP analysis was carried out with multiple sheath-flow gel capillary-array electrophoresis to analyze many samples simultaneously. CE-LIF is well suited for clinical analysis of SSCPs because of its high sensitivity, resolution, reproducibility and speed.
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