1993 Fiscal Year Final Research Report Summary
Investigation on dual PCR technique and its application to sex determination of forensic specimens
| Project/Area Number |
03807035
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Legal medicine
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| Research Institution | Shimane Medical University |
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Principal Investigator |
MATSUBARA Kazuo Shimane Medical University, School of Medicine, Associate Professor, 医学部, 助教授 (20127533)
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| Co-Investigator(Kenkyū-buntansha) |
SHIONO Hiroshi Asahikawa Medical College, School of Medicine, Professor, 医学部, 教授 (20112451)
AKANE Atsushi Shimane Medical University, School of Medicine, Instructor, 医学部, 助手 (70202520)
TAKAHASHI Setsunori Shimane Medical University, School of Medicine, Instructor, 医学部, 助手 (90032226)
KIMURA Kojiro Shimane Medical University, School of Medicine, Professor, 医学部, 教授 (30153191)
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| Project Period (FY) |
1991 – 1993
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| Keywords | DNA analysis / Sex determination / Polymerase chain reaction / Sex chromosomes / Amelogenin gene / Degradation of DNA / Hemoglobin / Hematin |
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| Research Abstract |
Sex determination by PCR amplification of X-Y homelogous amelogenin gene is quite informative since amplified X gene acts as an internal control. However, more than 250 ng templates are required to amplify single-copy amelogenin gene. By dual PCR amplification method, the gene was amplifiable from only 5 pg pure template DNA, but no PCR product was detected from contaminated and/or degraded forensic specimens.
When high molecular weight templates were recovered from such degraded samples by electroelution method, amelogenin gene was amplifiable by dual PCR method. The contaminant was identifide as the metabolite of hemoglobin like hematin, because pyridine hemochromogen analysis of the contaminant revealed the heme-specific absorbance. Furthermore, spectrophotometric and electrophoretic properties and inhibition ability of PCR indicated that the contaminant should be the proteinase K-digest of heme and some serum protein complex.
This contaminant and degraded DNA fragments were removable by Bio-Gel A-150m column chromatography. Following purification by the gel filtration, sexing was thus possible from such contaminatede and degraded forensic DNA samples.
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