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DNA polymorphic analysis from extremely small amounts of DNA samples using a semi-nested PCR

Research Project

Project/Area Number 04670352
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Legal medicine
Research InstitutionNagoya University

Principal Investigator

KATSUMATA Yoshinao  Nagoya University, Medicine, Professor, 医学部, 教授 (30109326)

Co-Investigator(Kenkyū-buntansha) UCHIHI Rieko  Nagoya University, Medicine, Assistant Professor, 医学部, 助手 (20223571)
Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1992: ¥1,600,000 (Direct Cost: ¥1,600,000)
KeywordsHLA-DQA1 / Semi-nested PCR / Sequence-specific Oligonucleotide Probe / Single Genome / Single Sperm / 法医鑑識 / 親子鑑定 / 犯罪搜査 / 白血球型 / 血液型 / 遺伝子増幅 / 二段階遺伝子増幅 / ドットブロットハイブリダイゼーション
Research Abstract

Since jeffreys et al. reported that DNA with hypervariable minisatellite loci could be used for identification of individuals, DNA typing systems have been developed. Large amounts(micrograms)of gemonic DNA must be analyzed in these methods, so application of these methods to forensic science samples, consisting of degraded or very small amounts of DNA, is not always possible. The polymerase chain reaction(PCR)procedure has provided a solution to this problem, enabling the amplification of a target DNA region by 100,000 times. However, one finds that the large number of PCR cycles required to amplify extremely small amounts of DNA such as a single sperm cell makes for even less efficiency of the reaction, often resulting in insufficient amounts of PCR products. The nested PCR method is known to increase the amplification sensitivity and can be applied to various DNA analyzes.
In the present study, we have improved the procedure of DNA purification or PCR to minimize the inhibition of the contaminated substances. Furthermore, we devised a new PCR system with a semi-nested set of primers in the second PCR to improve the amplification sensitivity of HLA-DQAI gene.
Using the semi-nested PCR, more than 2 or 3 pg of template DNA could be amplified and typed correctly. The semi-nested PCR technique was found to enhance the sensitivity and efficiency of the amplification reaction and allowed the successful typing of the HLA-DQA1 gene. This is helpful for genotyping from samples with extremely small amounts of DNA, such as forensic or ancient DNA samples.

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (15 results)

All Other

All Publications (15 results)

  • [Publications] Uchihi,R.et al.: "Deoxyribonucleic acid(DNA)typing of human leukocyte antigen(HLA)-DQA1 from single hairs in Japanese." Journal of Forensic Sciences. 37(3). 853-859 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 玉木敬二ら: "医学におけるバイオテクノロジー(17)-DNAによる個人識別法-." 現代医学. 40(1). 169-174 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Okajima,H.et al.: "Amplification of HLA-DQA1 gene from bloodstains by polymerase chain reaction." Japanese Journal of Legal Medicine. 47(1). 6-12 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 小島俊典ら: "唾液斑痕を試料としたHLAクラスII遺伝子のDNA型判定." 日本法医学雑誌. 47(5). 380-386 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Rieko Uchihi, Keiji Tamaki, Toshinori Kojima, Toshimichi Yamamoto and Yoshinao Katsumata: "Deoxyribonucleic acid(DNA)typing of human leukocyte antigen(HLA)-DQA1 from single hairs in Japanese" Journal of Forensic Sciences. 37(3). 853-859 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Keiji Tamaki, Rieko Uchihi, and Yoshinao Katsumata: "Biotechnology in medicine(17)-Personal identification by DNA-" Gendaiigaku. 40(1). 169-174 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hiroshi Okajima, Toshimichi Yamamoto, Toshinori Kojima, Rieko Uchihi and Yoshinao Katsumata: "Amplification of HLA-DQA1 gene from bloodstains by Polymerase chain reaction" Japanese Journal of Legal Medicine. 47(1). 6-12 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Toshinori Kojima, Rieko Uchihi, Toshimichi Yamamoto, Keiji Tamaki and Yoshinao Katsumata: "DNA typing of the three HLA-class II ioci from saliva stains" Japanese Journal of Legal Medicine. 47(5). 380-386 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Uchihi,R.et al.: "Deoxyribonucleic acid(DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs in Japanese." Journal of Forensic Sciences. 37(3). 853-859 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] 玉木敬二ら: "医学におけるバイオテクノロジー(17)-DNAによる個人識別法-" 現代医学. 40(1). 169-174 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Okajima,H.et al.: "Amplification of HLA-DQA1 gene from bloodstains by polymerase chain reaction." Japanese Journal of Legal Medicine. 47(1). 6-12 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 小島俊典ら: "唾液斑痕を試料としたHLAクラスII遺伝子のDNA型判定" 日本法医学雑誌. 47(5). 380-386 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 玉木 敬二: "日本におけるバイオテクノロジー(17)ーDNAによる個人識別法ー" 現代医学. 40. 169-174 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 玉木 敬二: "MVR-PCR法を用いた日本人におけるDIS8(MS32)のタイピングとアリルの解析" 日本法医学雑誌. 46. 474-482 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Hiroshi Okajima: "Amplification of HLA-DQAI gene form bloodstains by polymerase chain reaction" Jpn. J. Legal Med.47. 6-12 (1993)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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