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1997 Fiscal Year Final Research Report Summary

Assessment of genetic changes in the carcinogenesis of squamous cell lung cancer using in situ PCR.

Research Project

Project/Area Number 07457286
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Thoracic surgery
Research InstitutionTohoku University

Principal Investigator

SATO Masami  Tohoku University, Institute of Development, Aging and Cancer, Instructor, 加齢医学研究所, 助手 (30250830)

Project Period (FY) 1995 – 1997
Keywordsin situ PCR / K-ras / point mutation / PCR / in situ hybridization / direct in situ PCR / indirect in situ PCR
Research Abstract

In situ PCR is a new technique for the localization of low copy number sequences. We investigated to establish a method for the in situ visualization of a point mutation in K-ras codon 12 by indirect in situ PCR.Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and postfixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated : TGT) and NCI-H460 (wild type : GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the seme preparation confirmed desired amplification.We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations. We also applied direct in situ PCR,but this method failed to detect the point mutation. Huge non-specific incorporation of digoxigenin to several length of DNA fragments caused low specificity of this technique.

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] Sato, et al.: "Sequential loss of hereozygosity in the progression of squmous cell carcinoma of the lung." British Journal of Cancer. (in press). (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sagawa, et al.: "K-ras point mutation occurs in the early stage of carcinogenesis in lung cancer." British Journal of Cancer. 77(5). 720-723 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sagawa, et al.: "Detection of K-ras point mutation by insitu PCR in cell suspensions." Lung Cancer. 17. 181-195 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sato, et al.: "PTENI is frequently mutated in primary endometrial carcinomas." Nature Genetics. 17. 143-144 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sato, et al.: "Sequential loss of heterozygosity in the progression of squamous cell carcinoma of the lung" British Journal of Cancer. (in press). (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sagawa, et al.: "K-ras point mutation occurs in the early stage of carcinogenesis in lung cancer" British Journal of Cancer. 77-5. 720-723 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sagawa, et al.: "Detection of k-ras point mutation by in situ PCR in cell suspensions" Lung Cancer. 17. 181-195 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sato, et al.: "PTENI is frequently mutated in primary endometrial carcinomas" Nature Genetics. 17. 143-144 (1997)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-16  

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