A chemical mismatch cleavage method useful for the detection of point mutations in the p53 gene in lung cancer.

D. T. Curiel, D. L. Buchhagen, I. Chiba, D. D'Amico, T. Takahashi, J. D. Minna

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Abstract

Point mutations in genes can be etiologic of pulmonary diseases, as in the case of the inherited disorders alpha-1-antitrypsin deficiency and cystic fibrosis or in the context of dominant and recessive oncogenes in lung cancer. Various methodologies have been developed to screen for single-base mutations. These techniques include direct DNA sequencing, RNase protection, denaturing gradient gel electrophoresis, and chemical mismatch cleavage. The latter method offers the advantages of rapid and efficient analysis of genomic or cDNA and is thus ideally suited to screening applications. Furthermore, all possible single-base changes can theoretically be detected. In the present work, chemical mismatch cleavage was utilized to detect mutations in the p53 gene in small cell and non-small cell lung cancer. This technique was modified by using a two-step, hemi-nested PCR procedure for preparation of target genomic DNAs permitting an expanded target size for analysis. Evaluation by chemical mismatch cleavage of eight p53 cDNAs derived from lung tumors shown to have different mutations by DNA sequencing correctly detected the presence of a point mutation in all instances. Analysis of six additional tumor genomic DNAs with defined mutations in the corresponding p53 cDNAs accurately confirmed the mutation at the level of the genome. The technique also identified codon 72 and intron 6 polymorphisms. Using the intron 6 polymorphism, loss of heterozygosity at the p53 locus in tumor DNA was readily detected by chemical mismatch cleavage. Finally, utilizing this technique for scanning analysis of the p53 gene of uncharacterized lung tumor DNAs, additional mutations were identified in a prospective manner which were confirmed by sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Original languageEnglish (US)
Pages (from-to)405-411
Number of pages7
JournalAmerican journal of respiratory cell and molecular biology
Volume3
Issue number5
DOIs
StatePublished - Nov 1990

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ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

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