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Sensitive detection of loss of heterozygosity in the TP53 gene in pancreatic adenocarcinoma by fluorescence‐based single‐strand conformation polymorphism analysis using blunt‐end DNA fragments
Author(s) -
Sugano Kokichi,
Nakashima Yuki,
Yamaguchi Kensei,
Fukayama Noriko,
Maekawa Masato,
Ohkura Hisanao,
Kakizoe Tadao,
Sekiya Takao
Publication year - 1996
Publication title -
genes, chromosomes and cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.754
H-Index - 119
eISSN - 1098-2264
pISSN - 1045-2257
DOI - 10.1002/(sici)1098-2264(199603)15:3<157::aid-gcc2>3.0.co;2-1
Subject(s) - loss of heterozygosity , microbiology and biotechnology , biology , locus (genetics) , haeiii , dna , allele , gene , genotype , genetics , restriction fragment length polymorphism
We have developed a fluorescence‐based single‐strand conformation polymorphism analysis to detect Hae III‐sensitive polymorphic sites in intron I of the TP53 gene. It is important to treat the PCR products with Klenow fragment to remove a 3′‐protruding nucleotide from the amplified DNA fragments added during the reaction in order to obtain a single peak for each allele. A comparison of the signal profiles of two alleles with those of normal heterozygotes by data processing using computer software has enabled sensitive detection of loss of heterozygosity (LOH) from clinical materials with a fraction of tumor cells below 10%. In analysis of 14 pancreatic carcinomas in which the proportion of the tumor cells is usually low due to the abundance of the stromal component, 7 samples (50%) were informative and 5 of the 7 (71.4%) were positive for LOH at the TP53 locus. This approach would be useful for allelotyping tumors with low cellularity, as well as other clinical samples such as biopsied specimens and paraffin embedded tissues. Genes Chromosom Cancer 15:157–164 (1996). © 1996 Wiley‐Liss, Inc.