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

✍ Scribed by Kokichi Sugano; Yuki Nakashima; Kensei Yamaguchi; Noriko Fukayama; Masato Maekawa; Hisanao Ohkura; Tadao Kakizoe; Takao Sekiya

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 590 KB
• Volume: 15
• Category: Article
• ISSN: 1045-2257
• DOI: 10.1002/(sici)1098-2264(199603)15:3<157::aid-gcc2>3.0.co;2-1

✦ Synopsis

We have developed a fluorescence-based single-strand conformation polymorphism analytis to detect Haelll-sensitive polymorphic sites in intron I of the 7/53 gene. It is important t o treat the PCR products with Klenow fragment t o 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 prOnles of two alleles with those of normal heterozygotes by data processing using computer software has enabled sensitive detection o f 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 (7 I .4 %) were positive for LOH at the Tf53 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.