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

We have developed a fluorescence-based single-strand conformation poly morphism analysis to detect HaeIII-sensitive polymorphic sites in intr on I of the TP53 gene. It is important to treat the PCR products with Klenow fragment to remove a 3'-protruding nucleotide from the amplifie d DNA fragments added during the reaction in order to obtain a single peak for each allele. A comparison of the signal profiles of two allel es with those of normal heterozygotes by data processing using compute r 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 th e tumor cells is usually low due to the abundance of the stromal compo nent, 7 samples (50%) were informative and 5 of the 7 (71.4%) were pos itive for LOH at the TP53 locus. This approach would be useful for all elotyping tumors with low cellularity, as well as other clinical sampl es such as biopsied specimens and paraffin embedded tissues. (C) 1996 Wiley-Liss, Inc.