Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

• Published in: BMC biotechnology 2003-07, Vol.3 (1), p.11-11, Article 11
• Main Authors: Butz, James, Wickstrom, Eric, Edwards, Jeremy
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor; Codons; copy number; DNA Mutational Analysis; DNA, Neoplasm - chemistry; DNA, Neoplasm - genetics; Gene deletion; Genes, ras - genetics; Hot spots; Humans; immobilized PCR; K-ras2; Loss of Heterozygosity; Methodology; Mutation; mutations; p53; p53 protein; pancreatic cancer; Pancreatic Neoplasms - genetics; Pancreatic Neoplasms - pathology; Polony; Polymerase chain reaction; Polymerase Chain Reaction - methods; Q1; Tumor Suppressor Protein p53 - genetics
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/1472-6750-3-11

The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH), in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2. Using polony technology, Panc-1 was determined to possess only one copy of p53, which possessed a mutation in codon 273, and two copies of K-ras2, one wildtype and one with a mutation in codon 12. To further demonstrate the general approach of this method, polonies were also used to detect K-ras2 mutations in the pancreatic cell lines, AsPc-1 and CAPAN-1. In conclusion, we have developed an assay that can detect mutations in hotspots of p53 and K-ras2 as well as diagnose LOH in these same genes.