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Open Access Highly Accessed Methodology article

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

James Butz1, Eric Wickstrom2 and Jeremy Edwards1*

Author Affiliations

1 Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA

2 Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, PA 19107, USA

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BMC Biotechnology 2003, 3:11  doi:10.1186/1472-6750-3-11

Published: 23 July 2003

Abstract

Background

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.

Results

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.

Conclusions

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.

Keywords:
Polony; immobilized PCR; pancreatic cancer; p53; K-ras2; mutations; loss of heterozygosity