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

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.

Figure 1

The second position of codon 12 in K-ras2 of Panc-1 genomic DNA is heterozygous with roughly half the alleles being wildtype {G} and with the other half being mutant {A}. (A) All K-ras2 exon1 polonies are shown following Sybr Green I staining. (B) After the polonies were made single-stranded, a sequencing primer was hybridized to the polony and the position was shown to be heterozygous by performing independent single base extensions with Cy-5 labeled: dATP (blue) and dGTP (red). Extensions with Cy-5 labeled dCTP and dUTP did not yield any significant signal (data not shown). Colors are artificial in both images.

Figure 2

The second position of codon 273 in p53 of Panc-1 is mutated, possessing an "A" instead of a "G". (A) All p53 exon 8 polonies are shown following Sybr Green I staining. (B) The position was shown to harbor a G → A mutation by performing independent single base extensions with Cy-5 labeled: dATP (blue) and dGTP (red). Extensions with Cy-5 labeled dCTP and dUTP did not yield any significant signal (data not shown). Colors are artificial in both images.

Figure 3

Loss of heterozygosity analysis of K-ras2 and p53 in Panc-1. There are approximately twice as many (A) K-ras2 polonies than (B) p53 polonies following the polony amplification of equal amounts of genomic DNA, likely indicating that one of two p53 alleles was lost. These results, coupled with the sequencing of mutational hotspots in these same genes, demonstrate that p53 experienced LOH.