Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations.

Abstract

At present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed "constant denaturant gel electrophoresis" (CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was first tested with known p53 mutations in all four conserved regions. The CDGE technique was then used to screen 32 breast carcinomas that had been analyzed by immunohistochemical methods for altered p53 protein levels and whose DNA had already been shown to have loss of heterozygosity for a chromosome 17p marker. By immunostalning techniques, only 6 of the 32 tumors had elevated p53 expression. However, CDGE detected p53 mutations in 11 of the 32 tumors. DNA sequence analysis was performed to determine the nucleotide positions of these mutations in all 11 samples. Loss of heterozygosity for the pYNZ22 or pl44D6 markers did not associate with either the loss of heterozygosity at the p53 locus or the mutations detected by CDGE. We conclude that CDGE is a rapid and effective technique to screen for p53 mutations.A wide variety of human tumors have been shown to be associated with changes in the p53 gene (1-8). As the list of cancers with p53 mutations increases, there is a need for an accurate and rapid screening technique for these mutations. Unlike some of the dominant oncogenes, p53 can be inactivated by a diverse set of mutations scattered through several important regions of the gene (7). Many recent surveys of p53 mutations have used loss of heterozygosity (LOH) at polymorphic markers closely linked to p53 as an indirect assay for p53 inactivation (9-11). While such a screen is rapid, it cannot detect point mutations, and its accuracy is limited, since some of the nearby polymorphic markers might undergo deletions that do not extend into the p53 locus. Alternatively, because mutations in the p53 gene frequently result in a mutant p53 protein that is significantly overexpressed compared with the low levels of the wild-type protein, other p53 surveys have used immunostaining techniques to screen for mutations (12)(13)(14). Unfortunately, the p53 mutations that result in either a lack of protein or the same low levels of p53 as are present in cells containing the wild-type p53 cannot be detected by this method. The most sensitive screening technique is to sequence the genomic region encoding p53. Although sequencing is quite sensitive, it is also laborintensive. There are several new nucleic acid-based screening methods that can rapidly detect mutations within short fragments of DNA. These techniques include RNase protection assays (15), single-strand conformational polymorphisms (SSCP) (16, 17), denaturing gradient gel electrophoresis (DGGE) (18,19), and detection of base-pair mismatches with hydroxylamine and osmiu...

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“…Primer sequences, PCR and DGGE conditions used for each TP53 exon were those described by Agirre et al (2002) for exon 1-5 ASPP1 expression and regulation in ALL X Agirre et al and 9-11, Borresen et al (1991) for exons 7 and 8 and by Hamelin et al (1993) for exon 6. Negative controls (wild-type samples) and positive controls (known mutants) were included in each experiment.…”

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