Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations.
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 immunostaining 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 p144D6 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.
Department of Genetics, Norwegian Radium Hospital, Oslo.
A L
AL
Børresen
E
E
Hovig
B
B
Smith-Sørensen
D
D
Malkin
S
S
Lystad
T I
TI
Andersen
J M
JM
Nesland
K J
KJ
Isselbacher
S H
SH
Friend
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D002886:Chromosomes, Human, Pair 17; D004252:DNA Mutational Analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D016158:Genes, p53; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D009841:Oligonucleotides; D016159:Tumor Suppressor Protein p53