Børresen A L, Hovig E, Smith-Sørensen B, Malkin D, Lystad S, Andersen T I, Nesland J M, Isselbacher K J, Friend S H
Department of Genetics, Norwegian Radium Hospital, Oslo.
Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8405-9. doi: 10.1073/pnas.88.19.8405.
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.
目前，p53基因的突变似乎是人类癌症中最常见的基因改变。这些突变可发生在该基因的许多不同区域。我们开发了一种变性梯度凝胶电泳的改良方法，称为“恒定变性剂凝胶电泳”（CDGE），它提供了一种快速且灵敏的方法来筛查p53基因内的四个保守区域，据报道大多数p53突变都发生在这些区域。首先用所有四个保守区域中的已知p53突变来测试CDGE的灵敏度。然后使用CDGE技术对32例乳腺癌进行筛查，这些乳腺癌已通过免疫组化方法分析了p53蛋白水平的改变，并且其DNA已显示在17号染色体p标记处存在杂合性缺失。通过免疫染色技术，32例肿瘤中只有6例p53表达升高。然而，CDGE在32例肿瘤中的11例中检测到p53突变。对所有11个样本进行了DNA序列分析，以确定这些突变的核苷酸位置。pYNZ22或p144D6标记的杂合性缺失与p53基因座处的杂合性缺失或CDGE检测到的突变均无关。我们得出结论，CDGE是一种快速有效的筛查p53突变的技术。
