Analysis of p53 mutations in single cells obtained from histological tissue sections

We have previously reported on direct sequence analysis of the p53 gene in laser-dissected single cells from tissue sections, where each allele of two fragments (exons 7 and 8) could be accurately analyzed in only 14% of the cells due to the high frequency of exon and allele dropout. Here in an effo rt to minimize this problem, we have investigated various approaches for sa mple preparation and gene amplification. By pinpointing some critical steps in the procedure, we could increase the number of investigated exons and s ubstantially improve the genetic analysis of single cells obtained from his tochemically stained frozen tissue sections. The biggest improvement was ac hieved by minimizing DNA degradation using EDTA as a nuclease inhibitor in all sample preparation steps. Efforts to increase primer annealing, by incr easing the concentration of template and primers, in addition to prolonging the annealing and extension times, also improved the amplification efficie ncy. With these measures we can now amplify six individual exons of the p53 gene (exons 4-9) in 70% of the cells and in 50% of these cells both allele s are amplified. This allows application of the method in various investiga tions such as within the held of tumor pathology. (C) 2000 Academic Press.