Polymerase chain reaction and in situ hybridization: applications in toxicological pathology.
Polymerase chain reaction (PCR) and in situ hybridization (ISH) have revolutionized the study of genes and gene expression, and many of these molecular biology advances will greatly impact research in toxicological pathology. PCR is one of the most powerful tools in molecular biology and involves primer-mediated enzymatic in vitro amplification of specific target DNA sequences. Recent innovative methods utilizing PCR technology have been developed to detect mutations in neoplastic and small subpopulations of cells, to study biomarkers of genetic susceptibility and genes involved with carcinogen metabolism, to estimate mutation frequencies, to find novel genes induced by chemical exposure, and to characterize gene expression. ISH provides data on individual cells rather than an average of total cellular populations and allows analysis for heterogeneity. When combined with PCR, the sensitivity of ISH is elevated, and single-copy DNA sequences, single-base mutations, or low copies of messenger RNA (mRNA) can potentially be detected within individual cells. Herein are reviewed ISH- and PCR-based techniques such as single-strand conformation polymorphism analysis to detect point mutations, allelotypic analysis for loss of heterozygosity, differential display of mRNA to characterize gene expression, quantitative reverse transcriptase polymerase chain reaction, and in situ polymerase chain reaction with emphasis on current or potential applications in toxicological pathology. These new and evolving techniques offer tremendous potential in providing new insights into the molecular basis of toxicity and carcinogenesis.