Genetic analysis of multiple loci in microsamples of fixed paraffin-embedded tissue.
Molecular analysis of alterations in genomic DNA is essential for understanding mechanisms by which chemical agents induce or modify tumor development. The assessment of microsatellite polymorphisms, loss of heterozygosity, mutations, and gene rearrangement allows specific comparisons of tumors to premalignant lesions or normal tissue or between similar tumors seen in laboratory species and humans. Utilization of these techniques is frequently limited by minute quantities of available tissue, often restricted to small formalin-fixed tumors or biopsies in paraffin blocks. To address these limitations, we have combined recently developed methodologies for selective recovery, amplification, and analysis of DNA. These techniques provide sufficient materials of high quality for analysis of DNA alterations in microscale amounts of starting material. By combining whole genome amplification through primer extension preamplification with locus-specific heminested PCR, we are able to analyze multiple genetic loci from as little as 1 mm2 of a 3-micron-thick formalin-fixed paraffin section. From 10 to greater than 100 loci can be analyzed per tissue section, and locus-specific PCR products may be further evaluated by a variety of techniques (e.g., SSCP, sequencing). Integrating these methodologies into situations where evaluation of very small tissue samples is necessary provides a powerful approach for elucidating molecular events that may be causally related to chemically induced cellular transformation and tumorigenesis.
Department of Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Michigan 48105, USA.
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D000818:Animals; D004247:DNA / Q000032:analysis*; D066298:In Vitro Techniques; D008099:Liver / Q000737:chemistry; D008297:Male; D051379:Mice; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA
D004247:DNA