[PCR: DNA amplification from histological sections].
Specific DNA sequences can be amplified from tissue material by means of the polymerase chain reaction (PCR) using oligonucleotides homologous to upstream and downstream flanking regions as primers for repeated cycles of Taq polymerase-mediated DNA synthesis (primer extension) in vitro. The amplification product provides the unique possibility to analyze genomic alterations (mutations, deletions, translocations) which may play a role during pathogenetic processes, or to detect heterologous (viral, bacterial) nucleic acids with maximum sensitivity. PCR with morphologically defined material from histologic sections gives the chance to bridge the gap between morphological description of a disease and the underlying molecular alteration. PCR from sections can be performed even from paraffin-embedded material of archival specimens. As an example a ras gene mutation analysis of human colorectal cancers and their metastasis and of human seminomas is presented. Only minute amounts of biological material are required for PCR, as exemplified with material punched from defined preneoplastic areas in rat liver cryostat sections. Using this material, not only a thorough mutational analysis of DNA of preneoplastic foci is possible after a simultaneous PCR amplification of various genomic sequences, but also an investigation of transcription activity after reverse transcription of mRNA into cDNA, as shown for c-myc expression during preneoplasia. The extremely high sensitivity of the method requires severe precaution with respect to contamination, and product control by Southern blots or sequencing. PCR from histological sections will become a valuable tool for analyzing molecular mechanisms of disease based on the classical morphological parameters of pathology.
Rabes
H
H
Pathologisches Institut, Universität München.
Suchy
B
B
Ostermayr
R
R
Zietz
C
C
Waldmann
V
V
D000818:Animals; D002872:Chromosome Deletion; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D006801:Humans; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D014178:Translocation, Genetic
D004273:DNA, Neoplasm; D015345:Oligonucleotide Probes; D004247:DNA
PCR: DNA-Amplifikation am Schnittpräparat.