PCR amplification of DNA from archival specimens. A methodological approach.
The experiments were designed to assess whether DNA could be recovered from archival, fixed, paraffin embedded specimens, 1-39 years old, for use in PCR and Southern blot analyses. Specimen fixed in either 10% formalin, Carnoy's or AMeX fixative produced almost equally abundant 318 bp long beta-actin fragment, after 2 x 120 min deparaffinization time, proteinase K DNA extraction and 40, or more, amplification cycles. Prolonged deparaffinization time and phenol/chloroform extraction did not influence DNA quality for PCR. Formalin fixed tissues can be successfully used for DNA/PCR of shorter fragments (318 bp) even if they are up to 39 years old. Longer fragment (720 bp) was successfully amplified from 1 and 10 years old specimens, also investigated whether DNA suitable for hybridization studies could be prepared from fixed tissue. Formalin caused irreversible DNA damages which were greater with prolonged fixation time making it unsuitable for hybridization studies, but still suitable for PCR amplification. Carnoy's and AMeX fixation resulted in consistently high yield of high molecular size DNA suitable for use in hybridization studies and PCR respectively. DNA from Papanicolaou stained smears was successfully amplified by PCR as well. Of all stains used in the preparation of smears, only eosin was detectable as a greenish band in agarose gels under ultraviolet illumination.
Pavelić
J
J
Laboratory of Molecular Oncology, Ruder Boković Institute, Zagreb, Croatia.
Gall-Troselj
K
K
Bosnar
M H
MH
Kardum
M M
MM
Pavelić
K
K
D001923:Brain Chemistry; D001932:Brain Neoplasms / Q000737:chemistry / Q000235:genetics; D002584:Cervix Uteri / Q000737:chemistry / Q000821:virology; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D016158:Genes, p53; D006801:Humans; D008579:Meningioma / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D065006:Papanicolaou Test; D027383:Papillomaviridae; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D014626:Vaginal Smears
D004273:DNA, Neoplasm