Southern and dot blot analysis of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas.
Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffin-embedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA. Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.
Moerkerk
P T
PT
Department of Pathology, University of Limburg, Maastricht, The Netherlands.
Kessels
H J
HJ
ten Kate
J
J
de Goeij
A F
AF
Bosman
F T
FT
D015139:Blotting, Southern; D003110:Colonic Neoplasms / Q000235:genetics*; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000032:analysis*; D003720:Densitometry; D004587:Electrophoresis, Agar Gel; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D015151:Immunoblotting; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010232:Paraffin; D015183:Restriction Mapping; D014021:Tissue Preservation / Q000379:methods
D015342:DNA Probes; D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde; D010232:Paraffin