The role of DNase and EDTA on DNA degradation in formaldehyde fixed tissues.
Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.
Yagi
N
N
Department of Pathology, Kobe University School of Medicine, Japan.
Satonaka
K
K
Horio
M
M
Shimogaki
H
H
Tokuda
Y
Y
Maeda
S
S
D000818:Animals; D002843:Chromatin; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology*; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D011905:Genes, ras; D051381:Rats; D016707:Tissue Fixation
D002843:Chromatin; D005404:Fixatives; D005557:Formaldehyde; D004247:DNA; D004492:Edetic Acid; D003851:Deoxyribonucleases