DNA extraction from formalin-fixed, paraffin-embedded tissues: protein digestion as a limiting step for retrieval of high-quality DNA.
Several DNA extraction methods have been used for formalin-fixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 micrograms/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the beta-globin gene tested the suitability of the retrieved DNA for PCR amplification.
Díaz-Cano
S J
SJ
Department of Pathology, New England Medical Center Hospitals, Boston, Massachusetts 02111, USA.
Brady
S P
SP
D000311:Adrenal Glands / Q000737:chemistry; D001940:Breast / Q000737:chemistry; D003106:Colon / Q000737:chemistry; D004247:DNA / Q000302:isolation & purification*; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D016612:Paraffin Embedding / Q000379:methods*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012867:Skin / Q000737:chemistry; D013961:Thyroid Gland / Q000737:chemistry; D016707:Tissue Fixation / Q000379:methods; D001743:Urinary Bladder / Q000737:chemistry
D011506:Proteins; D005557:Formaldehyde; D004247:DNA