Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues.
A problem associated with use of the polymerase chain reaction to amplify specific DNA fragments from formalin fixed, paraffin wax embedded tissues is the not infrequent failure of amplification. One possible reason for this could be the presence of inhibitor(s), which interfere with the activity of the reaction. It has been shown that such inhibitor(s) exist when amplifying the human beta globin gene (which exists in human genomic DNA as a single copy gene) from routine clinical samples. A variety of methods to remove such inhibitor(s) were investigated. The results indicate that inhibitor(s) are removed by proteinase K digestion, followed by purification with phenol/chloroform, and centrifugation through a Centricon-30 membrane (30,000 molecular weight cut off). Other factors, including the length and concentration of the DNA sequence to be amplified, can also affect amplification.
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Nuffield Department of Pathology and Bacteriology, University of Oxford, John Radcliffe Hospital.
Fleming
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KA
D001483:Base Sequence; D002498:Centrifugation; D004247:DNA / Q000302:isolation & purification; D019286:Endopeptidase K; D005557:Formaldehyde; D005914:Globins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012697:Serine Endopeptidases / Q000378:metabolism; D016707:Tissue Fixation
D005557:Formaldehyde; D005914:Globins; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K