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 fl 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. The polymerase chain reaction (PCR) permits specific and rapid amplification of nucleic acid sequences from a variety of materials, including blood, hair and fresh or frozen tissues.' In view of the large amount of archival material stored in pathology departments, retrospective analysis of formalin fixed, paraffin wax embedded tissues has also been performed.2' Several reports, however, have indicated that the PCR on such material is not reliable, the failure rate varying between 2-31%.' We have also noted this intermittent failure of amplification from such tissues.9 The purpose of this study was, therefore, to investigate the causes of non-amplification by the PCR when using formalin fixed paraffin wax embedded tissues and to devise a reliable reproducible method of amplification of nucleic acid from such tissues. NuffieldDepartmentof Pathology and Bacteriology, Methods University of Oxford, Blocks of formalin fixed paraffin wax embedJohn Radcliffe ded tissue were chosen at random from the oX3 9DU files of Nuffield Department of Pathology and S F An Bacteriology, Uiiiversity of Oxford, John K A Fleming Radcliffe Hospital, Oxford. The material had Dr K A Fleming been processed between 1988-1990. A variety of tissue types were amplified (table). Fixation Accepted for publicwation 22 May 1991 was in neutral formol/saline. The time of fixation varied between 24 and 48 hours. Processing was by standard methods-that is, dehydration in graded alcohols, clearing in xylene, and embedding in Paraplast paraffin using a VIPII (Tissue Tek) processor. Paraffin wax sections (10 yM) were cut and placed in a 1-5 ml Eppendorf tube. The number of sections varied according to the surface area of the tissue block. About 7 mm3 was taken for digestion. The paraffin was removed by washing with Citroclear (HD Supplies, England) for five minutes and 95% ethanol for five minutes, following by drying at 95°C for 90 minutes. Five methods were tried for DNA extraction: (1) No additional procedure after dewaxing, the PCR reaction mix being added to the tissue. (2) After dewaxing, the tissue was boiled for 20 minutes in 200 pl of distilled water and a variable amount of the supernatant used for amplification. Sometimes this supernatant was passed through a Sephadex G50 column before attempting amplification. (3) Treated as method 2, but additionally digested with proteinase K (Boehringer Mannheim, East Sussex, England) final concentration 1 mg/ml in 100 mM phosphate buffered saline (pH 7.2). Digestion was performed at 37°C overnight. The supernatant was boiled to inactivate the proteinase K and then used for amplification. (4) Treated as method 3, but the supernatant was additionally extracted once with an equal volume of phenol (equilibrated with 100 mM TRIS (pH 8-0) 0X1% hydroxyquinoline), once with 1:1 mixture of phenol and chloroform (a 24:1 (v/v) mixture of chloroform and iso-amyl List offormalinfixed paraffin wax embedded tissues Tissues No of samples Liver 3 Skin 7 Rectum 1 Ovary 2 Bladder 1 Omentum 1 Cervix 1 Uterus 1 Rectal Polyp 2 Appendix 1 Stomach I Thyroid 1 Heart 1 Prostate 1 Gum I Lymph node 1 Kidney 1 Total 27 924 group.bmj.com on July 7, 2017 Published by http://jcp.bmj.com/ Downloaded from