THE IMPORTANCE OF FIXATION PROCEDURES ON DNA-TEMPLATE AND ITS SUITABILITY FOR SOLUTION-PHASE POLYMERASE CHAIN-REACTION AND PCR IN-SITU HYBRIDIZATION

Conventional solution-phase polymerase chain reaction (PCR) and in sit u PCR/PCR in situ hybridization are powerful tools for retrospective a nalysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archi val fixed tissues. Such 'failures' may not only be due to absent targe t DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence ampl ification results. This is particularly true with in situ PCR/PCR in s itu hybridization. To examine these effects in solution-phase PCR, P-g lobin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and gluta raldehyde at 0 to 4 degrees C, room temperature and 37 degrees C fixat ion temperatures and for fixation periods of 6, 24, 48 and 72 hours an d 1 week. DNA extraction procedures used were simple boiling and 5 day s' proteinase K digestion at 37 degrees C. Amplified product was visib le primarily yet variably from tissue fixed in neutral buffered formal dehyde and Carnoy's, whereas fixation in mercuric chloride-based fixat ives produced consistently negative results. Room temperature and 37 d egrees C fixation temperature appeared most conducive to yielding ampl ifiable DNA template. Fixation times of 24 and 48 hours in neutral buf fered formaldehyde and Camoy's again favoured amplification. Fixed SiH a cells (containing 1-2 copies of HPV 16) were examined using PCR in s itu hybridization for the amplification of HPV 16. Discrete and diffus e amplification signals were obtained. Neutral buffered formaldehyde f ixation for 12-24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within c ellular preparations was 40%, with non-specific background staining al so seen. Possible technical problems encountered with PCR in situ hybr idization are discussed.