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 situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such 'failures' may not only be due to absent target 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 amplification results. This is particularly true with in situ PCR/PCR in situ hybridization. To examine these effects in solution-phase PCR, beta-globin 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 glutaraldehyde at 0 to 4 degrees C, room temperature and 37 degrees C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37 degrees C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37 degrees C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pathology, University of Leeds, UK.
J J
JJ
O'Leary
G
G
Browne
R J
RJ
Landers
M
M
Crowley
I B
IB
Healy
J T
JT
Street
A M
AM
Pollock
J
J
Murphy
M I
MI
Johnson
F A
FA
Lewis
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D019286:Endopeptidase K; D005557:Formaldehyde; D005914:Globins / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D014066:Palatine Tonsil / Q000737:chemistry*; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000737:chemistry; D013698:Templates, Genetic; D016707:Tissue Fixation
D004279:DNA, Viral; D005557:Formaldehyde; D005914:Globins; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K