Aims: Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that can be performed on a variety of cervical sample types including Pap-stained cervical smears. However, since the quality of DNA from such specimens is inferior to that from fresh tissue, the correct processing methods are required. This study evaluates three commercial isolation methods and one digestion procedure for their ability to obtain DNA suitable for PCR from fixed and stained Pap smears.
Methods: The High Pure PCR Template Preparation kit, the NucliSENS easyMAG system, the QIAamp DNA Mini Kit and crude proteinase K digestion were used to obtain DNA for subsequent PCR applications. Amplification of β-globin was performed to verify the presence and integrity of target DNA. The influence of PCR inhibitors and extent of DNA fragmentation were analysed.
Results: All commercial DNA isolation techniques provided DNA suitable for PCR amplification, and DNA isolated from 10-year-old archival smears yielded amplicons up to 400 base pairs. Conversely, crude proteinase K digestion limited the amplicon size to 300 bp and did not consistently yield amplifiable digests, as these were contaminated with PCR-inhibiting factors and debris.
Conclusion: The study indicates that commercial DNA isolation techniques are suitable for PCR amplification of DNA isolated from archival smears, yielding amplicons up to 400 base pairs. Proteinase K digestion is not suitable to obtain amplifiable DNA from fixed and stained Pap-stained smears.
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Competing interests: None.
Funding: GAVB is supported by the Fund for Scientific Research Flanders (FWO-Vlaanderen). CAJH is supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen). JJB is supported by the Fund for Scientific Research Flanders (FWO-Vlaanderen, G.0205.04) and the Belgian Cancer Foundation (Belgische Stichting tegen Kanker). MA received funding from the 6th Framework Programme (European Commission, DG Research, Brussels, Belgium) through the CCPRB Network (University of Lund, Malmö, Sweden), the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen) and Belgian Cancer Foundation (Belgische Stichting tegen Kanker).
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