Recovery and analysis of DNA from fixed tissue
thesisposted on 15.12.2014, 10:31 by Anthony. Warford
This research was undertaken to: 1) Develop techniques for the recovery of DNA from fixed and paraffin wax embedded tissues. 2) Assess the potential for use of this DNA for the diagnosis of lymphoma. 3) Contribute to the understanding of the effects of fixation on DNA.;Initial investigations used pure DNA and lymphocytes to determine the effect of five fixatives on the integrity, recovery and restriction endonuclease digestion of the nucleic acid. Two fixatives, Bouin and formol sublimate, proved unsuitable for further analysis. DNA recovery and Southern analysis was then attempted using Carnoy, formol saline and neutral buffered formalin fixed and paraffin wax embedded tissue. From this an optimised method featuring prolonged incubation of tissue with Proteinase K and SDS at 37Â°C followed by purification was developed. Within limits imposed by fixation this DNA was suitable for Southern analysis and amplification by PCR. A simplified DNA recovery method involving overnight incubation of paraffin wax sections in Proteinase K at 55Â°C without purification was also evaluated. This gave satisfactory results by PCR but the maximum product size obtained was 500 bp compared with 1250 bp using the optimised method.;DNA was better preserved after Carnoy than following formalin fixation and this was reflected in consistently superior Southern and PCR results. Formalin fixation induced degradation of DNA, which increased as fixation time was extended. This made Southern analysis for the identification of B and T cell rearrangements in lymphomas unreliable. However, the t(14:18) translocation was demonstrated successfully by PCR in formalin fixed follicular lymphomas.;The results of these investigations show the importance of the pH of fixatives on the preservation of DNA. They also suggest that chemical interactions with DNA and associated proteins occur using fixatives containing formalin and mercury..