Inter-laboratory comparison of DNA preservation in archival paraffin-embedded human brain tissue from participating centres on four continents.

Published

Journal Article

DNA extracted from formalin-fixed and paraffin-embedded brain tissue is known to contain as yet ill-characterized inhibitors of the PCR process. As part of a project that aims to clarify the role of mitochondrial DNA sequence variation in human neurodegenerative diseases using DNA from various ethnic backgrounds, we have investigated factors that influence the preservation of archival DNA and its suitability for PCR. In this study, neuropathological tissue samples were analysed that had been routinely processed in 18 international centres on four continents. Following DNA extraction, PCR amplification of mitochondrial and nuclear DNA sequences was performed with and without additional purification of the template DNA. In addition, the DNA used for PCR was analysed by HPLC. Phosphate-buffered formalin proved to be a superior fixative compared with unbuffered aldehyde: DNA extraction resulted in greater yields, the molecular weight of the isolated DNA was higher and PCR was more successful. PCR inhibitors were identified as (1) high concentrations of small (<300 bp) DNA fragments that competitively compete with template DNA and (2) contaminants of the DNA template solution including denatured protein that cannot be completely removed by phenolic extraction. HPLC analysis did not reveal significant qualitative differences between DNA isolated from fresh-frozen tissue samples and DNA recovered from formalin-fixed, paraffin-embedded brain tissue. The fact that DNA could be amplified from the majority of tissue specimens in this study suggests that rare diseases and diseases where ethnic background plays an important role can be sampled for genetic polymorphism analysis on a global scale using archival neuropathological collections.

Full Text

Duke Authors

Cited Authors

  • Kösel, S; Grasbon-Frodl, EM; Arima, K; Chimelli, L; Hahn, M; Hashizume, Y; Hulette, C; Ikeda, K; Jacobsen, PF; Jones, M; Kobayashi, M; Love, S; Mizutani, T; Rosemberg, S; Sasaki, A; Smith, TW; Takahashi, H; Vortmeyer, AO; Graeber, MB

Published Date

  • July 2001

Published In

Volume / Issue

  • 3 / 3

Start / End Page

  • 163 - 170

PubMed ID

  • 11523568

Pubmed Central ID

  • 11523568

International Standard Serial Number (ISSN)

  • 1364-6745

Language

  • eng

Conference Location

  • United States