ISPO

Partial restoration of degraded DNA from archival paraffin embedded tissues

E.N. Imyanitov, PhD MD, MY Grigoriev, MD, V.M. Gorodinskaya, MD, E.S. Kuligina, PhD, K.M. Pozharisski, PhD MD, A.V. Togo, PhD, and K.P. Hanson, PhD MD

N.N. Petrov Institute of Oncology, St.-Petersburg 197758, Russia

AIMS: The use of paraffin-embedded tissues (PET) for molecular analysis is limited due to the degradation of archival DNA. As the poor quality of PET-derived DNA is at least in part attributed to the single-strand breaks, this template can be substantially restored by filling the nicks in the polymerase reaction. Here we present the protocol allowing in situ DNA reconstruction. METHODS: The availability of chromosome-embedded DNA for enzymatic reaction was achieved by limited proteinase K hydrolysis. After the subsequent removal of the proteinase K, Taq polymerase-driven DNA restoration was carried out in PCR-like conditions. Finally, the standard DNA-releasing procedure was applied, which includes intensive protein digestion and heating of the tissue samples. The obtained lysates were added directly to the PCR reactions. RESULTS: The proposed modification increased the success rate of PCR and allowed to amplify larger fragments from PET-derived DNA. CONCLUSIONS: The proposed method has a potential significance for all applications related to the genetic analysis of formalin-fixed paraffin-embedded archival tissues.

For more information, contact imyanit@mail.axon.ru

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Molecular Pathology.

http://www.cancerprev.org/Journal/Issues/26/101/991/4173