S Bonin1, F Petrera, B Niccolini, G Stanta. 1. ICGEB, International Centre for Genetic Engineering and Biotechnology, 99 Padriciano, 34012 Trieste, Italy.
Abstract
BACKGROUND: Formalin fixed and paraffin wax embedded tissues of necropsy origin are an important source for molecular analysis especially in rare diseases, neuropathology, or molecular epidemiology studies. Because of DNA degradation, only short sequences can be amplified from this type of tissue, very often less than 100 bases. This poses problems because studies on polymorphism and mutations occurring in large genes often require the analysis of long sequences. METHODS: The development of a simple treatment to obtain longer fragments of DNA for the analysis of archival postmortem paraffin wax embedded tissues. RESULTS: It was possible to amplify longer sequences ranging up to 300 bases from postmortem tissues, with no modification to the usual DNA extraction procedures. To obtain longer stretches of DNA, a pre-PCR restoration treatment was required, by filling single strand breaks, followed by a vigorous denaturation step. CONCLUSIONS: The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.
BACKGROUND:Formalin fixed and paraffin wax embedded tissues of necropsy origin are an important source for molecular analysis especially in rare diseases, neuropathology, or molecular epidemiology studies. Because of DNA degradation, only short sequences can be amplified from this type of tissue, very often less than 100 bases. This poses problems because studies on polymorphism and mutations occurring in large genes often require the analysis of long sequences. METHODS: The development of a simple treatment to obtain longer fragments of DNA for the analysis of archival postmortem paraffin wax embedded tissues. RESULTS: It was possible to amplify longer sequences ranging up to 300 bases from postmortem tissues, with no modification to the usual DNA extraction procedures. To obtain longer stretches of DNA, a pre-PCR restoration treatment was required, by filling single strand breaks, followed by a vigorous denaturation step. CONCLUSIONS: The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.
Authors: E N Imyanitov; M Y Grigoriev; V M Gorodinskaya; E S Kuligina; K M Pozharisski; A V Togo; K P Hanson Journal: Biotechniques Date: 2001-11 Impact factor: 1.993
Authors: D Jane Hata; Seanne P Buckwalter; Bobbi S Pritt; Glenn D Roberts; Nancy L Wengenack Journal: J Clin Microbiol Date: 2008-05-14 Impact factor: 5.948