Extração de DNA de biópsias de pele fixadas em formalina tamponada e embebidas em parafina e amplificação por PCR

Ana Carla Pereira; Vânia Brito de Souza; Ida Maria Foschiani Dias-Baptista

doi:10.47878/hi.2008.v33.36239

Authors

Ana Carla Pereira Doutor em Ciências Farmacêuticas; Equipe de Farmacologia-Bioquímica, Instituto Lauro de Souza Lima
Vânia Brito de Souza Doutor em Genética e Biologia Molecular; Equipe de Imunologia, Instituto Lauro de Souza Lima
Ida Maria Foschiani Dias-Baptista Doutor em Biologia Celular e Molecular; Equipe de Microbiologia, Instituto Lauro de Souza Lima

DOI:

https://doi.org/10.47878/hi.2008.v33.36239

Keywords:

molecular epidemiology, DNA databank, PCR, dermatology, leprosy

Abstract

Skin biopsies from leprosy diagnostic services can be great sources of material for retrospective studies
concerning human and Mycobacterium leprae genetic. However, fixation and paraffin embedding procedures make difficult to obtain good quality DNA to PCR amplification. Thus, paraffin-embedded samples require special protocols to DNA extraction. The aim of this paper is to present an alternative method for DNA extraction based on the combination of heat and enzymatic digestion. The sections were heated at 120ºC at pH 9.0, submitted to enzymatic digestion with proteinase K and the DNA was extracted by using phenol:chlorophorm:isoamilic alcohol. PCR amplification for regions in TNF and LTA human genes was successful for 85.4 % of specimens. Regarding M. leprae DNA, we obtained amplification in 67.6%, 48.5%, 36.7% and 64.8% for the TA18, GTA9, TTC and RLEP markers, respectively. We conclude that this is
an inexpensive method which provided a satisfactory yield of a good quality DNA for PCR from paraffin- em-bedded skin biopsies.

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