DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification

Detalhes bibliográficos
Autor(a) principal: Pereira, Ana Carla
Data de Publicação: 2008
Outros Autores: Souza, Vânia Brito de, Dias-Baptista, Ida Maria Foschiani
Tipo de documento: Artigo
Idioma: por
Título da fonte: Hansenologia Internationalis (Online)
Texto Completo: https://periodicos.saude.sp.gov.br/hansenologia/article/view/36239
Resumo: Skin biopsies from leprosy diagnostic services can be great sources of material for retrospective studiesconcerning 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 isan inexpensive method which provided a satisfactory yield of a good quality DNA for PCR from paraffin- em-bedded skin biopsies.
id ILSL-1_135f4f339a0c3cb4388675f4df9ba5cf
oai_identifier_str oai:ojs.periodicos.saude.sp.gov.br:article/36239
network_acronym_str ILSL-1
network_name_str Hansenologia Internationalis (Online)
repository_id_str
spelling DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplificationExtração de DNA de biópsias de pele fixadas em formalina tamponada e embebidas em parafina e amplificação por PCRepidemiologia molecularbanco de DNAPCRdermatologiahanseníasemolecular epidemiologyDNA databankPCRdermatologyleprosySkin biopsies from leprosy diagnostic services can be great sources of material for retrospective studiesconcerning 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 isan inexpensive method which provided a satisfactory yield of a good quality DNA for PCR from paraffin- em-bedded skin biopsies.Biópsias de pele oriundas dos serviços de diagnóstico da hanseníase podem ser grande fonte de material para estudos retrospectivos em genética humana e do Mycobacterium leprae. No entanto, os procedimentos de fixação e inclusão em parafina podem dificultar a obtenção de DNA de qualidade para amplificação por PCR. Assim, estas amostras requerem protocolos especiais para a extração do material genético. O objetivo deste trabalho é apresentar um método alternativo para extração de DNA com base na combinação de calor e digestão enzimática. Para tanto, os cortes foram aquecidos a 120º C em solução tampão de pH 9,0, submetidos à digestão enzimática com proteinase K e o DNA foi extraído por meio de solução de fenol:clorofórmio:álcool isoamílico. A amplificação por PCR para as regiões dos genes humanos TNF e LTA foi bem sucedida para 85,4% dos espécimes. Considerando o DNA do M. leprae, obtivemosamplificação em 67,6%, 48,5%, 36,7% e 64,8% para os marcadores TA18, GTA9, TTC e RLEP, respectivamente.Concluímos que este é um método de baixo custo que proporcionou um rendimento satisfatório de DNA deboa qualidade para emprego em PCR a partir de biópsias parafinadas de pele.Instituto Lauro de Souza Lima da Secretaria de Estado da Saúde de São Paulo2008-11-30info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://periodicos.saude.sp.gov.br/hansenologia/article/view/3623910.47878/hi.2008.v33.36239Hansenologia Internationalis: leprosy and other infectious diseases; Vol. 33 No. 2 (2008); 25-30Hansenologia Internationalis: hanseníase e outras doenças infecciosas; v. 33 n. 2 (2008); 25-301982-5161reponame:Hansenologia Internationalis (Online)instname:Instituto Lauro de Souza Lima (ILSL)instacron:ILSLporhttps://periodicos.saude.sp.gov.br/hansenologia/article/view/36239/34541https://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessPereira, Ana CarlaSouza, Vânia Brito deDias-Baptista, Ida Maria Foschiani2023-02-17T18:23:48Zoai:ojs.periodicos.saude.sp.gov.br:article/36239Revistahttps://periodicos.saude.sp.gov.br/hansenologiaPRIhttps://periodicos.saude.sp.gov.br/hansenologia/oaihansen_int@ilsl.br || hansenologia.internationalis@gmail.com || periodicossp@saude.sp.gov.br1982-51610100-3283opendoar:2023-02-17T18:23:48Hansenologia Internationalis (Online) - Instituto Lauro de Souza Lima (ILSL)false
dc.title.none.fl_str_mv DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification
Extração de DNA de biópsias de pele fixadas em formalina tamponada e embebidas em parafina e amplificação por PCR
title DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification
spellingShingle DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification
Pereira, Ana Carla
epidemiologia molecular
banco de DNA
PCR
dermatologia
hanseníase
molecular epidemiology
DNA databank
PCR
dermatology
leprosy
title_short DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification
title_full DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification
title_fullStr DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification
title_full_unstemmed DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification
title_sort DNA extraction from formalin-fixed and paraffin-embedded skin biopsies and PCR amplification
author Pereira, Ana Carla
author_facet Pereira, Ana Carla
Souza, Vânia Brito de
Dias-Baptista, Ida Maria Foschiani
author_role author
author2 Souza, Vânia Brito de
Dias-Baptista, Ida Maria Foschiani
author2_role author
author
dc.contributor.author.fl_str_mv Pereira, Ana Carla
Souza, Vânia Brito de
Dias-Baptista, Ida Maria Foschiani
dc.subject.por.fl_str_mv epidemiologia molecular
banco de DNA
PCR
dermatologia
hanseníase
molecular epidemiology
DNA databank
PCR
dermatology
leprosy
topic epidemiologia molecular
banco de DNA
PCR
dermatologia
hanseníase
molecular epidemiology
DNA databank
PCR
dermatology
leprosy
description Skin biopsies from leprosy diagnostic services can be great sources of material for retrospective studiesconcerning 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 isan inexpensive method which provided a satisfactory yield of a good quality DNA for PCR from paraffin- em-bedded skin biopsies.
publishDate 2008
dc.date.none.fl_str_mv 2008-11-30
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://periodicos.saude.sp.gov.br/hansenologia/article/view/36239
10.47878/hi.2008.v33.36239
url https://periodicos.saude.sp.gov.br/hansenologia/article/view/36239
identifier_str_mv 10.47878/hi.2008.v33.36239
dc.language.iso.fl_str_mv por
language por
dc.relation.none.fl_str_mv https://periodicos.saude.sp.gov.br/hansenologia/article/view/36239/34541
dc.rights.driver.fl_str_mv https://creativecommons.org/licenses/by/4.0
info:eu-repo/semantics/openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/4.0
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Instituto Lauro de Souza Lima da Secretaria de Estado da Saúde de São Paulo
publisher.none.fl_str_mv Instituto Lauro de Souza Lima da Secretaria de Estado da Saúde de São Paulo
dc.source.none.fl_str_mv Hansenologia Internationalis: leprosy and other infectious diseases; Vol. 33 No. 2 (2008); 25-30
Hansenologia Internationalis: hanseníase e outras doenças infecciosas; v. 33 n. 2 (2008); 25-30
1982-5161
reponame:Hansenologia Internationalis (Online)
instname:Instituto Lauro de Souza Lima (ILSL)
instacron:ILSL
instname_str Instituto Lauro de Souza Lima (ILSL)
instacron_str ILSL
institution ILSL
reponame_str Hansenologia Internationalis (Online)
collection Hansenologia Internationalis (Online)
repository.name.fl_str_mv Hansenologia Internationalis (Online) - Instituto Lauro de Souza Lima (ILSL)
repository.mail.fl_str_mv hansen_int@ilsl.br || hansenologia.internationalis@gmail.com || periodicossp@saude.sp.gov.br
_version_ 1796797580987858944

Registros relacionados