Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

Detalhes bibliográficos
Autor(a) principal: PASSAMANI,PAULO Z.
Data de Publicação: 2018
Outros Autores: CARVALHO,CARLOS R., SOARES,FERNANDA A.F.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Anais da Academia Brasileira de Ciências (Online)
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652018000100041
Resumo: ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.
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spelling Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniquesDOP-PCRFISHmicromanipulationPRINSABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.Academia Brasileira de Ciências2018-03-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652018000100041Anais da Academia Brasileira de Ciências v.90 n.1 2018reponame:Anais da Academia Brasileira de Ciências (Online)instname:Academia Brasileira de Ciências (ABC)instacron:ABC10.1590/0001-3765201720160089info:eu-repo/semantics/openAccessPASSAMANI,PAULO Z.CARVALHO,CARLOS R.SOARES,FERNANDA A.F.eng2018-04-25T00:00:00Zoai:scielo:S0001-37652018000100041Revistahttp://www.scielo.br/aabchttps://old.scielo.br/oai/scielo-oai.php||aabc@abc.org.br1678-26900001-3765opendoar:2018-04-25T00:00Anais da Academia Brasileira de Ciências (Online) - Academia Brasileira de Ciências (ABC)false
dc.title.none.fl_str_mv Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
title Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
spellingShingle Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
PASSAMANI,PAULO Z.
DOP-PCR
FISH
micromanipulation
PRINS
title_short Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
title_full Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
title_fullStr Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
title_full_unstemmed Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
title_sort Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques
author PASSAMANI,PAULO Z.
author_facet PASSAMANI,PAULO Z.
CARVALHO,CARLOS R.
SOARES,FERNANDA A.F.
author_role author
author2 CARVALHO,CARLOS R.
SOARES,FERNANDA A.F.
author2_role author
author
dc.contributor.author.fl_str_mv PASSAMANI,PAULO Z.
CARVALHO,CARLOS R.
SOARES,FERNANDA A.F.
dc.subject.por.fl_str_mv DOP-PCR
FISH
micromanipulation
PRINS
topic DOP-PCR
FISH
micromanipulation
PRINS
description ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.
publishDate 2018
dc.date.none.fl_str_mv 2018-03-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652018000100041
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652018000100041
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/0001-3765201720160089
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Academia Brasileira de Ciências
publisher.none.fl_str_mv Academia Brasileira de Ciências
dc.source.none.fl_str_mv Anais da Academia Brasileira de Ciências v.90 n.1 2018
reponame:Anais da Academia Brasileira de Ciências (Online)
instname:Academia Brasileira de Ciências (ABC)
instacron:ABC
instname_str Academia Brasileira de Ciências (ABC)
instacron_str ABC
institution ABC
reponame_str Anais da Academia Brasileira de Ciências (Online)
collection Anais da Academia Brasileira de Ciências (Online)
repository.name.fl_str_mv Anais da Academia Brasileira de Ciências (Online) - Academia Brasileira de Ciências (ABC)
repository.mail.fl_str_mv ||aabc@abc.org.br
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