Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/24221
Type: Artigo de periódico
Title: Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism
Author: Jorge, S.B.
Melo, M.B.
Costa, F.F.
Sonati, M.F.
Abstract: Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem™ and GenePhor™, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem™ and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.
Subject: Alpha-globin genes
Nonradioactive SSCP
Alpha-globin structural variants
Hemoglobinopathies
Mutation screening
Editor: Associação Brasileira de Divulgação Científica
Rights: aberto
Identifier DOI: 10.1590/S0100-879X2003001100004
Address: http://dx.doi.org/10.1590/S0100-879X2003001100004
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2003001100004
Date Issue: 1-Nov-2003
Appears in Collections:Unicamp - Artigos e Outros Documentos

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