Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer
Genet. mol. res. (Online)
; 6(2): 250-255, 2007. ilus, graf
Artigo
em Inglês
| LILACS
| ID: lil-482046
Biblioteca responsável:
BR1.1
ABSTRACT
Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.
Texto completo:
Disponível
Coleções:
Bases de dados internacionais
Contexto em Saúde:
Doenças Negligenciadas
Problema de saúde:
Doenças Negligenciadas
/
Zoonoses
Base de dados:
LILACS
Assunto principal:
Análise de Sequência de DNA
/
Primers do DNA
/
Fluoresceína
/
DNA Polimerase I
/
Escherichia coli
Idioma:
Inglês
Revista:
Genet. mol. res. (Online)
Assunto da revista:
Biologia Molecular
/
Genética
Ano de publicação:
2007
Tipo de documento:
Artigo
País de afiliação:
Brasil
/
França
Instituição/País de afiliação:
Institut de Pharmacologie et de Biologie Structurale/FR
/
Universidade Federal de Minas Gerais/BR