Highly sensitive and selective colorimetric genotyping of single-nucleotide polymorphisms based on enzyme-amplified ligation on magnetic beads.
Biosens Bioelectron
; 36(1): 89-94, 2012.
Article
em En
| MEDLINE
| ID: mdl-22534103
ABSTRACT
Herein we report a new strategy for highly sensitive and selective colorimatric assay for genotyping of single-nucleotide polymorphisms (SNPs). It is based on the use of a specific gap ligation reaction, horseradish peroxidase (HRP) for signal amplification, and magnetic beads for the easy separation of the ligated product. Briefly, oligonucleotide capture probe functionalized magnetic beads are first hybridized to a target DNA. Biotinylated oligonucleotide detection probes are then allowed to hybridize to the already captured target DNA. A subsequent ligation at the mutation point joins the two probes together. The introduction of streptavidin-conjugated HRP and a simple magnetic separation allow colorimetric genotyping of SNPs. The assay is able to discriminate one copy of mutant in 1000 copies of wild-type KRAS oncogene at 30 picomolar. The detection limit of the assay is further improved to 1 femtomolar by incorporating a ligation chain reaction amplification step, offering an excellent opportunity for the development of a simple and highly sensitive diagnostic tool.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
/
Proteínas Proto-Oncogênicas
/
Colorimetria
/
Proteínas ras
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Técnicas de Genotipagem
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article