Partially double-stranded linear DNA probes: novel design for sensitive detection of genetically polymorphic targets.
J Virol Methods
; 144(1-2): 1-11, 2007 Sep.
Article
em En
| MEDLINE
| ID: mdl-17434605
ABSTRACT
Genetically polymorphic targets present a significant challenge to the reliability of detection and quantification by nucleic acid-based assays. A probe system with enhanced mismatch tolerance would be advantageous for such applications. The present study introduces a novel class of DNA probes, designated as partially double-stranded linear probes, composed of a long target-specific strand 5' labeled with a fluorophore and a markedly shorter quencher strand, complementary to the 5' end of the target-specific strand, that is 3' end-labeled with a quencher moiety. The utility of this probe system for sensitive detection of amplification products was demonstrated in a real-time PCR format. Comparison of multiple partially double-stranded linear probe combinations revealed that increased asymmetry in strand length was associated with improved mismatch tolerance. Notably, for a 45-mer/11-mer combination, the difference in threshold cycle values obtained for a perfectly matched target and one containing six mismatches was <1.5 cycles. The capacity for superior mismatch tolerance, ease of design, simplicity and flexibility of application are characteristics that make this new class of probes a desirable alternative for homogeneous detection of targets with a high level of genetic heterogeneity.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sondas de DNA
/
Reação em Cadeia da Polimerase
/
Hibridização de Ácido Nucleico
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article