Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis
| Autor: | Mikael Kubista, Neven Zoric, Kristina Lind, Anders Ståhlberg |
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| Rok vydání: | 2006 |
| Předmět: |
Chromatography
Chemistry DNA Sequence Analysis DNA Nucleic Acid Denaturation Polymerase Chain Reaction General Biochemistry Genetics and Molecular Biology Melting curve analysis law.invention chemistry.chemical_compound Real-time polymerase chain reaction Microscopy Fluorescence Biochemistry law TaqMan Nucleic acid Transition Temperature Locked nucleic acid DNA Probes In Situ Hybridization Fluorescence Polymerase chain reaction Fluorescent Dyes Biotechnology |
| Zdroj: | BioTechniques. 40:315-319 |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/000112101 |
| Popis: | Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan® probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products. |
| Databáze: | OpenAIRE |
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