Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis

Research output: Contribution to journalArticle

Abstract

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 tire advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplifcation, 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 swine reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan((R)) 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-dinter formation and the presence of any other anomalous products.

Details

Authors
  • K Lind
  • Anders Stålberg
  • N Zoric
  • M Kubista
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Cell and Molecular Biology
Original languageEnglish
Pages (from-to)315-319
JournalBioTechniques
Volume40
Issue number3
Publication statusPublished - 2006
Publication categoryResearch
Peer-reviewedYes

Bibliographic note

The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Stem Cell and Pancreas Developmental Biology (013212044)