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

Chalmers University of Technology, Gothenburg, Sweden.
BioTechniques (Impact Factor: 2.75). 04/2006; 40(3):315-9. DOI: 10.2144/000112101
Source: PubMed

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 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.

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    • "Results showed that BOXTO can be used together with both TaqMan probes and locked nucleic acid (LNA) probes without any inhibitory effect on PCR. Signals acquired by the probe were used to quantify the PCR product while that acquired by BOXTO dsDNA binding was used to generate a melting curve (Lind et al 2006). Since the past decade, SG is considered the best fl uorescent dye that can be used to quantify PCR products in real-time format (Wittwer et al 1997; Morrison et al 1998); therefore, SG is considered the standard and reference dye in the present study, which focused on studying the ability of BOXTO and its derivative BOXTO-PRO to be used in detecting and quantifying amplifi ed DNA during PCR in a real-time format, and how close the new dye's effi ciency and dynamic range was to that of SG. "
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    ABSTRACT: The unsymmetrical cyanine dyes BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2- methylidene)]-1-methyl-quinolinium chloride)and its positive divalent derivative BOXTO-PRO (4-[(3-methyl-6-(benzoxazole-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-(3-trimethylammonium-propyl)-quinolinium dibromide) were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG)(2-[N- (3-dimethylaminopropyl)-N-propylamino] -4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl- quinolinium).Unmodified BOXTO showed no inhibitory effects on real-time PCR,while BOXTO-PRO showed complete inhibition. Sufficient fluorescent signal was acquired when 0.5-1.0 meu M BOXTO was used with RotorGene and iCycler platforms.Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO and SG.BOXTO stock solution (1.5 mM) was stable at -20 degree C for more than one year and 40 meu M BOXTO solution was more stable than 5x SG when both were stored at 4 degree C for 45 days.
    Journal of Biosciences 04/2007; 32(2):229-39. · 1.94 Impact Factor
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    ABSTRACT: The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.
    Molecular Aspects of Medicine 04/2006; 27(2-3):95-125. DOI:10.1016/j.mam.2005.12.007 · 10.30 Impact Factor
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