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Földes-Papp, Z; Kinjo, M; Tamura, M; Birch-Hirschfeld, E; Demel, U; Tilz, GP.
A new ultrasensitive way to circumvent PCR-based allele distinction: direct probing of unamplified genomic DNA by solution-phase hybridization using two-color fluorescence cross-correlation spectroscopy.
Exp Mol Pathol. 2005; 78(3):177-189 Doi: 10.1016/j.yexmp.2005.01.005
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Führende Autor*innen der Med Uni Graz
Földes-Papp Zeno
Co-Autor*innen der Med Uni Graz
Demel Ulrike
Tilz Gernot
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Abstract:
Single-molecule fluorescence methods enable a new class of nucleic acid assays to be performed that are not possible with PCR-based methods. In this basic study, the methylene tetrahydrofolate reductase (MTHFR)-genotypes (normal, homozygous mutated, as well as heterozygous mutated) were directly detected for the first time onto unamplified double-stranded genomic DNA in solution down to femtomolar allele concentrations (10(-15) M) in a homogeneous assay format. This was accomplished by taking advantage of the decrease by a factor of 40 to 100 in fluorescence background signals of the non-bound nonlinear hybridization probes in two colors and two-color fluorescence cross-correlation spectroscopy. The designed 'intelligent' probes contained the built-in 5'-fluorescent dyes rhodamine green and Alexa633, respectively, and the 3'-non-fluorescent quenchers BHQ1 and BHQ3, respectively, with perfectly matched spectral overlaps for both dye-quencher combinations. Upon binding of two appropriate probes that were sequence-specific for the genotype, the steady-state fluorescence in two colors increased by about two orders of magnitude. The obtained allele sensitivity of femtomolar and the specificity of the described molecular interactions allow PCR-based allele distinction to be circumvented. Furthermore, the results present an alternative to existing hybridization approaches that are currently used with and without amplification at the 'many-molecule' level and the 'single-molecule' level.
Find related publications in this database (using NLM MeSH Indexing)
Alleles -
Chromatography, High Pressure Liquid -
DNA - analysis
Electrophoresis, Gel, Two-Dimensional - analysis
Fluorescent Dyes - diagnostic use
Humans - diagnostic use
Methylenetetrahydrofolate Reductase (NADPH2) - genetics
Nucleic Acid Hybridization - methods
Polymerase Chain Reaction - methods
Polymorphism, Single Nucleotide - genetics
Sensitivity and Specificity - genetics
Spectrum Analysis - methods

Find related publications in this database (Keywords)
two-color fluorescence cross-correlation spectroscopy
single-nucleotide polymorphisms
unamplified genomic DNA
double-stranded DNA
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