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.
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.
Földes-Papp
Zeno
Z
Clinical Immunology and Jean Dausset Laboratory, Medical University of Graz, Auenbruggerplatz 8, A-8036 Graz, LKH, Austria. Zeno.Foldes-Papp@meduni-graz.at
Kinjo
Masataka
M
Tamura
Mamoru
M
Birch-Hirschfeld
Eckhard
E
Demel
Ulrike
U
Tilz
Gernot P
GP
D000483:Alleles; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods
D005456:Fluorescent Dyes; D004247:DNA; D042965:Methylenetetrahydrofolate Reductase (NADPH2)