Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis.
Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.
Földes-Papp
Zeno
Z
Clinical Immunology and Jean Dausset Laboratory, Graz University Medical School and Hospital, A-8036 Graz, LKH, Austria. Zeno.Foldes-Papp@uni-graz.at
Costa
Jean M
JM
Demel
Ulrike
U
Tilz
Gernot P
GP
Kinjo
Masataka
M
Saito
Kenta
K
Kii
Hiroaki
H
Takagi
Takuya
T
Tamura
Mamoru
M
Thyberg
Per
P
Birch-Hirschfeld
Eckhard
E
D004247:DNA / Q000097:blood / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005456:Fluorescent Dyes; D005500:Follow-Up Studies; D005838:Genotype; D006801:Humans; D008433:Mathematics; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000097:blood / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA; D042965:Methylenetetrahydrofolate Reductase (NADPH2)