A homogeneous high-throughput genotyping method based on competitive hybridization.
OBJECTIVE
A reliable high-throughput assay system is necessary for the analysis of the ever-increasing numbers of single-nucleotide polymorphisms (SNP) relevant to genetic screening studies. We describe an assay suitable also for large-scale screening programs.


METHODS
The one-step assay is based on asymmetric PCR amplification of the target sequence and subsequent time-resolved fluorescence measurement. Asymmetric amplification results in a single-stranded PCR product that is detected in the amplification vessel with a highly sensitive, homogeneous hybridization method.


RESULTS
A dual label, homogeneous high-throughput platform for nucleic acid sequence analysis was developed and validated using a C/T single-nucleotide polymorphism in the insulin gene as a model analyte and applied also to two other SNP-assays (poliovirus receptor A/G-polymorphism and CD86-gene exon 2 A/G-polymorphism).


CONCLUSIONS
The described high-throughput genotyping technology is very competitive in price, simple in design and easily applied to any analyte sequence.
Kiviniemi
Minna
M
Department of Virology, University of Turku, Tykistökatu 6 A, 4th floor, 20520 Turku, Finland. minna.kiviniemi@utu.fi
Nurmi
Jussi
J
Turpeinen
Hannu
H
Lövgren
Timo
T
Ilonen
Jorma
J
D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006801:Humans; D007328:Insulin / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D007328:Insulin