title: New separation-free assay technique for SNPs using two-photon excitation fluorometry.
abstract: A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia trade mark TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot trade mark assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.
authors.affiliation: Laboratory of Biophysics, Institute of Biomedicine, University of Turku, Finland.
authors.forename: Jonne
authors.initials: J
authors.lastname: Vaarno
authors.forename: Emmi
authors.initials: E
authors.lastname: Ylikoski
authors.forename: Niko J
authors.initials: NJ
authors.lastname: Meltola
authors.forename: Juhani T
authors.initials: JT
authors.lastname: Soini
authors.forename: Pekka
authors.initials: P
authors.lastname: Hänninen
authors.forename: Riitta
authors.initials: R
authors.lastname: Lahesmaa
authors.forename: Aleksi E
authors.initials: AE
authors.lastname: Soini
mesh_terms: D017931:DNA Primers; D005470:Fluorometry / Q000379:methods*; D005838:Genotype; D006801:Humans; D008863:Microspheres; D017785:Photons; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers