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Nucleic Acids Research 2004 32(13):e108; doi:10.1093/nar/gnh102
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Published online 19 July 2004

Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved

New separation-free assay technique for SNPs using two-photon excitation fluorometry

Jonne Vaarno1, Emmi Ylikoski2,3, Niko J. Meltola4, Juhani T. Soini4, Pekka Hänninen1, Riitta Lahesmaa3 and Aleksi E. Soini4,*

1 Laboratory of Biophysics, Institute of Biomedicine, University of Turku, Finland, 2 Turku Graduate School for Biomedicine (TuBS), Turku, Finland, 3 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland and 4 Arctic Diagnostics Oy, 20521 Turku, Finland

* To whom correspondence should be addressed. Tel: +358 40 5510424; Fax: +358 2 2637 1250; Email: info{at}arcdia.com

Received May 7, 2004; Revised June 1, 2004; Accepted June 24, 2004

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 ArcDiaTM 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 SNaPshotTM assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.


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