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Nucleic Acids Research Advance Access originally published online on June 5, 2008
Nucleic Acids Research 2008 36(12):e75; doi:10.1093/nar/gkn357
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Nucleic Acids Research, 2008, Vol. 36, No. 12 e75
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Kaarel Krjutskov1,2, Reidar Andreson3, Reedik Mägi3, Tiit Nikopensius1, Andrey Khrunin4, Evelin Mihailov1, Veronika Tammekivi1, Helena Sork1, Maido Remm3 and Andres Metspalu1,2,5,*

1Department of Biotechnology, IMCB, University of Tartu, 2Estonian Biocentre, 3Department of Bioinformatics, IMCB, University of Tartu, Tartu, Estonia, 4Department of Molecular Bases of Human Genetics, Institute of Molecular Genetics, RAS, Moscow, Russia and 5Estonian Genome Project of University of Tartu, Tartu, Estonia

*To whom correspondence should be addressed. Tel: +372 7375029; Fax: +372 7420286; Email: andres{at}ebc.ee

Received February 28, 2008. Revised May 15, 2008. Accepted May 20, 2008.

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems.


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