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Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays
Authors:Krjutskov Kaarel  Andreson Reidar  Mägi Reedik  Nikopensius Tiit  Khrunin Andrey  Mihailov Evelin  Tammekivi Veronika  Sork Helena  Remm Maido  Metspalu Andres
Affiliation:Department of Biotechnology, IMCB, University of Tartu, Tartu, Estonia.
Abstract: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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