Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays.
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.
Krjutskov
Kaarel
K
Department of Biotechnology, IMCB, University of Tartu, Tartu, Estonia.
Andreson
Reidar
R
Mägi
Reedik
R
Nikopensius
Tiit
T
Khrunin
Andrey
A
Mihailov
Evelin
E
Tammekivi
Veronika
V
Sork
Helena
H
Remm
Maido
M
Metspalu
Andres
A
D017931:DNA Primers; D005838:Genotype; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers