Journal: Genome Biology
Article Title: A low-cost open-source SNP genotyping platform for association mapping applications
Figure Lengend Snippet: Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.
Article Snippet: OLA and OLA amplification reaction conditions The OLA reactions are just 3 μl in volume, and contain 1 × OLA buffer (50 mM Tris-HCl pH 8.5, 50 mM KCl, 7.5 mM MgCl2 , 1 mM NAD), 2.5 mM dithiothreitol, 1.6 units Taq (Thermus aquaticus ) DNA ligase (NEB), and 0.03 pmol of each genotyping oligo.
Techniques: Polymerase Chain Reaction, Ligation, Amplification, Sequencing, Labeling