From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

Figure 3

Schematic diagrams for the Oligonucleotide Ligation Assay (OLA). DNA fragments from the normal type and the mutant type (at the single nucleotide polymorphism (SNP) site) are shown as reaction templates. (1) Denature: samples are heated to 85–95°C for one to several minutes to denature (separate into single strands) the target DNA. (2) Hybridization: two oligonucleotide probes, 5′-biotin labeled probes and 3′-fluorescent (or radiolabeled) probes, which are complementary to the normal-type target, hybridize to the target DNA fragment. (3) Ligation: adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase. (4) Binding to support: the fluorescently labeled ligation samples are immobilized to streptavidin and detected by fluorography only if ligated to biotinylated oligonucleotides that can be bound to streptavidin on a solid support (left).