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

Figure 5

Schematic diagrams for the Ligation Chain Reaction (LCR) and Ligation Amplification Reaction (LAR) processes. (Here, we describe LCR.) DNA fragments from the normal type and the mutant type (at the single nucleotide polymorphism (SNP) site) are shown as reaction templates. (1) The denaturation process is the same as in Figure 3 (1). (2) Hybridization: four oligonucleotide probes, complementary to the normal-type target, hybridize to the target DNA fragment, and (3) Ligation: adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase. Ligated LCR probes from the first round of the ligation become the targets for the subsequent round using another probe set (dotted lines) complementary to the first set. Thus, the amount of ligated LCR probes increases exponentially. In the case of the mutant type template, the presence of a single-base mismatch at the junction inhibits the ligation, and therefore no ligated LCR probes are formed (right).