[Development of novel clinical tests and their contribution to laboratory diagnosis--ligase chain reaction]

Abstract

There are several methods that have been developed for the detection of a small amount of nucleic acids. Ligase chain reaction(LCR) is a highly sensitive assay for the detection of a specific DNA sequence. LCR utilizes four oligodeoxynucleotides(probes) complementary to each of target DNA strands and repeats a thermalcycling amplification step. Two pairs of oligodeoxynucleotides renature adjacent to one another on each of the separated target DNA strands, resulting in nick formation. A thermostable DNA ligase joins the nick covalently. Each ligated product can serve as a template sequence in subsequent rounds of thermalcycling (denaturation, annealing/ligation). In this manner LCR accumulates the ligated products exponentially by repeating thermalcycling. One of the distinctive features of LCR is to be able to detect a known point-mutation easily. Gap-LCR, a modified LCR, has been developed to improve the sensitivity and the specificity of the standard LCR by setting up gaps (3 < or = nucleotides) at the ligatable 3' and 5' termini of the probes. The gap-LCR probes form a short gap after annealing of the probes to the target DNA. The gap is filled in by a thermostable DNA polymerase and the resultant nick is sealed by a thermostable DNA ligase to generate the ligated probes. Gap-LCR repeats this step on thermalcycling and accumulates the LCR products in an exponential fashion.