Ligase Chain Reaction

Ligase chain reaction (LCR)53,54 is initiated when a mixture of target DNA, thermostable DNA ligase, four oligonucleotide probes, and NAD+ or ATP is heated to denature dsDNA (both target and complementary probes) in the reaction mixture. Two pairs of complementary probes are used, and, of necessity, their correct design demands a priori knowledge of the sequence of the DNA target. After denaturation and subsequent reaction cooling, the four probes present in the reaction mixture hybridize to their complementary sequences on each target DNA sister strand. The two probes that hybridize to one sister strand and the two probes that bind to the other sister strand are designed such that when hybridized, the 3' hydroxyl end of the upstream probe is immediately adjacent to the 5' phosphate end of the downstream probe. Thermostable DNA ligase enzymatically ligates the two bound probes, thus achieving a "doubling" of the mass of target DNA in the reaction. As the temperature cycling proceeds, a theoretical exponential amplification of the mass of target DNA in the original reaction occurs because the resultant ligated amplicons also serve as targets for probe hybridizations. In practice, amplification is less than exponential, but sufficient to achieve target DNA identification by various detection methods.

There is a tendency for target-independent blunt-end ligation of the probes in the reaction to occur in LCR, which can cause unacceptably high levels of background signal, limiting the assay's sensitivity and specificity. This problem has been solved by use of gap LCR (G-LCR). In G-LCR, the probes are designed such that they cannot be ligated in a target-independent manner because they are not blunt ended. When G-LCR probes hybridize to target DNA, a gap of one or more bases exists between the probes hybridized to the same target strand. This gap is then biochemically "filled" in vitro, thus providing a suitable substrate for DNA ligase, which then performs target-dependent ligation.

Examples of Applications of LCR

1. Chlamydia trachomatis detection

2. Neisseria gonorrhoeae detection

Was this article helpful?

0 0

Post a comment