Ligase Chain Reaction And Ligase Detection Reaction

Ligase chain reaction utilizes two complementary oligo-nucleotide probe pairs, which are specific for a DNA target sequence. In the presence of the target sequence, each probe of a pair hybridizes to adjacent positions on the template such that the 5'-phosphate of one oligonucleotide abuts the 3'-hydroxyl of the other. The resulting nick is subsequently sealed by a DNA ligase. By temperature cycling, bound ligated units dissociate from the target and the released ligated unit becomes the target for the next round of ligation which is initiated by aligning the opposite sense adjacent probes. After repeated cycles of denaturation, annealing, and ligation the target sequence is amplified exponentially (Fig. 1). Ligase detection reaction employs only one set of oligonucleotide probes in a thermal cycling reaction similar to the one described above, resulting in linear product increase. Attaching biotin on the first probe and a suitable reporter group on the second probe allows for product capture and detection in a manner amenable to automation.1-3,4-1 Ligase chain reaction and ligase detection reaction are very sensitive assays for discriminating DNA sequences that differ by only one single base pair. A mismatch in the ligation junction inhibits the ligation reaction thus conferring an excellent resolution power to hybridization, decreasing the need for high stringency conditions and thus providing excellent conditions for multiplexing reactions.[3,5] A great potential of LCR is its compatibility with other nucleic acid amplification techniques such as PCR, 3 SR, and the Qp replicase assay. Real-time applications,1-11,12-1 multiplexing assays,[13,14] competitive LCR and multiplex-competitive LCR,[15] quantitative LCR,[16] and nested LCR[4] have been described. The sensitivity of LCR is dependent on the fidelity of the ligase. Four thermostable ligases are commercially available: 1) Taq ligase (New England Biolabs, Beverly, MA); 2) Tth (Abbott Diagnostics, Abbott Park, IL); 3) Pfu (Stratagene, La Jolla, CA); and 4) Ampligase (Epicenter Technologies, Madison, WI). Pfu was advertised to have higher ligation specificity and lower background than Tth.[7] Although

Was this article helpful?

0 0
Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment