What role does Ligase play in Multiplex Ligation-Dependent Probe Amplification?

In Multiplex Ligation-Dependent Probe Amplification (MLPA), ligase plays a critical role in connecting adjacent primers or probes. This process is fundamental because MLPA relies on the specific hybridization of probes to unique regions of target DNA. Once the probes have annealed to their complementary sequences on the target, the ligase enzyme facilitates the covalent joining of the ends of these probes. This ligation step is essential as it ensures that only those probes that are perfectly matched with their target sites are joined together, leading to a more specific and efficient amplification of the desired DNA fragments.

The successful ligation then allows for subsequent amplification of these joined probes, which is harnessed to generate a detectable signal, thus providing quantitative information about the target DNA. This specificity is crucial for obtaining accurate results in applications such as copy number variation analysis and genotyping.