A single mismatch can affect the hybridisation of the probe oligonucleotides to their targets. Mismatches at the ligation site always disrupt the ligation of oligonucleotides (see this article about detecting point mutations with MLPA), although the extent of the disruption depends on the type of mismatch. Furthermore, ligation speed is decreased by mismatches close (within approximately 5 nt) to the ligation site, resulting in a decreased signal for that probe. Mismatches at other places in the target sequence can also reduce the peak signal as they may disrupt the stability of the probe’s hybridization to the sample DNA. 

Background

An advantage of the sensitivity of MLPA probes to mismatches is that it allows for the possibility to detect known point mutations. However, this means that a (non-pathogenic) polymorphism can sometimes result in a decreased MLPA probe signal, which can mimic a deletion. When probes are designed by MRC-Holland, we try to avoid known SNPs found in a variety of public genetic databases (including NCBI GenBank). However, as new polymorphisms are continuously being discovered, and as some regions have a lot of known SNPs, it is impossible to exclude all SNPs. This is why MRC-Holland recommends confirming all MLPA findings with another method, especially in the case of single probe deletions.