Sample purity is very important for MLPA while sample quality is relatively unimportant for successful MLPA reactions.
Sample purity is of utmost importance in MLPA. As it is a multiplex reaction, MLPA has higher sensitivity to sample impurities than conventional PCR. Impurities such as ethanol, metal ions, salt, or phenol/Trizol remnants can decrease the activity of one of the enzymes or alter the properties of a probe. This can lead to variable results—for example, some probes are more sensitive than others to polymerase activity, and longer amplicons are more sensitive to polymerase activity than shorter fragments in general.
Furthermore, the presence of ionic impurities such as small amounts of Mg2+ ions can result in incomplete denaturation of the sample DNA. For instance, the presence of 1 mM Mg2+ is sufficient to impede CpG islands from being completely denatured during the 5 minute 98°C heat treatment step. Thus, probes detecting sequences in or close to CpG islands can have reduced signals when DNA denaturation is incomplete.
In contrast, the quality of the DNA itself (e.g. fragmentation) does not strongly influence relative probe signals in MLPA because the target sequences of SALSA MLPA probes are very short (typically 58-82 nt). Therefore, DNA samples that are degraded to such an extent that a 1000 nt PCR is no longer possible can still yield excellent MLPA patterns in most cases.