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Principle of MLPA
MLPA probemixes have probes that target a specific genomic sequence. An MLPA probe consists of two parts: a left and a right probe oligonucleotide (LPO and RPO). The LPO and RPO contain PCR primer and DNA hybridisation sequences. An additional stuffer sequence in the RPO gives each probe a unique length.
Sample denaturation and probe hybridisation
In the first step, purified sample DNA is denatured. This is followed by overnight incubation with MLPA probe oligos. The LPO and RPO parts of each probe hybridise to immediately adjacent target DNA sequences.
The second step is the ligation of probe oligos that hybridised to immediately adjacent target sequences. No mismatches around the ligation site are permitted, making the ligation reaction highly specific. The number of probe ligation products is a measure for the number of target sequences in the sample.
In the third step, ligated probes are amplified in a multiplex PCR using a single universal primer pair. Only ligated probes are exponentially amplified, making the removal of unbound and non-ligated probes unnecessary.
The fourth step is fragment separation by capillary electrophoresis. PCR products are loaded onto a capillary electrophoresis device and separated by length. Each fragment corresponds to a specific MLPA probe.
The final step is data analysis by Coffalyser.Net. Relative copy numbers are determined by comparing the relative peak heights of reference probes and target probes in the test samples with those in reference samples with a known normal copy number. Advanced quality checks help to recognize unreliable data.