Principle of the MLPA® technique:
MLPA (Multiplex Ligation-dependent Probe Amplification) is a multiplex PCR method detecting copy numbers of up to 60 different genomic DNA sequences, which is able to distinguish sequences differing in only one nucleotide. MLPA reactions require only 50 ng of human chromosomal DNA. It can be used for a wide variety of disorders including: the detection of exon deletions/duplications in e.g. the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples, SNP and mutation detection, DNA methylation analysis. The MLPA technique is easy to use, as it only requires a thermocycler and capillary electrophoresis equipment.
The MLPA reaction can be divided in five major steps (see figure 1):
- DNA denaturation/hybridisation: DNA is denatured and incubated overnight with a mixture of MLPA probes. MLPA probes consist of two separate oligonucleotides(LPO and RPO), each containing one of the PCR primer sequences. The two probe oligonucleotides hybridise to immediately adjacent target sequences.
- Ligation reaction: when the two probe oligonucleotides are both hybridised to their adjacent targets, they can be ligated during the ligation reaction.
- PCR amplification reaction: Because only ligated probes will be exponentially amplified during the PCR reaction, the number of probe ligation products is a measure for the number of target sequences in the sample.
- Fragment Separation: The amplification products are separated using capillary electrophoresis.
- Analysis and report: The analysis of the samples can be done easily with the Coffalyser® software, which is free of charge.
Figure 1: schematic overview of the MLPA technique
Principle of the MS-MLPA® technique:
With the MS-MLPA® technique a second reaction is added specific for methylation profiling. After the denaturation and hybridisation step, the DNA sample is divided in two: one tube for the copy number calculation, which is basically the standard MLPA® reaction, and one tube for the methylation profiling. For the methylation profiling, an additional restriction enzyme HhaI is added. This will determine the methylation status of the target sequence. The amplification of the products and the fragment separation will be done in the same manner as described before for MLPA®. With the Coffalyser® Software, the analysis of the copy number calculation and the methylation profiling is determined in a clear manner (see figure 2).
Figure 2: schematic overview of the MS-MLPA technique
The advantages of MLPA are:
Although for most hereditary conditions, (partial) gene deletions or duplications account for less than 10% of all disease-causing mutations, for many other disorders this is even higher (e.g. up to 36% of Hereditary Predisposition to Breast Cancer caused by deletions/duplications, 70% of Duchenne Muscular Dystrophy caused by deletions).
The inclusion of MLPA in clinical settings can therefore significantly increase the detection rate of many genetic disorders.