1. INTENDED USE

Copy Number Variations (CNVs) are a prominent source of genetic variation in human DNA and play a role in a wide

range of disorders. Multiplex Ligation-dependent Probe Amplification (MLPA) is a semi-quantitative technique that is

used to determine the relative copy number of up to 60 DNA sequences in a single multiplex PCR-based reaction.

MRC-Holland manufactures and sells MLPA reagents and a wide range of MLPA probemixes. Together, these can be

used to detect deletions and duplications in a DNA sample. Details on the intended use are specified in the MLPA

probemix-specific Product Description.

2. MLPA ASSAY PRINCIPLE

As outlined in Figure 1, the principle of MLPA is based on the amplification (by use of a single PCR primer pair) of up

to 60 probes, each of which detecting a specific DNA sequence of approximately 60 nt in length. After denaturation of

the sample DNA, a mixture of MLPA probes is added to the sample. Each MLPA probe consists of two oligonucleotides

that must hybridise to immediately adjacent target sequences in order to be ligated into a single probe. Each probe in

an MLPA probemix has a unique amplicon length, typically ranging between 130-500 nt. During the subsequent PCR

reaction, all ligated probes are amplified simultaneously using the same PCR primer pair. One PCR primer is

fluorescently labelled, enabling the amplification products to be visualised during fragment separation. This is done on

a capillary electrophoresis instrument, yielding a specific electropherogram (Figure 2, left). The relative height of each

individual probe peak, as compared to the relative probe peak height in various reference DNA samples, reflects the

relative copy number of the corresponding target sequence in the sample. A deletion of one or more target sequence

thus becomes apparent as a relative decrease in peak height (Figure 2, right), while an increase in relative peak

height reflects an amplification.