PCR Mutation Detection Protocols

Petra G. C. Eijk - Van Os, Jan P. Schouten, Os, Petra G. C. Eijk - Van, Schouten, Jan P.

Multiplex Ligation-dependent Probe Amplification (MLPA®) is a high-throughput method developed to determine the copy number of up to 50 genomic DNA sequences in a single multiplex PCR-based reaction. MLPA is easy to perform, requires only 20 ng of sample DNA and can distinguish sequences differing in only a single nucleotide. The MLPA reaction results in a mixture of amplification fragments ranging between 100 and 500 nt in length which can be separated and quantified by capillary electrophoresis. The equipment necessary for MLPA is identical to that for performing standard sequencing reactions: a thermocycler and a fluorescent capillary electrophoresis system. Comparison of the peak pattern obtained on a DNA sample to that of a reference sample indicates which sequences show aberrant copy numbers.Fundamental for the MLPA technique is that it is not the sample DNA that is amplified during the PCR reaction, but MLPA probes that hybridise to the sample DNA. Each MLPA probe consists of two probe oligonucleotides, which should hybridise adjacent to the target DNA for a successful ligation. Only ligated probes can be exponentially amplified by PCR. In contrast to standard multiplex PCR, only one pair of PCR primers is used for the MLPA PCR reaction, resulting in a more robust system. This way, the relative number of fragments present after the PCR reaction depends on the relative amount of the target sequence present in a DNA sample.