We describe a new method for relative quantification of 40 different DNA sequences
in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown
of this multiplex ligation-dependent probe amplification (MLPA) technique include
the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1
genes, detection of trisomies such as Down's syndrome, characterisation of chromosomal
aberrations in cell lines and tumour samples and SNP/mutation detection. Relative
quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic
acids but probes added to the samples are amplified and quantified. Amplification
of probes by PCR depends on the presence of probe target sequences in the sample.
Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that
hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides
are ligated, permitting subsequent amplification. All ligated probes have identical
end sequences, permitting simultaneous PCR amplification using only one primer pair.
Each probe gives rise to an amplification product of unique size between 130 and 480
bp. Probe target sequences are small (50-70 nt). The prerequisite of a ligation reaction
provides the opportunity to discriminate single nucleotide differences.