Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification
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.
Samples containing ∼100 ng DNA were analysed by MLPA using probe mix P001. Male and female control DNA was obtained from Promega. Blood-derived DNA from triple 21 and triple X individuals as well as DNA from a triple 13 and a triple 18 cell line were provided by the Department of Clinical Genetics, Free University of Amsterdam. Reactions were analysed by capillary electrophoresis on a Beckman CEQ2000. Peak area of each probe amplification product was divided by the combined peak area of all 40 probes. The resulting relative peak area of each probe amplification product was divided by the relative peak area of that probe obtained on female control DNA. The presence of two copies of a probe target sequence/diploid genome should therefore result in a relative signal of 1.00. The presence of three copies of a probe target sequence/diploid genome should result in a 1.50 relative probe signal. The exact sequence recognised by each probe of the P001 probe mix can be found at the www.mrc-holland.com website.
The authors thank Maria Worsham (Henry Ford Hospital, Detroit) for providing DNA from the SkBr3 cell line, M. Spaargaren and S. T. Pals (AMC, Amsterdam) for providing DLBCL DNA samples and Tibor van Welsem and Petra Nederlof (Netherlands Cancer Institute, Amsterdam) for providing DNA samples from primary breast tumours. We thank Rahanna Nasrullah, Peter den Harder and Yordi Coffa for excellent technical assistance. C.J.M. is a recipient of a Marie Curie Industrial Host Fellowship. J.P.S. thanks Jesus Calmero for many years of excellent technical assistance and the Instrumentmakerij Biologie and the Biology faculty of the Free University of Amsterdam for 17 years of support.
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