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.
Schouten
Jan P
JP
MRC-Holland, Hudsonstraat 68, 1057SN Amsterdam, The Netherlands. schouten@mrc-holland.com
McElgunn
Cathal J
CJ
Waaijer
Raymond
R
Zwijnenburg
Danny
D
Diepvens
Filip
F
Pals
Gerard
G
D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D002352:Carrier Proteins; D002460:Cell Line; D002869:Chromosome Aberrations; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins; D005091:Exons; D005260:Female; D019398:Genes, BRCA1; D006801:Humans; D008297:Male; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D014314:Trisomy; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein