Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.
Journal: 2002/June - Nucleic Acids Research
ISSN: 1362-4962
PUBMED: 12060695
Abstract:
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.
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Nucleic Acids Res 30(12): e57

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification

MRC-Holland, Hudsonstraat 68, 1057SN Amsterdam, The Netherlands and Department of Clinical Genetics, Free University of Amsterdam, v.d. Boechorststraat 7, 1081BT Amsterdam, The Netherlands
To whom correspondence should be addressed. Tel: +31 20 4447218; Fax: +31 20 6891149; Email: schouten@mrc-holland.com
Received 2002 Feb 22; Revised 2002 Apr 20; Accepted 2002 Apr 20.

Abstract

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.

Abstract

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.

ACKNOWLEDGEMENTS

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.

ACKNOWLEDGEMENTS

REFERENCES

REFERENCES

References

  • 1. Norman A.M., Thomas,N.S.T., Kingston,H.M. and Harper,P.S. (1990) Becker muscular dystrophy: correlation of deletion type with clinical severity. J. Med. Genet., 27, 236–239.
  • 2. Petrij-Bosch A., Peelen,T., van Vliet,M., van Eijk,R., Olmer,R., Drusedau,M., Hogervorst,F.B., Hageman,S., Arts,P.J., Ligtenberg,M.J. et al. (1997) BRCAI genomic deletions are major founder mutations in Dutch breast cancer patients. Nature Genet., 17, 341–345. [[PubMed]
  • 3. Wijnen J., van der Klift,H., Vasen,H., Khan,P.M., Menko,F., Tops,C., Meijers Heijboer,H., Lindhout,D., Moller,P. and Fodde,R. (1998) MSH2 genomic deletions are a frequent cause of HNPCC. Nature Genet., 20, 326–328. [[PubMed]
  • 4. Kauraniemi P., Barlund,M., Monni,O. and Kallioniemi,A. (2001) New amplified and highly expressed genes discovered in the ERBB2 amplicon in breast cancer by cDNA microarrays. Cancer Res., 61, 8235–8240. [[PubMed]
  • 5. Leyland-Jones B. and Smith,I. (2001) Role of herceptin in primary breast cancer. Oncology, 61 (Suppl. 2), 83–91. [[PubMed]
  • 6. Kallioniemi A., Kallioniemi,O.P., Rutovitz,D., Gray,J.W., Waldman,F. and Pinkel,D. (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science, 258, 818–821. [[PubMed]
  • 7. Klinger K., Landes,G., Shook,D., Harvey,R., Lopez,L., Locke,P., Lerner,T., Osathanondh,R., Leverone,B., Houseal,T. et al. (1992) Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH). Am. J. Hum. Genet., 51, 55–65.
  • 8. Snijders A.M., Nowak,N., Segraves,R., Blackwood,S., Brown,N., Conroy,J., Hamilton,G., Hindle,A.K., Huey,B., Kimura,K. et al. (2001) Assembly of microarrays for genome-wide measurement of DNA copy number. Nature Genet., 29, 263–264. [[PubMed]
  • 9. Devilee P., Cleton-Jansen,A.-M. and Cornelisse,C.J. (2001) Ever since Knudson. Trends Genet., 17, 569–573. [[PubMed]
  • 10. Vos P., Hogers,R., Bleeker,M., Reijans,M., van de Lee,T., Hornes,M., Frijters,A., Pot,J., Peleman,J. and Kuiper,M. (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res., 23, 4407–4414.
  • 11. Liang P. and Pardee,A.B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 257, 967–971. [[PubMed]
  • 12. Armour J.A., Sismani,C., Patsalis,P.C. and Cross,G. (2000) Measurement of locus copy number by hybridisation with amplifiable probes. Nucleic Acids Res., 28, 605–609.
  • 13. Hsuih T.C.H., Park,Y.N., Zaretski,C., Wu,F., Tyagi,S., Kramer,F.R., Sperling,R. and Zhang,D.Y. (1996) Novel, ligation-dependent PCR assay for detection of hepatitis C virus in serum. J. Clin. Microbiol., 34, 501–507.
  • 14. Carrino J.J. (1996) Patent application WO9615271A1. Multiplex ligations-dependent amplification.
  • 15. Barany F. and Lubin,M. (1997) Patent application WO9745559A1. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions.
  • 16. Pauletti G., Godolphin,W., Press,M.F. and Slamon,D.J. (1996) Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization. Oncogene, 13, 63–72. [[PubMed]
  • 17. Feo S., Di Liegro,C., Jones,T., Read,M. and Fried,M. (1994) The DNA region around the c-myc gene and its amplification in human tumor cell lines. Oncogene, 9, 955–961. [[PubMed]
  • 18. Schouten J.P. (1985) Hybridization selection of covalent nucleic acid-protein complexes. 2. Cross-linking of Escherichia coli mRNAs and DNA sequences by formaldehyde treatment of intact cells. J. Biol. Chem., 260, 9929–9935. [[PubMed]
  • 19. Pollack J.R., Perou,C.M., Alizadeh,A.A., Eisen,M.B., Pergamenschikov,A., Williams,C.F., Jeffrey,S.S., Botstein,D. and Brown,P.O. (1999) Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nature Genet., 23, 41–46. [[PubMed]
  • 20. Tong J., Cao,W. and Barany,F. (1999) Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D. Nucleic Acids Res., 27, 788–794.
  • 21. Housby J.H. and Southern,E.M. (1998) Fidelity of DNA ligation. Nucleic Acids Res., 26, 4259–4266.
  • 22. BRCA1 Exon 13 Duplication Screening Group (2000) The exon 13 duplication in the BRCA1 gene is a founder mutation present in geographically diverse populations. Am. J. Hum. Genet., 67, 207–212.
  • 23. Jones C., Payne,J., Wells,D., Delhanty,J.D., Lakhany,S.R. and Kortenkamp,A. (2000) Comparative genomic hybridization reveals extensive variation among different MCF-7 cell stocks. Cancer Genet. Cytogenet., 117, 153–158. [[PubMed]
  • 24. Messing J., Gronenborn,B., Muller-Hill,B. and Hans Hopschneider,P. (1977) Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc. Natl Acad. Sci. USA, 74, 3642–3646.
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