Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

High level multiplex genotyping by MALDI-TOF mass spectrometry

Nature Biotechnology volume 16, pages 1347–1351 (1998)Cite this article

Abstract

A primer extension assay is used to perform highly multiplexed genotyping of single nucleotide polymorphisms (SNPs) present in genomic DNA amplified by a multiplex PCR. The assay uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to accurately measure the masses of short oligonucleotide primers extended by a single dideoxynucleotide. The multiplexed genotyping assays rely on the natural molecular weight differences of DNA bases. By careful analysis of primer composition complementary to the target, or by judicious addition of one or more noncomplementary 5´ bases to the genotyping primers, mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment. Using a model multiplex PCR system, we demonstrate the ability to perform 12-fold multiplex SNP analysis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mass compression for a sixfold multiplex genotyping assay.
Figure 2: (A) Twelve-fold multiplex spectrum expanded in three regions, illustrating primer-extended primer pairs.

Similar content being viewed by others

References

  1. Vestal, M.L., Juhasz, P., and Martin, S.A. 1995. Delayed extraction matrix-assisted laser desorption time-of-flight mass spectrometry. Rapid. Commun. Mass. Spectrom. 8: 865–868.

    Google Scholar 

  2. Juhasz, P., Roskey, M.T., Smirnov, I.P., Haff, L.A., Vestal, M.L., and Martin, S.A. 1996 Applications of delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to oligonucleotide analysis. Anal. Chem. 68: 941–946.

    Article  Google Scholar 

  3. Dai, Y., Whittal, R.M., Weinberger, S.R., and Li, L. 1996. Accurate mass measurement of oligonucleotides using a time-lag focusing matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Rapid. Commun. Mass Spectrom. 9: 1792– 1796.

    Article  Google Scholar 

  4. Wu, K.J., Steding, A., and Becker, C.H. 1993. Matrix-assisted laser desorption time-of-flight mass spectrometry of oligonucleotides using 3-hydroxypicolinic acid as an ultraviolet sensitive matrix. Rapid. Commun. Mass Spectrom . 4: 99–102.

    Google Scholar 

  5. Ross, P.L., Davis, P.A., and Belgrader, P. 1998. Analysis of PCR products from conventional and microfabricated thermal cyclers using delayed extraction MALDI-TOF mass spectrometry. Anal. Chem. 70: 2067–2073.

    Article  Google Scholar 

  6. Mouradian, S., Rank, D.R., and Smith, L.M. 1996. Analyzing sequencing reactions from bacteriophage M13 by matrix-assisted laser desorption/ionization mass spectrometry. Rapid. Commun. Mass Spectrom. 10: 1475–1478.

    Article  Google Scholar 

  7. Braun, A., Little, D.P., and Koster, H. 1997, Detecting CFTR gene mutations using primer oligo base extension and mass spectrometry. Clin. Chem. 43: 1151–1158.

    Google Scholar 

  8. Little, D.P., Cornish, T.J., O'Donnell, M.J., Braun, A., Cotter, R.J., and Koster, H. 1997. MALDI on a chip: analysis of arrays of low- to sub-femtomole quantities of synthetic oligonucleotides and DNA diagnostic products dispensed by a piezoelectric pipette. Anal. Chem. 69: 4540–4546.

    Article  Google Scholar 

  9. Fu, D.-J., Tang, K., Braun, A., Reuter, D., Demar, B.D., Little, D.P. et al. 1998. Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry. Nat. Biotechnol. 16: 381–384.

    Article  Google Scholar 

  10. Roskey, M.T., Juhasz, P., Smirnov, I.P., Takach, E.J., Martin, S.A., and Haff, L.A. 1996. DNA sequencing by delayed extraction matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proc. Natl. Acad. Sci. USA 93: 4724– 4729.

    Article  Google Scholar 

  11. Haff, L.A. and Smirnov, I.P. 1997. Single nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry. Genome Res . 7: 378–388.

    Article  Google Scholar 

  12. Haff, L.A. and Smirnov, I.P. 1997. Multiplex genotyping of PCR products with mass tag-labeled primers. Nucleic Acids Res. 25: 3749–3750.

    Article  Google Scholar 

  13. Ross, P.L., Lee, K., and Belgrader, P. 1997. Discrimination of single nucleotide polymorphisms in human DNA using peptide nucleic acid probes detected by MALDI-TOF mass spectrometry. Anal. Chem. 69: 4197– 4202.

    Article  Google Scholar 

  14. Griffin, T.J., Tang, W., and Smith, L.M. 1997. Genetic analysis using peptide nucleic acid affinity mass spectrometry. Nat. Biotechnol. 15: 1368–1372.

    Article  Google Scholar 

  15. Belgrader, P., Marino, M.A., Lubin, M., and Barany, F. A 1996 . A multiplex PCR–ligase detection reaction assay for human identity testing. Genome Sci. and Technol. 1: 77–87.

    Article  Google Scholar 

  16. Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X.C., Stern, D. et al. 1996. Accessing genetic information with high density DNA arrays. Science 274: 610–614.

    Article  Google Scholar 

  17. Wang, D.G., Fan, J.B., Siao, C.J., Berno, A., Young, P., Sapolsky, R. et al. 1998. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 280: 1077– 1082.

    Article  Google Scholar 

  18. Zhao, L.P., Aragaki, C., Hsu, L., and Quiaoit, F. 1998 . Mapping of complex traits by single nucleotide polymorphisms. Am. J. Hum. Genet. 63: 225– 240.

    Article  Google Scholar 

  19. Armstrong, M., Idle, J.R., and Daley, A.K. 1993. A polymorphic Cfo I site in exon 6 of the human cytochrome P450 CYPD6 gene detected by the polymerase chain reaction. Hum. Genet. 91: 616– 617.

    Article  Google Scholar 

  20. Bock, S.C., Marrinan, J.A., and Radziejewska, E. 1991. Antithrombin III utah: proline 407 to leucine mutation in a highly conserved region near the inhibitor reactive site. Biochemistry 28: 3628.

    Article  Google Scholar 

  21. Dewald, G., Nothen, M.M., and Cichon, S. 1993. Polymorphism of human complement component C6: an amino acid substitution (glu/ala) within the second thrombospondin repeat differentiates between the two common allotypes C6 A and C6 B. Biochem. Biophys. Res. Commun. 194: 458– 464.

    Article  Google Scholar 

  22. Velden, P.A. and Reitsman, P.R. 1993. Amino acid dimorphism in IL-1A is detectable by PCR amplification. Hum. Mol. Genet. 2: 1753.

    Article  Google Scholar 

  23. Cawthon, R.M., O'Connel, P., Buchberg, A.M., Viskochil, D., Weiss, R.B., Culver, M. et al. 1990. Identification and characterization of transcripts from the neurofibromatosis 1 region. The sequence and genomic structure of EV12 and mapping of other transcripts. Genomics 7: 555–565.

    Article  Google Scholar 

  24. Brooks, C.C. and Tolan, D.R. 1993. Association of the widespread A149P hereditary fructose intolerance mutation with newly identified sequence polymorphisms in the aldolase B gene. Am. J. Hum. Genet. 52: 835–840.

    Google Scholar 

  25. Poller, W., Faber, J.P., and Olek, K. 1991. Sequence polymorphism in the human alpha 2 macroglobulin (A2M) gene. Nucleic Acids Res. 19: 198.

    Article  Google Scholar 

  26. Gloudemans, T., Pospiech, I., Van der Ven, L.T.M., Lips, C.J.M., Otter, W.D., and Sussenbach, J.S. 1993. An Ava II restriction fragment length polymorphism in the insulin-like growth factor gene and the occurrence of smooth muscle tumors. Cancer Res. 53: 5754–5758.

    Google Scholar 

  27. Diepstraten, C.M., Amstel, J.K., and Bertina, R.M. 1991. A CCA/CCG neutral dimorphism in the codon for pro 626 of the protein S gene Psa (PROS1). Nucleic Acids Res. 19: 5091.

    Article  Google Scholar 

  28. Reina, M. and Deeb, S. 1992. SSCP polymorphism in the human hepatic triglyceride lipase (LIPC) gene. Hum. Mol. Genet. 1: 453.

    Article  Google Scholar 

  29. Mastuura, S. and Kishi, F. 1994. Investigation of the polymorphic AvaII site by a PCR based assay at the human CD18 gene locus. Hum. Genet. 93: 721.

    Article  Google Scholar 

  30. Warnich, L., Kotze, M.J., Langenhoven, E., and Retief, A.E. 1992. Detection of a frequent polymorphism in exon 10 of the low-density liprprotein receptor gene. Hum. Genet. 89: 362.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. PerSeptive Biosystems, 500 Old Connecticut Path, Framingham, 01701, MA

    Philip Ross, Laura Hall, Igor Smirnov & Larry Haff

Authors
  1. Philip Ross
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laura Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Igor Smirnov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Larry Haff
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Philip Ross.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ross, P., Hall, L., Smirnov, I. et al. High level multiplex genotyping by MALDI-TOF mass spectrometry. Nat Biotechnol 16, 1347–1351 (1998). https://doi.org/10.1038/4328

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/4328

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing