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:

Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips

Nature Biotechnology volume 17, pages 365–370 (1999)Cite this article

Abstract

We have developed a rapid assay for single nucleotide polymorphism (SNP) detection that utilizes electronic circuitry on silicon microchips. The method was validated by the accurate discrimination of blinded DNA samples for the complex quadra-allelic SNP of mannose binding protein. The microchip directed the transport, concentration, and attachment of amplified patient DNA to selected electrodes (test sites) creating an array of DNA samples. Through control of the electric field, the microchip enabled accurate genetic identification of these samples using fluorescently labeled DNA reporter probes. The accuracy of this approach was established by internal controls of dual labeled reporters and by using mismatched sequences in addition to the wild-type and variant reporter sequences to validate the SNP-genotype. The ability to customize this assay for multiple genes has advantages over other existing approaches.

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: Microchip image, assay overview, and MBP sequences.
Figure 2: Cy3/Cy5 images of a representative chip.
Figure 3: Quantification and scoring of blinded samples.
Figure 4: MBP blinded samples (allele D) and IL-1β T/T homozygote.

Similar content being viewed by others

References

  1. Schafer, A.J. & Hawkins, J.R. DNA variation and the future of human genetics. Nat. Biotechnol. 16, 33– 39 (1998).

    Article  CAS  Google Scholar 

  2. Jeffreys, A.J. DNA sequence variants in the Gγ-, Aγ-, δ- and β-globin genes in of man. Cell 18, 1– 10 (1979).

    Article  CAS  Google Scholar 

  3. Cooper, D.N., Smith, B.A., Cooke, H.J., Niemann, S. & Schmidtke J. An estimate of unique DNA sequence heterozygosity in the human genome. Hum. Genet. 69, 201– 205 (1985).

    Article  CAS  Google Scholar 

  4. Collins, F.S., Guyer, M.S. & Chakravarti, A. Variations on a theme: cataloging human DNA sequence variation. Science 278, 1580– 1581 (1997).

    Article  CAS  Google Scholar 

  5. Housman, D. & Ledley, F.D. Why pharmacogenomics? Why now? Nat. Biotechnol. 16, 492– 493 (1998).

    Article  CAS  Google Scholar 

  6. Vesell, E.S. Therapeutic lessons from pharmacogenetics. Ann. Intern. Med. 126, 653–655 (1997).

    Article  CAS  Google Scholar 

  7. Pennisi, E. DNA sequencers' trial by fire. Science 280, 814–817 (1998).

    Article  CAS  Google Scholar 

  8. Marshall, E. Snipping away at genome patenting. Science 277, 1752–1753 (1998).

    Article  Google Scholar 

  9. Wang, D.G. et al. Large-scale identification and genotyping of single-nucleotide polymorphisms in the genome. Science 280, 1077– 1082 (1998).

    Article  CAS  Google Scholar 

  10. Fu, D.J. et al. Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry. Nat. Biotechnol. 16, 381–384 (1998).

    Article  CAS  Google Scholar 

  11. Tyagi, S., Bratu, D.P. & Kramer, F.R. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49– 53 (1998).

    Article  CAS  Google Scholar 

  12. Piatek, A.S. et al. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat. Biotechnol. 16, 359–363 (1998).

    Article  CAS  Google Scholar 

  13. Nielsen, P.E., Egholm, M., Berg, R.H. & Buchardt, O. Sequence selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497–1500 (1991).

    Article  CAS  Google Scholar 

  14. Weiler, J., Gausepohl, H., Hauser, N., Jensen, O.N. & Hoheisel, J.D. Hybridization based DNA screening on peptide nucleic acid (PNA) oligomer arrays. Nucleic Acids Res. 25, 2792–2799 ( 1997).

    Article  CAS  Google Scholar 

  15. Zhang, Y., Coyne, M.Y., Will, S.G., Leveson, C.H. & Kawaski, E.S. Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides. Nucleic Acids Res. 19, 3929–3933 (1991).

    Article  CAS  Google Scholar 

  16. Livak, K.J., Marmaro, J. & Todd, J.A. Towards fully automated genome-wide polymorphism screening. Nat. Genet. 9, 341–342 (1995).

    Article  CAS  Google Scholar 

  17. Edman, C.F. et al. Electric field directed nucleic acid hybridization on microchips. Nucleic Acid Res. 25, 4907– 4914 (1997).

    Article  CAS  Google Scholar 

  18. Marshall, A. & Hodgson, J. DNA chips: an array of possibilities. Nat. Biotechnol. 16, 27– 31 (1998).

    Article  CAS  Google Scholar 

  19. Sosnowski, R., Tu, E., Butler, W.B., O'Connell, J.P. & Heller, M.J. Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control. Proc. Natl. Acad. Sci. 94, 1119–1123 ( 1997).

    Article  CAS  Google Scholar 

  20. Sumiya, M. et al. Molecular basis of opsonic defect in immunodeficient children. Lancet 337, 1569–1570 ( 1991).

    Article  CAS  Google Scholar 

  21. Turner, M.W. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol. Today 17, 532– 540 (1996).

    Article  CAS  Google Scholar 

  22. Madsen, H.O. et al. A new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein. Immunogenetics 40, 37–44 (1994).

    Article  CAS  Google Scholar 

  23. Epstein, J., Eichbaum, Q., Sheriff, S. & Ezekowitz, R.A. The collectins in innate immunity. Curr. Opin. Immunol. 8, 29–35 (1996).

    Article  CAS  Google Scholar 

  24. Garred, P. et al. Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin. Lancet 349, 239–240 (1997).

    Google Scholar 

  25. Sullivan, K.E., Wooten, C., Goldman, D. & Petri, M. Mannose-binding protein genetic polymorphisms in black patients with systemic lupus erythematosus. Arthritis Rheum. 39, 2046– 2051 (1996).

    Article  CAS  Google Scholar 

  26. Ezekowitz, A. mannose-binding protein and susceptibility to chronic hepatitis B infection. Lancet 348, 1396–1397 (1996).

    Article  CAS  Google Scholar 

  27. Thomas, H.C. et al. Mutation of gene of mannose-binding protein associated with chronic hepatitis B viral infection. Lancet 348, 1417–1419 (1996).

    Article  CAS  Google Scholar 

  28. Summerfield, J.A., Sumiya, M., Levin, M. & Turner, M.W. Association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series. Br. Med. J. 314, 1229– 1232 (1997).

    Article  CAS  Google Scholar 

  29. Moss, T. in Handbook on Semiconductors 3rd edn (ed Mahajan, S.) (North-Holland, Amsterdam, The Netherlands, 1993).

    Google Scholar 

  30. Foster, C.B. et al. Host defense molecule polymorphisms influence risk for immune-mediated complications in chronic granulomatous disease. J. Clin. Invest. 102, 2146–2155 ( 1998).

    Article  CAS  Google Scholar 

  31. Kornman, K.S. et al. The interleukin-1 genotype as a severity factor in adult peridontal disease. J. Clin. Peridontol. 24, 72– 77 (1997).

    Article  CAS  Google Scholar 

  32. Malmgren, H., Gustavsson, J., Tuvemo, T. & Dahl, N. Rapid detection of a mutation hot-spot in the human androgen receptor. Clin. Genet. 50, 202–205 (1996).

    Article  CAS  Google Scholar 

  33. Winzeler, E.A. et al. Direct allelic variation scanning of the yeast genome. Science 281, 1194–1197 ( 1998).

    Article  CAS  Google Scholar 

  34. Cheng, J. et al. Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips. Nat. Biotechnol. 16, 541–546 (1998).

    Article  CAS  Google Scholar 

  35. Cheng, J., Sheldon, E.L., Wei, L., Heller, M.J. & O'Connell, J.P. Isolation of cultured cervical carcinoma cells mixed with peripheral blood cells on a bioelectronic chip. Anal. Chem. 70, 2321–2326 ( 1998).

    Article  CAS  Google Scholar 

  36. Van Zant, P. Microchip Fabrication: A Practical Guide To Semiconductor Processing (McGraw Hill, New York, 1997).

    Google Scholar 

  37. Yates, C. et al. Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azthiopurine and mercaptopurine intolerance. Ann. Intern. Med. 126, 608–614 ( 1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Michael Heller, James O'Connell, Mark Collins, Elaine Weidenhammer, and Tina Nova for critical reading of the manuscript, and Daniel Raymond, Lori Westin, and Don Ackley for technical assistance.

Author information

Author notes

  1. Patrick N. Gilles & Patrick J. Dillon

    Present address: Invitrogen Corporation, 1600 Faraday, Carlsbad, CA, 92008

Authors and Affiliations

  1. Nanogen Inc., 10398 Pacific Center Ct. , San Diego, 92121, CA

    Patrick N. Gilles, David J. Wu & Patrick J. Dillon

  2. Pediatric Oncology Branch, National Cancer Institutes, National Institute of Health, 9000 Rockville Pike, Bethesda, 20892, MD

    Charles B. Foster & Stephen J. Chanock

Authors
  1. Patrick N. Gilles
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David J. Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Charles B. Foster
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patrick J. Dillon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stephen J. Chanock
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David J. Wu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gilles, P., Wu, D., Foster, C. et al. Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips. Nat Biotechnol 17, 365–370 (1999). https://doi.org/10.1038/7921

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/7921

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