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Digital genotyping using molecular affinity and mass spectrometry

Nature Reviews Genetics volume 4, pages 1001–1008 (2003)Cite this article

Abstract

The goal of DNA sequencing and genotyping is to efficiently generate accurate high-throughput digital genetic information that unambiguously identifies sources of genetic variation and clearly distinguishes heterozygous from homozygous variants. Recent advances in mass-spectrometry-based DNA sequencing and genotyping bode well for meeting these criteria. Pilot studies show that these recently developed approaches allow unambiguous multiplex detection of heterozygous variants and the identification of deletion and insertion variants.

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Figure 1: Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Figure 2: Solid-phase capture (SPC) sequencing.
Figure 3: Mass-sequencing spectrum from a DNA template that contains a polymorphic site.
Figure 4: Schematic representation of mutation detection using solid-phase capture (SPC) sequencing.
Figure 5: The SPC–SBE approach for multiplex SNP analysis using biotinylated ddNTPs and MALDI-TOF MS.
Figure 6: Simultaneous detection of nucleotide variations in 30 codons of the p53 gene using SPC–SBE.

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References

  1. Collins, F. S., Green, E. D., Guttmacher, A. E. & Guyer, M. S. A vision for the future of genomics research. Nature 422, 835–847 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Smith, L. M. et al. Fluorescence detection in automated DNA sequence analysis. Nature 321, 674–679 (1986).

    Article  CAS  PubMed  Google Scholar 

  3. Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N. & Mathies, R. A. Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis. Proc. Natl Acad. Sci. USA 92, 4347–4351 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ju, J., Glazer, A. N. & Mathies, R. A. Cassette labeling for facile construction of energy transfer fluorescent primers. Nucleic Acids Res. 24, 1144–1148 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kheterpal, I. et al. DNA sequencing using a four-color confocal fluorescence capillary array scanner. Electrophoresis 17, 1852–1859 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Bowling, J. M., Bruner, K. L., Cmarik, J. L. & Tibbetts, C. Neighboring nucleotide interactions during DNA sequencing gel electrophoresis. Nucleic Acids Res. 19, 3089–3097 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Yamakawa, H. & Ohara, O. A DNA cycle sequencing reaction that minimizes compressions on automated fluorescent sequencers. Nucleic Acids Res. 25, 1311–1312 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Fitzgerald, M. C., Zhu, L. & Smith, L. M. The analysis of mock DNA sequencing reactions using matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 7, 895–897 (1993).

    Article  CAS  Google Scholar 

  9. Roskey, M. T. et al. DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry. Proc. Natl Acad. Sci. USA. 93, 4724–4729 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kirpekar, F. et al. DNA sequence analysis by MALDI mass spectrometry. Nucleic Acid Res. 26, 2554–2559 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Monforte, J. & Becker, C. High-throughput DNA analysis by time-of-flight mass spectrometry. Nature Med. 3, 360–362 (1997).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Sanger, F., Nicklen, S. & Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74, 5463–5467 (1977).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Langer, P. R., Waldrop, A. A. & Ward, D. C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl Acad. Sci. USA. 78, 6633–6637 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hawkins, T. L., O'Connor-Morin, T., Roy, A. & Santillan, C. DNA purification and isolation using a solid-phase. Nucleic Acids Res. 22, 4543–4544 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Uhlen, M. Magnetic separation of DNA. Nature 340, 733–734 (1989).

    Article  CAS  PubMed  Google Scholar 

  17. Ju, J. DNA sequencing with solid phase capturable dideoxynucleotides and energy transfer primers. Anal. Biochem. 309, 35–39 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Edwards, J. R., Itagaki, Y. & Ju, J. DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry. Nucleic Acids Res. 29, 1–5 (2001).

    Article  Google Scholar 

  19. Prober, J. et al. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238, 336–341 (1987).

    Article  CAS  PubMed  Google Scholar 

  20. Gebeyehu, G., Rao, P., SooChan, P., Simms, D. A. & Klevan, L. Novel biotinylated nucleotide analogs for labeling and calorimetric detection of DNA. Nucleic Acid Res. 15, 4513–4534 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Herman, T., Lefever, E. & Shimkus, M. Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs. Anal. Biochem. 156, 48–55 (1986).

    Article  CAS  PubMed  Google Scholar 

  22. Ronaghi, M., Uhlén, M. & Nyrén, P. A sequencing method based on real-time pyrophosphate. Science 281, 363–365 (1998).

    Article  CAS  PubMed  Google Scholar 

  23. Fakhrai-Rad, H., Pourmand, N. & Ronaghi, M. Pyrosequencing™: an accurate detection platform for single nucleotide polymorphisms. Hum. Mutat. 19, 479–485 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Ruparel, H., Ulz, M. E., Kim, S. & Ju, J. Digital detection of genetic mutations using SPC–sequencing. Genome Res. (in the press).

  25. Friedman, L. S. et al. Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nature Genet. 8, 399–404 (1994).

    Article  CAS  PubMed  Google Scholar 

  26. Steele, R. J. C., Thompson, A. M., Hall, P. A. & Lane, D. P. The p53 tumour suppressor gene. Br. J. Surgery 85, 1460–1467 (1998).

    Article  CAS  Google Scholar 

  27. Kwok, P -Y. High-throughput genotyping assay approaches. Pharmacogenomics 1, 95–100 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Roses, A. Pharmacogenetics and the practice of medicine. Nature 405, 857–865 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. The International SNP Map Working Group. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409, 928–933 (2001).

  30. Beavis, R. C. & Chait, B. T. Matrix-assisted laser-desorption mass spectrometry using 355 nm radiation. Rapid Commun. Mass Spectrom. 3, 436–439 (1989).

    Article  CAS  PubMed  Google Scholar 

  31. Stoerker, J., Mayo, J. D., Tetzlaff, C. N., Sarracino, D. A., Schwope, I. & Richert, C. Rapid genotyping by MALDI-monitored nuclease selection from probe libraries. Nature Biotechnol. 18, 1213–1216 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  33. Griffin, T. J., Hall, J. G., Prudent, J. R. & Smith, L. M. Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proc. Natl Acad. Sci. USA, 96, 6301–6306 (1999).

    Article  CAS  Google Scholar 

  34. Lyamichev, V. et al. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nature Biotechnol. 17, 292–296 (1999).

    Article  CAS  Google Scholar 

  35. Haff, L. A. & Smirnov, I. P. Multiplex genotyping of PCR products with mass tag-labeled primers. Nucleic Acids Res. 25, 3749–3750 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tang, K. et al. Chip-based genotyping by mass spectrometry. Proc. Natl Acad. Sci. USA 96, 10016–10020 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ross, P., Hall, L., Smirnov, I. P. & Haff, L. High level multiplex genotyping by MALDI-TOF mass spectrometry. Nature Biotechnol. 16, 1347–1351 (1998).

    Article  CAS  Google Scholar 

  38. Fei, Z., Ono, T. & Smith, L. M. MALDI-TOF mass spectrometric typing of single nucleotide polymorphisms with mass-tagged ddNTPs. Nucleic Acids Res. 26, 2827–2828 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Griffin, T. J. & Smith, L. M. Single-nucleotide polymorphism analysis by MALDI-TOF mass spectrometry. Trends. Biotechnol. 18, 77–84 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Ding, C. & Cantor, C. R. A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. Proc. Natl Acad. Sci. USA, 100, 3059–3064 (2003).

    Article  CAS  Google Scholar 

  41. Ding, C. & Cantor, C. R. Direct molecular haplotyping of long-range genomic DNA with M1-PCR. Proc. Natl Acad. Sci. USA 100, 7449–7453 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kim, S., Edwards, J. R., Deng, L., Chung, W. & Ju, J. Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry. Nucleic Acids Res. 30, 1–6 (2002).

    Article  Google Scholar 

  43. Kim, S. et al. Multiplex genotyping of the human β2-adrenergic receptor gene using solid phase capturable dideoxynucleotides and mass spectrometry. Anal. Biochem. 316, 251–258 (2003).

    Article  CAS  PubMed  Google Scholar 

  44. Hanson, E. H., Imperatore, G. & Burke, W. HFE gene and hereditary hemochromatosis: A HuGE review. Am. J. Epidem. 154, 193–206 (2001).

    Article  CAS  Google Scholar 

  45. Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C. C. p53 mutations in human cancers. Science 253, 49–53 (1991).

    Article  CAS  PubMed  Google Scholar 

  46. Bardelli, A. et al. Mutational analysis of the tyrosine kinome in colorectal cancers. Science 300, 949 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Kim, S. et al. Thirty fold multiplex genotyping of the p53 gene using solid phase capturable dideoxynucleotides and mass spectrometry Genomics (in the press).

  48. Malkin, D., Sexsmith, E., Yeger, H., Williams, B. R. G. & Coppes, M. J. Mutations of the p53 tumor suppressor gene occur infrequently in Wilms' tumor. Cancer Res. 54, 2077–2079 (1994).

    CAS  PubMed  Google Scholar 

  49. Lahoti, C., Thorner, P., Malkin, D. & Yeger, H. Immunohistochemical detection of p53 in Wilms' tumors correlates with unfavorable outcome. Am. J. Pathol. 148, 1577–1589 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Min, B. M. et al. Inactivation of the p53 gene by either mutation or HPV infection is extremely frequent in human oral squamous cell carcinoma cell lines. Oral Oncol. Eur. J. Cancer Part B 30, 338–345 (1994).

    Article  Google Scholar 

  51. Popanda, O. et al. Mutation analysis of replicative genes encoding the large subunits of DNA polymerase-α and replication factors A and C in human sporadic colorectal cancers. Int. J. Cancer 86, 318–324 (2000).

    Article  CAS  PubMed  Google Scholar 

  52. Lin-Lee, Y. C., Tatebe, S., Savaraj, N., Ishikawa, T. & Kuo, M. T. Differential sensitivities of the MRP gene family and γ-glutamylcysteine synthetase to prooxidants in human colorectal carcinoma cell lines with different p53 status. Biochem. Pharmacol. 61, 555–563 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Rodi, C. P., Darnhofer-Patel, B., Stanssens, P., Zabeau, M. and van den Boom, D. A strategy for the rapid discovery of disease markers using the massARRAY™ system. Biotechniques 32 (Suppl.), 62–69 (2002).

    Article  Google Scholar 

  54. Chen, X., Levine, L. & Kwok, P -U. Fluorescence polarization in homogeneous nucleic acid analysis. Genome Res. 9, 492–498 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Hardenbol, P. et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nature Biotechnol. 21, 673–678 (2003).

    Article  CAS  Google Scholar 

  56. Dressman, D., Yan, H., Traverso, G., Kinzler, K. W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variation. Proc. Natl Acad. Sci. USA 100, 8817–8822 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by a Packard Fellowship for Science and Engineering (J.J.) and the Columbia University Genomics Initiative.

Author information

Authors and Affiliations

  1. Columbia Genome Center and the Department of Chemical Engineering, Columbia University College of Physicians and Surgeons, New York, 10032, USA

    Sobin Kim, Hameer D. Ruparel & Jingyue Ju

  2. Columbia Genome Center and the Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, 10032, USA

    T. Conrad Gilliam

  3. Room 405A, Russ Berrie Medical Science Pavilion, Columbia University College of Physicians and Surgeons, 1150 Saint Nicholas Avenue, New York, 10032, USA

    T. Conrad Gilliam & Jingyue Ju

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Correspondence to T. Conrad Gilliam or Jingyue Ju.

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Glossary

ANALYTE

A molecule that is of interest for analysis in a particular study.

COMPRESSION

A phenomenon in gel electrophoresis that occurs as a result of the thermodynamic stability of hairpin formations in DNA sequences that terminate in a string of G and C nucleotides.

FLOW CYTOMETRY

The analysis of single cells or subcellular particles by the detection of their light absorption, scattering and/or fluorescence properties as they pass through a laser beam in a directed fluid stream.

INVASIVE CLEAVAGE

The excision of redundant portions of DNA by DNA repair enzymes such as 5′ to 3′ exonuclease or 5′ nuclease. These redundancies are caused when two oligonucleotides, which are hybridized to the same DNA template, overlap along some of their terminal bases. The cleavage of the overlapped portion causes the formation of a nick at the position of redundancy that can later be repaired by ligation.

LASER-INDUCED FLUORESCENCE DETECTION

The measurement of emitted fluorescence signals from molecules that are excited by laser radiation.

PHARMACOGENOMICS

The study of the influence of genetic differences on the variability in the response of individuals to drugs.

PHLEBOTOMY

The removal of blood from a vein for diagnostic therapeutic purposes.

SANGER DNA SEQUENCING

A sequencing method that involves the enzymatic synthesis of DNA chains of different length using dideoxynucleotides, the separation of the DNA fragments by size and the identification of the fragments to reveal the sequences.

TAG SNPS

A small subset of SNPs that is needed to uniquely identify a complete haplotype.

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Kim, S., Ruparel, H., Gilliam, T. et al. Digital genotyping using molecular affinity and mass spectrometry. Nat Rev Genet 4, 1001–1008 (2003). https://doi.org/10.1038/nrg1230

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