Abstract
The ability to analyze multiple polymorphic sites rapidly and accurately is crucial in all areas of genetic analysis. We have developed an approach for the detection of multiple point mutations, using allele-specific, mass-labeled, peptide nucleic acid (PNA) hybridization probes, and direct analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The composite mass spectra produced contain peaks of distinct masses corresponding to each allele present, resulting in a mass spectral “fingerprint” for each DNA sample. The hybridization characteristics of PNA:DNA duplexes were found to be highly dependent on both base content and sequence. Results from the analysis of four polymorphic sites contained in exon 4 of the human tyrosinase gene show that this approach is simple, rapid, and accurate with potential applications in many areas of genetic analysis.
Similar content being viewed by others
References
Forrest, S., Cotton, R., Landegren, U., and Southern, E. 1995. How to find all those mutations. Nature Genet. 10: 375–376.
Caskey, C.T. 1987. Disease diagnosis by recombinant DNA methods. Science 236: 1223–1229.
Landegren, U., Kaiser, R., Caskey, C.T., and Hood, L. 1988. DNA diagnostics-molecular techniques and automation. Science 242: 229–237.
Cotton, R. 1989. Detection of single base changes in nucleic acids. J. Biochem. 263: 1–10.
Saiki, R., Walsh, P.S., Levenson, C., and Erlich, H. 1989. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Nat. Acad. Sci. USA 86: 6230–6234.
Wallace, B., Shaffer, J., Murphy, R.F., Bonner, J., Hirose, T., and Ikatura, K. et al. 1979. Hybridization of synthetic oligodeoxy-ribonucleotides to φχ 174 DNA: the effect of a single base pair mismatch. Nucl. Acids. Res. 6: 3543–3557.
Zhang, Y., Coyne, M., Will, S., Levenson, C., and Kawasaki, E. 1991. Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides. Nucl. Acids Res. 19: 3929–3933.
Guo, Z., Guilfoyle, R., Thiel, A., Wang, R., and Smith, L.M. 1994. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucl. Acids Res. 22: 5456–5465.
Hillenkamp, F., Karas, M., Beavis, R., and Chait, B. 1991. Matrix-assisted laser desorption-ionization mass spectrometry of biopolymers. Anal. Chem. 63: 1193A–1203A.
Karas, M., Bachmann, D., Bahr, U., and Hillenkamp, F. 1987. Matrix-assisted ultraviolet laser desorption of non-volatile compounds. Int. J. Mass Spectrom. Ion Phys. 78: 53–68.
Chou, C., Bingham, S., and Williams, P. 1996. Affinity methods for purification of DNA sequencing reaction products for mass spectrometric analysis. Rapid Commun. Mass Spectrom. 10: 1410–1414.
Köster, H., Tang, K., Fu, D., Braun, A., van den Boom, D., Smith, C.L., et al. 1996. A strategy for rapid and efficient DNA sequencing by mass spectrometry. Nature Biotechnology 14: 1123–1128.
Girault, S., Chassaing, G., Blais, J., Brunot, A., and Bolbach, G. 1996. Coupling of MALDI-TOF mass analysis to the separation of biotinylated peptides by magnetic streptavidin beads. Anal. Chem. 68: 2122–2126.
Tang, K., Fu, D., Kotter, S., Cotter, R.J., Cantor, C., and Koster, H. 1995. Matrix-assisted laser desorption/ionization mass spectrometry of immobilized duplex DNA probes. Nucl. Acids Res. 23: 3126–3131.
Jurinke C., van den Boom, B., Jacob, A., Tang, K., Worl, R., and Koster, H. 1996. Analysis of ligase chain reaction products via matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry. Anal. Biochem. 237: 174–181.
Nielsen, P., Egholm, M., Berg, R., and Buchardt, O. 1991. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254: 1497–1500.
Egholm, M., Buchardt, O., Nielsen, P., and Berg, R. 1992. Peptide nucleic acids (PNA): oligonucleotide analogues with an achiral peptide backbone. J. Am. Chem. Soc. 114: 1895–1897.
Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S.M., Driver, D., et al. 1993. PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules. Nature 365: 566–568.
Tomac, S., Sarkar, M., Ratilainen, T., Wittung, P., Nielsen, P., Borden, B., and Graslund, A. 1996. Ionic effects on the stability and conformation of peptide nucleic acid complexes. J. Am. Chem. Soc. 118: 5544–5552.
Butler, J., Jiang-Baucom, P., Huang, M., Belgrader, P., and Girard, J. 1996. Peptide nucleic acid characterization by MALDI-TOF mass spectrometry. Anal. Chem. 68: 3283–3287.
Giebel, L., Strunk, K., and Spritz, R. 1991. Organization and nucleotide sequence of the human tyrosinase gene and a truncated tyrosinase-related segment. Genomics 9: 435–445.
Tripathi, R., Strunk, K., Giebel, L., Weleber, R., and Spritz, R. 1992. Tyrosinase gene mutations in type I (tyrosinase-deficient) oculocutaneous albinism define two clusters of missense substitutions. Am. J. Med. Genet. 43: 865–871.
Wu, K., Steding, A., and Becker, C. 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. 7: 142–146.
Zhu, L., Parr, G., Fitzgerald, M., Nelson, C., and Smith, L.M. 1995. Oligonucleotide fragmentation in MALDI/TOF mass spectrometry using 355 nm radiation. J. Am. Chem. Soc. 117: 6048–6056.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Griffin, T., Tang, W. & Smith, L. Genetic analysis by peptide nucleic acid affinity MALDI-TOF mass spectrometry. Nat Biotechnol 15, 1368–1372 (1997). https://doi.org/10.1038/nbt1297-1368
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt1297-1368
- Springer Nature America, Inc.
This article is cited by
-
PNAs as novel cancer therapeutics
International Journal of Peptide Research and Therapeutics (2003)
-
Progress in high throughput SNP genotyping methods
The Pharmacogenomics Journal (2002)
-
Accessing genetic variation: genotyping single nucleotide polymorphisms
Nature Reviews Genetics (2001)
-
Peptide Nucleic Acids and Biosensor Technology for Real-Time Detection of the Cystic Fibrosis W1282X Mutation by Surface Plasmon Resonance
Laboratory Investigation (2001)