Ligase Chain Reaction for the Detection of Specific DNA Sequences and Point Mutations

  • R. Bruce Wallace
  • Ching-I P. Lin
  • Antonio A. Reyes
  • Jimmie D. Lowery
  • Luis Ugozzoli


DNA diagnostics is a new field that utilizes the techniques of molecular biology to identify and characterize the specific genetic material associated with genetic, neoplastic, and infectious diseases. DNA diagnostics grew out of the discoveries made possible using molecular cloning and DNA sequencing techniques beginning in the mid-1970s. Despite the availability of exquisitely specific reagents such as restriction endonucleases, synthetic oligonucleotides, and cloned DNA and cDNA probes, as well as technologies such as electrophoretic methods to fractionate DNA and novel DNA hybridization methods (Southern and Northern blots, dot blot, etc.), DNA diagnostic procedures were not immediately adopted by clinical laboratories. This failure was due in part to the long turnaround times, the lack of sensitivity, and the lack of reliable nonradioactive detection procedures.


Human Papilloma Virus Cystic Fibrosis Transmembrane Conductance Regulator Chlamydia Trachomatis Ligation Product Cystic Fibrosis Transmembrane Regulator 
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  1. Abravaya, K., Carrino, J. J., Muldoon, S., and Lee, H. H. (1995). Detection of point mutations with a modified ligase chain reaction (gap-LCR). Nucleic Acids Res. 23:675–682.PubMedCrossRefGoogle Scholar
  2. Balles, J., and Plfugfelder, G. O. (1994). Facilitated isolation of rare recombinants by ligase chain reaction: Selection for intragenic crossover events in the Drosophila optomotor-blind gene. Mol. Gen. Genet. 245:734–740.PubMedCrossRefGoogle Scholar
  3. Barany, F. (1991a). Genetic disease and DNA amplification using cloned thermostable ligase. Proc. Natl. Acad. Sci. USA 88:189–193.PubMedCrossRefGoogle Scholar
  4. Barany, F. (1991b). The ligase chain reaction in a PCR world. PCR Methods Appl. 1:5–16.PubMedCrossRefGoogle Scholar
  5. Barany, F., and Gelfand, D. H. (1991). Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene. Gene 109:1–11.PubMedCrossRefGoogle Scholar
  6. Bassiri, M., Hu, H. Y., Domeika, M. A., Burczak, J., Svensson, L.-O., Lee, H. H. and Mardh, P.-A. (1995). Detection of Chlamydia trachomatis in urine specimens from women by ligase chain reaction. J. Clin. Microbiol. 33:898–900.PubMedGoogle Scholar
  7. Batt, C. A., Wagner, P., Wiedmann, M., Luo, J., and Gilbert, R. (1994). Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction. Anim. Genet. 25:95–98.PubMedGoogle Scholar
  8. Birkenmeyer, L., and Armstrong, A. S. (1992). Preliminary evaluation of the ligase chain reaction for specific detection of Neisseria gonorrhoeae. J. Clin. Microbiol. 30:3089–3094.Google Scholar
  9. Chernesky, M. A., Jang, D., Lee, H., Burczak, J. D., Hu, H., Sellors, J., Tomazic-Allen, S. J., and Mahony, J. B. (1994a). Diagnosis of Chlamydia trachomatis infections in men and women by testing first-void urine by ligase chain reaction. J. Clin. Chem. 32:2682–2685.Google Scholar
  10. Chemesky, M. A., Lee, H, Schachter, J., Burczak, J. D., Stamm, W. E., McCormack, W. M., and Quinn, T. C. (1994b). Diagnosis of Chlamydia trachomatis urethral infection in symptomatic and asymptomatic men by testing first-void urine in a ligase chain reaction assay. J. Infect. Dis. 170:1308–1311.CrossRefGoogle Scholar
  11. Chetverin, A. B., and Kramer, F. R. (1994). Oligonucleotide arrays: New concepts and possibilities. Bio/ Technology 12:1093–1099.PubMedCrossRefGoogle Scholar
  12. Delahunty, C. M., Ankener, W., Brainerd, S., Nickerson, D. A., and Monomen, I. T. (1995). Finnish-type aspartylglucosaminuria detected by oligonucleotide ligation assay. Clin. Chem. 41:59–61.PubMedGoogle Scholar
  13. Dille, B. J., Butzen, C. C., and Birkenmeyer, L. G. (1993). Amplification of Chlamydia trachomatis DNA by ligase chain reaction. J. Clin. Microbiol. 31:729–731.PubMedGoogle Scholar
  14. Eckert, K. A., and Kunkel, T. A. (1991). DNA polymerase fidelity and the polymerse chain reaction. PCR Methods Appl. 1:17–24.PubMedCrossRefGoogle Scholar
  15. Eggerding, F.fA., Iovannisci, D. M., Brinson, E., Grossman, P., and Winn-Deen, E. S. (1995). Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations. Hum. Mutat. 5:153–165.PubMedCrossRefGoogle Scholar
  16. Grimberg, J., Lin, C. P., Sheridan, K., Tackney, C., and Waksal, J. (1993). Detection of serum hepatitis B virus DNA sequence by the repair chain reaction DNA amplification system. Clin. Chem. 39:738.Google Scholar
  17. Grossman, P. D., Bloch, W., Brinson, E., Chang, C. C., Eggerding, F. A., Fung, S., Iovannisci, D.A., Woo, S., and Winn-Deen, E. S. (1994). High-density multiplex detection of nucleic acid sequences: Oligonucleotide ligation assay and sequence-coded separation. Nucleic Acids Res. 22:4527–4534.PubMedCrossRefGoogle Scholar
  18. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993). Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Bio/Technology 11:1026–1030.PubMedCrossRefGoogle Scholar
  19. Jou, C., Rhoads, J., Bouma, S., Ching, S. F., Hoijer, J., Schroeder-Poliak, P., Zaun, P., Smith, S., Richards, S., Caskey, C. T., and Gordon, J. (1995). Deletion detection in the dystrophin gene by multiplex gap ligase chain reaction and immunochromatographic strip technology. Hum. Mutat. 5:86–93.PubMedCrossRefGoogle Scholar
  20. Kleppe, K., Ohtsuka, E., Kleppe, R., Molineux, I., and Khorana, H. G. (1971). Studies on polynucleotides. XCVI. Repair replication of short synthetic DNAs as catalyzed by DNA polymerases. J. Mol. Biol. 56:341–361.PubMedCrossRefGoogle Scholar
  21. Landegren, U., Kaiser, R., Sanders, J., and Hood, L. (1988). A ligase-mediated gene detection technique. Science 241:1077–1080.PubMedCrossRefGoogle Scholar
  22. Lawyer, F C., Stoffel, S., Saiki, R. K., Myambo, K., Drummond, R., and Gelfand, D. H. (1989). Isolation, characterization, and expression in E. coli of the DNA polymerase gene from the extreme thermophile, Thermus aquaticus. J. Biol. Chem. 264:6427–6437.PubMedGoogle Scholar
  23. Leckie, G., Cao, J., Davis, A., Facey, I., Lin, B.-C, and Lee, H. (1994). Ligase chain reaction (LCR) DNA amplification for direct detection of Mycobacterium tuberculosis (MTB) in clinical specimens, in:Abstracts of the General Meeting of the American Society for Microbiology, 189.Google Scholar
  24. Lee, H. H., Chernesky, M. A., Schachter, J., Burczak, J. D., Andrews, W. W, Muldoon, S., Leckie, G., and Stamm, W. E. (1995). Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine. Lancet 345:213–216.PubMedCrossRefGoogle Scholar
  25. Lin, C. P., Goldbard, S., Grills, G., Sheridan, K., Waksal, H. W, Tackney, C., and Segev. D. (1991). Amplification and nonradioactive detection of the human papilloma virus 16 DNA by the repair chain reaction, in Abstracts of the Annual San Diego Conference on Nucleic Acids..Google Scholar
  26. Ling, L. L., Keohavong, P., Dias, C., and Thilly, W. G. (1991). Optimization of polymerase chain reaction with regard to fidelity: Modified T7, Taq and Vent DNA polymerases. PCR Methods Appl. 1:63–69.PubMedCrossRefGoogle Scholar
  27. Liu, M. S., Zang, J., Evangelista, R. A., Rampai, S., and Chen, F.-T. A. (1995). Double-stranded DNA analysis by capillary electrophoresis with laser-induced fluorescence using ethidium bromide as an intercalator. Bio-Techniques 18:316–323.Google Scholar
  28. Livak, K. J., Marmaro, J., and Todd, J. A. (1995). Towards fully automated genome-wide polymorphism screening. Nature Genet. 9:341–342.PubMedCrossRefGoogle Scholar
  29. Marshall, R. L., Laffler, T. G., Cerney, M. B., Sustachek, J. C., Kratochvil, J. D., and Morgan, R. L. (1994). Detection of HCV RNA by the asymmetric gap ligase chain reaction. PCR Methods Appl. 4:80–84.PubMedCrossRefGoogle Scholar
  30. Mattila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991). Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19:4967–4973.PubMedCrossRefGoogle Scholar
  31. Nakazawa, H., English, D., Randell, P. L., Nakazawa, K., Martel, N., Armstrong, B. K., and Yamasaki, H. (1994) UV and skin cancer: Specific p53 gene mutation in normal skin as a biologically relevant exposure measurement. Proc. Natl. Acad. Sci. USA 91:360–364.PubMedCrossRefGoogle Scholar
  32. Nickerson, D. A., Kaiser, R., Lappin, S., Stewart, J., Hood, L., and Landegren, U. (1990). Automated DNA diagnostics using an EUSA-based oligonucleotide ligation assay. Proc. Natl. Acad. Sci. USA 87:8923–8927.PubMedCrossRefGoogle Scholar
  33. Pfeffer, M., Meyer, H., and Wiedmann, M. (1994). A ligase chain reaction targeting two adjacent nucleotides allows the differentiation of cowpox virus from other Orthopoxvirus species. J. Virol. Methods 49:353–360.PubMedCrossRefGoogle Scholar
  34. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, F. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Ehrlich, H. A. (1988). Primer-directed enzymatic amplification of cDNA with a thermal stable DNA polymerase. Science 239:487–491.PubMedCrossRefGoogle Scholar
  35. Schachter, J., Stamm, W. E., Quinn, T. C., Andrews, W. W., Burzak, J. D., and Lee, H. H. (1994). Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix. J. Clin. Microbiol. 32:2540–2543.PubMedGoogle Scholar
  36. Schwartz, H. E., and Ulfelder, K. J. (1992). Capillary electrophoresis with laser-induced fluorescence detection of PCR fragments using thiazole orange. Anal. Chem. 64:1737–1740.CrossRefGoogle Scholar
  37. Segev, D. (1992). Amplification of nucleic acid sequence by the repair chain reaction, in:Nonradioactive Labeling and Detection of Biomolecules (C. Kessler, ed.), Springer-Verlag, Berlin, pp. 212–218.Google Scholar
  38. Takahashi, M., Yamaguchi, E., and Uchida, T. (1984). Thermophilic DNA ligase—Purification and properties of the enzyme from Thermus thermophilus HB8. J. Biol. Chem. 259:10041–10047.PubMedGoogle Scholar
  39. Tavormina, P. L., Shiang, R., Thompson, L. M., Zhu, Y.-Z., Wilkin, D. J., Lachman, R. S., Wilcox, W. R., Rimoin, D. L., Cohn, D. H., and Wasmuth, J. J. (1995). Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nature Genet. 9:321–328.PubMedCrossRefGoogle Scholar
  40. Ugozzoli, L., and Wallace, R. B. (1991). Allele specific polymerase chain reaction. Methods: A Companion to Methods in Enzymology 2:42–48.CrossRefGoogle Scholar
  41. Wallace, R. B., Ugozzoli, L., Lowery, J., and Reyes, A. A. (1994). The application of LCR to neonatal testing of variant hemoglobin genes. Proceedings, 10th National Neonatal Screening Symposium, Seattle, Washington, pp. 127–130.Google Scholar
  42. Ward, L. J., Brown, J. C., and Davey, G. P. (1994). Application of the ligase chain reaction to the detection of nisinZ genes in Lactococcus lactis ssp. lactis. FEMS Microbiol. Lett. 117:29–34.PubMedCrossRefGoogle Scholar
  43. Wiedmann, M., Wilson, W. J., Czajka, J., Luo, J., Barany, F., and Batt, C. A. (1994). Ligase chain reaction (LCR)—Overview and applications. PCR Methods Appl. 3:S51–S64.PubMedCrossRefGoogle Scholar
  44. Wu, D. Y., and Wallace, R. B. (1989a). The ligation amplification reaction (LAR)—Amplification of specific DNA sequences using sequential rounds of template-dependent ligation. Genomics 4:560–569.PubMedCrossRefGoogle Scholar
  45. Wu, D. Y., and Wallace, R. B. (1989b). Specificity of the nick-closing activity of bacteriophage T4 DNA ligase. Gene 76:245–254.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • R. Bruce Wallace
    • 1
  • Ching-I P. Lin
    • 1
  • Antonio A. Reyes
    • 1
  • Jimmie D. Lowery
    • 1
  • Luis Ugozzoli
    • 1
  1. 1.DNA Diagnostics DivisionBio-Rad LaboratoriesHerculesUSA

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