Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Jan 1;88(1):189–193. doi: 10.1073/pnas.88.1.189

Genetic disease detection and DNA amplification using cloned thermostable ligase.

F Barany 1
PMCID: PMC50775  PMID: 1986365

Abstract

Polymerase chain reaction, using thermostable DNA polymerase, has revolutionized DNA diagnostics. Another thermostable enzyme, DNA ligase, is harnessed in the assay reported here that both amplifies DNA and discriminates a single-base substitution. This cloned enzyme specifically links two adjacent oligonucleotides when hybridized at 65 degrees C to a complementary target only when the nucleotides are perfectly base-paired at the junction. Oligonucleotide products are exponentially amplified by thermal cycling of the ligation reaction in the presence of a second set of adjacent oligonucleotides, complementary to the first set and the target. A single-base mismatch prevents ligation/amplification and is thus distinguished. This method was exploited to detect 200 target molecules as well as to discriminate between normal beta A- and sickle beta S- globin genotypes from 10-microliters blood samples.

Full text

PDF
189

Images in this article

191Image
on p.191
192Image
on p.192
193Image
on p.193

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barringer K. J., Orgel L., Wahl G., Gingeras T. R. Blunt-end and single-strand ligations by Escherichia coli ligase: influence on an in vitro amplification scheme. Gene. 1990 Apr 30;89(1):117–122. doi: 10.1016/0378-1119(90)90213-b. [DOI] [PubMed] [Google Scholar]
  2. Chamberlain J. S., Gibbs R. A., Ranier J. E., Nguyen P. N., Caskey C. T. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 1988 Dec 9;16(23):11141–11156. doi: 10.1093/nar/16.23.11141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chehab F. F., Kan Y. W. Detection of specific DNA sequences by fluorescence amplification: a color complementation assay. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9178–9182. doi: 10.1073/pnas.86.23.9178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Conner B. J., Reyes A. A., Morin C., Itakura K., Teplitz R. L., Wallace R. B. Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides. Proc Natl Acad Sci U S A. 1983 Jan;80(1):278–282. doi: 10.1073/pnas.80.1.278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cotton R. G., Rodrigues N. R., Campbell R. D. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4397–4401. doi: 10.1073/pnas.85.12.4397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cui X. F., Li H. H., Goradia T. M., Lange K., Kazazian H. H., Jr, Galas D., Arnheim N. Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9389–9393. doi: 10.1073/pnas.86.23.9389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guatelli J. C., Whitfield K. M., Kwoh D. Y., Barringer K. J., Richman D. D., Gingeras T. R. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1874–1878. doi: 10.1073/pnas.87.5.1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Horvath S. J., Firca J. R., Hunkapiller T., Hunkapiller M. W., Hood L. An automated DNA synthesizer employing deoxynucleoside 3'-phosphoramidites. Methods Enzymol. 1987;154:314–326. doi: 10.1016/0076-6879(87)54082-4. [DOI] [PubMed] [Google Scholar]
  9. Ishino Y., Shinagawa H., Makino K., Tsunasawa S., Sakiyama F., Nakata A. Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli. Mol Gen Genet. 1986 Jul;204(1):1–7. doi: 10.1007/BF00330179. [DOI] [PubMed] [Google Scholar]
  10. Kornher J. S., Livak K. J. Mutation detection using nucleotide analogs that alter electrophoretic mobility. Nucleic Acids Res. 1989 Oct 11;17(19):7779–7784. doi: 10.1093/nar/17.19.7779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kwok S., Kellogg D. E., McKinney N., Spasic D., Goda L., Levenson C., Sninsky J. J. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 1990 Feb 25;18(4):999–1005. doi: 10.1093/nar/18.4.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Landegren U., Kaiser R., Caskey C. T., Hood L. DNA diagnostics--molecular techniques and automation. Science. 1988 Oct 14;242(4876):229–237. doi: 10.1126/science.3051381. [DOI] [PubMed] [Google Scholar]
  13. Landegren U., Kaiser R., Sanders J., Hood L. A ligase-mediated gene detection technique. Science. 1988 Aug 26;241(4869):1077–1080. doi: 10.1126/science.3413476. [DOI] [PubMed] [Google Scholar]
  14. Lander E. S. DNA fingerprinting on trial. Nature. 1989 Jun 15;339(6225):501–505. doi: 10.1038/339501a0. [DOI] [PubMed] [Google Scholar]
  15. Larder B. A., Kemp S. D. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. doi: 10.1126/science.2479983. [DOI] [PubMed] [Google Scholar]
  16. Miyada C. G., Wallace R. B. Oligonucleotide hybridization techniques. Methods Enzymol. 1987;154:94–107. doi: 10.1016/0076-6879(87)54072-1. [DOI] [PubMed] [Google Scholar]
  17. Myers R. M., Larin Z., Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science. 1985 Dec 13;230(4731):1242–1246. doi: 10.1126/science.4071043. [DOI] [PubMed] [Google Scholar]
  18. Orita M., Iwahana H., Kanazawa H., Hayashi K., Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2766–2770. doi: 10.1073/pnas.86.8.2766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  20. Saiki R. K., Walsh P. S., Levenson C. H., Erlich H. A. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6230–6234. doi: 10.1073/pnas.86.16.6230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Studier F. W. A strategy for high-volume sequencing of cosmid DNAs: random and directed priming with a library of oligonucleotides. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6917–6921. doi: 10.1073/pnas.86.18.6917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Takahashi M., Yamaguchi E., Uchida T. Thermophilic DNA ligase. Purification and properties of the enzyme from Thermus thermophilus HB8. J Biol Chem. 1984 Aug 25;259(16):10041–10047. [PubMed] [Google Scholar]
  24. Wilson G. G., Murray N. E. Molecular cloning of the DNA ligase gene from bacteriophage T4. I. Characterisation of the recombinants. J Mol Biol. 1979 Aug 15;132(3):471–491. doi: 10.1016/0022-2836(79)90270-5. [DOI] [PubMed] [Google Scholar]
  25. Wu D. Y., Wallace R. B. Specificity of the nick-closing activity of bacteriophage T4 DNA ligase. Gene. 1989;76(2):245–254. doi: 10.1016/0378-1119(89)90165-0. [DOI] [PubMed] [Google Scholar]
  26. Wu D. Y., Wallace R. B. The ligation amplification reaction (LAR)--amplification of specific DNA sequences using sequential rounds of template-dependent ligation. Genomics. 1989 May;4(4):560–569. doi: 10.1016/0888-7543(89)90280-2. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES