Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1979 Nov 24;7(6):1485–1495. doi: 10.1093/nar/7.6.1485

Specific chemical labeling of DNA fragments.

H Eshaghpour, D Söll, D M Crothers
PMCID: PMC342322  PMID: 228251

Abstract

We describe a simple method for specific chemical labeling of DNA fragments at their 3'-termini. The procedure includes enzymatic addition of 4-thiouridine, followed by reaction in mild non-denaturing conditions with the highly reactive alpha-haloacetamido derivatives of several chemical labels. The attached reporter molecule can be removed by extended treatment with beta-mercaptoethanol. Among the potential applications of this labeling method is the study of specific protein-DNA interactions in solution.

Full text

PDF
1485

Images in this article

1493Image
on p.1493

Selected References

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

  1. Beardsley K., Cantor C. R. Studies of transfer RNA tertiary structure by singlet-singlet energy transfer. Proc Natl Acad Sci U S A. 1970 Jan;65(1):39–46. doi: 10.1073/pnas.65.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bollum F. J., Chang L. M., Tsiapalis C. M., Dorson J. W. Nucleotide polymerizing enzymes from calf thymus gland. Methods Enzymol. 1974;29:70–81. doi: 10.1016/0076-6879(74)29010-4. [DOI] [PubMed] [Google Scholar]
  3. Eshaghpour H., Crothers D. M. Preparative separation of the complementary strands of DNA restriction fragments by alkaline RPC-5 chromatography. Nucleic Acids Res. 1978 Jan;5(1):13–21. doi: 10.1093/nar/5.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hara H., Horiuchi T., Saneyoshi M., Nishimura S. 4-Thiouridine-specific spin-labeling of E. coli transfer RNA. Biochem Biophys Res Commun. 1970 Jan 23;38(2):305–311. doi: 10.1016/0006-291x(70)90713-8. [DOI] [PubMed] [Google Scholar]
  5. Hudson E. N., Weber G. Synthesis and characterization of two fluorescent sulfhydryl reagents. Biochemistry. 1973 Oct 9;12(21):4154–4161. doi: 10.1021/bi00745a019. [DOI] [PubMed] [Google Scholar]
  6. Klevan L., Crothers D. M. Isolation and characterization of a spacerless dinucleosome from H1-deleted chromatin. Nucleic Acids Res. 1977 Dec;4(12):4077–4089. doi: 10.1093/nar/4.12.4077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kruse T. A., Clark B. F. The effect of specific structural modification on the biological activity of E. coli arginine tRNA. Nucleic Acids Res. 1978 Mar;5(3):879–892. doi: 10.1093/nar/5.3.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  9. Ofengand J. The function of pseudouridylic acid in transfer ribonucleic acid. I. The specific cyanoethylation of pseudouridine, inosine, and 4-thiouridine by acrylonitrile. J Biol Chem. 1967 Nov 10;242(21):5034–5045. [PubMed] [Google Scholar]
  10. Roychoudhury R., Jay E., Wu R. Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 1976 Apr;3(4):863–877. doi: 10.1093/nar/3.4.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Rozovskaia T. A., Bibilashvili P. Sh, Tarusova N. B., Gurskii G. V., Strel'tsov S. A. Vvedenie fluorestsentnoi metki na 3'OH-konets DNK i 3'-OH-konets rastushchei tsepi RNK. Mol Biol (Mosk) 1977 May-Jun;11(3):598–610. [PubMed] [Google Scholar]
  12. Schwartz I., Ofengand J. Photo-affinity labeling of tRNA binding sites in macromolecules. I. Linking of the phenacyl-p-azide of 4-thiouridine in (Escherichia coli) valyl-tRNA to 16S RNA at the ribosomal P site. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3951–3955. doi: 10.1073/pnas.71.10.3951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sollner-Webb B., Melchior W., Jr, Felsenfeld G. DNAase I, DNAase II and staphylococcal nuclease cut at different, yet symmetrically located, sites in the nucleosome core. Cell. 1978 Jul;14(3):611–627. doi: 10.1016/0092-8674(78)90246-5. [DOI] [PubMed] [Google Scholar]
  14. Sprinzl M., Siboska G. E., Pedersen J. A. Properies of tRNAPhe from yeast carrying a spin label on the 3'-terminal. Interaction with yeast phenylalanyl-tRNA Synthetase and elongation factor Tu from Escherichia coli. Nucleic Acids Res. 1978 Mar;5(3):861–877. doi: 10.1093/nar/5.3.861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Szabo L., Kalman T. I., Bardos T. J. The reaction of 5-bromouracil derivatives with sulfur nucleophiles, and a novel synthetic route to 5-sulfur-substituted uracils and nucleotides. J Org Chem. 1970 May;35(5):1434–1437. doi: 10.1021/jo00830a038. [DOI] [PubMed] [Google Scholar]
  16. Wetzel R., Söll D. Analogs of methionyl-tRNA synthetase substrates containing photolabile groups. Nucleic Acids Res. 1977;4(5):1681–1694. doi: 10.1093/nar/4.5.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Yang C. H., Söll D. Studies of transfer RNA tertiary structure of singlet-singlet energy transfer. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2838–2842. doi: 10.1073/pnas.71.7.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES