Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Oct 1;24(19):3806–3810. doi: 10.1093/nar/24.19.3806

Immobilized metal affinity chromatography of DNA.

C Min 1, G L Verdine 1
PMCID: PMC146165  PMID: 8871562

Abstract

Many of the most widely employed operations in molecular biology hinge upon the use of single-stranded DNA as a probe or template. Here we report a straightforward method by which to produce long single-stranded DNA molecules using the polymerase chain reaction (PCR) in combination with immobilized metal affinity chromatography (IMAC). We demonstrate that a tag consisting of six successive 6-histaminylpurine (H) residues (H6-tag) endows a DNA strand with selective retentivity onto a Ni2+-NTA-agarose chromatography matrix. The H6-tagged strand can then be eluted from the resin using 200 mM imidazole. Quantitative phosphorimaging analysis revealed that the PCR/IMAC procedure typically yields unmodified strands comprising >90% of the unbound DNA and H6-tagged strands comprising >95% of the bound fractions. DNA strands generated in this manner are shown to be excellent substrates for template-directed polymerization. The chemistry reported herein should facilitate a wide variety of operations in molecular biology, including automated DNA sequencing, hybridization screening of DNA libraries, assembly of gene cassettes, run-off transcription, site-directed mutagenesis and footprinting of protein-DNA complexes by template-directed interference footprinting.

Full Text

The Full Text of this article is available as a PDF (125.4 KB).

Selected References

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

  1. Allerson C. R., Verdine G. L. Synthesis and biochemical evaluation of RNA containing an intrahelical disulfide crosslink. Chem Biol. 1995 Oct;2(10):667–675. doi: 10.1016/1074-5521(95)90030-6. [DOI] [PubMed] [Google Scholar]
  2. Arnold F. H. Metal-affinity separations: a new dimension in protein processing. Biotechnology (N Y) 1991 Feb;9(2):151–156. doi: 10.1038/nbt0291-151. [DOI] [PubMed] [Google Scholar]
  3. Bowman B. H., Palumbi S. R. Rapid production of single-stranded sequencing template from amplified DNA using magnetic beads. Methods Enzymol. 1993;224:399–406. doi: 10.1016/0076-6879(93)24030-x. [DOI] [PubMed] [Google Scholar]
  4. Chen L., Oakley M. G., Glover J. N., Jain J., Dervan P. B., Hogan P. G., Rao A., Verdine G. L. Only one of the two DNA-bound orientations of AP-1 found in solution cooperates with NFATp. Curr Biol. 1995 Aug 1;5(8):882–889. doi: 10.1016/s0960-9822(95)00178-3. [DOI] [PubMed] [Google Scholar]
  5. Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hayashibara K. C., Verdine G. L. Template-directed interference footprinting of cytosine contacts in a protein-DNA complex: potent interference by 5-aza-2'-deoxycytidine. Biochemistry. 1992 Nov 24;31(46):11265–11273. doi: 10.1021/bi00161a002. [DOI] [PubMed] [Google Scholar]
  7. Mitchell L. G., Merril C. R. Affinity generation of single-stranded DNA for dideoxy sequencing following the polymerase chain reaction. Anal Biochem. 1989 May 1;178(2):239–242. doi: 10.1016/0003-2697(89)90631-3. [DOI] [PubMed] [Google Scholar]
  8. Porath J., Carlsson J., Olsson I., Belfrage G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 1975 Dec 18;258(5536):598–599. doi: 10.1038/258598a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  11. Smith M. C., Furman T. C., Ingolia T. D., Pidgeon C. Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins. J Biol Chem. 1988 May 25;263(15):7211–7215. [PubMed] [Google Scholar]
  12. Sulkowski E. The saga of IMAC and MIT. Bioessays. 1989 May;10(5):170–175. doi: 10.1002/bies.950100508. [DOI] [PubMed] [Google Scholar]

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

RESOURCES