Immobilized metal affinity chromatography of DNA.
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.
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Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
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D002846:Chromatography, Affinity / Q000379:methods*; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D008670:Metals; D016133:Polymerase Chain Reaction
D004277:DNA, Single-Stranded; D008670:Metals