Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography

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dc.contributor.authorKanakaraj, Indhu
dc.contributor.authorJewell, David L.
dc.contributor.authorMurphy, Jason C.
dc.contributor.authorFox, George E.
dc.contributor.authorWillson, Richard C.
dc.date.accessioned2020-03-11T17:16:32Z
dc.date.available2020-03-11T17:16:32Z
dc.date.issued2011-1
dc.description.abstractImmobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and 揾istidine tags� genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu2+-iminodiacetic acid (IDA) agarose spin column, 94�% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu2+-IDA agarose) can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.
dc.identifier.citationCopyright 2012 PLoS ONE. Recommended citation: Kanakaraj, Indhu, David L. Jewell, Jason C. Murphy, George E. Fox, and Richard C. Willson. "Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography." PloS one 6, no. 1 (2011): e14512. DOI:10.1371/journal.pone.0014512. URL: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0014512. Reproduced in accordance with the original publisher's licensing terms and with permission from the authors.
dc.identifier.urihttps://hdl.handle.net/10657/6221
dc.language.isoen_US
dc.publisherPLoS ONE
dc.subjectpolymerase chain reaction
dc.subjectDNA purification
dc.subjectElution
dc.subjectImidazole
dc.subjectNucleotide sequencing
dc.subjectsequence alignment
dc.subjectolgonucleotides
dc.subjecthistidine
dc.titleRemoval of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography
dc.typeArticle

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