removal of pcr error products and unincorporated primers by metal-chelate affinity chromatography删除产品和非公司引物pcr错误metal-chelate亲和色谱法.pdfVIP

Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography 1 2¤a 2¤b 1 1,2 Indhu Kanakaraj , David L. Jewell , Jason C. Murphy , George E. Fox , Richard C. Willson * 1 Department of Biology and Biochemistry, University of Houston, Houston, Texas, United States of America, 2 Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, United States of America Abstract Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and ‘‘histidine 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, 94–99% 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