Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography.
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 Cu(2+)-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 matrices (specifically Cu(2+)-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.
Department of Biology and Biochemistry, University of Houston, Houston, Texas, United States of America.
Indhu
I
Kanakaraj
David L
DL
Jewell
Jason C
JC
Murphy
George E
GE
Fox
Richard C
RC
Willson
D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers / Q000302:isolation & purification*; D007098:Imino Acids; D008722:Methods; D009711:Nucleotides / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
D002614:Chelating Agents; D017931:DNA Primers; D007098:Imino Acids; D009711:Nucleotides; C008109:iminodiacetic acid
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