Separation of Genomic DNA from Plasmid DNA by Selective Renaturation with Immobilized Metal Affinity Capture

Abstract

In contrast to proteins, many nucleic acids can undergo reversible modification of their conformations, and this flexibility can be used to facilitate purification. Selective renaturation with capture is a novel method of removing contaminating genomic DNA from plasmid samples. Plasmid DNA quickly renatures after thermal denaturation and cooling (or alkaline denaturation followed by neutralization), whereas genomic DNA remains locally denatured after rapid cooling in mismatch-stabilizing high ionic strength buffer. Partially denatured genomic DNA can be selectively bound to a metal chelate affinity adsorbent through exposed purine bases, while double-stranded renatured plasmid DNA is not bound. Using this method we have readily achieved 1,000,000-fold clearance of 71 wt % contaminating E. coli genomic DNA from plasmid samples.

Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry

“…Strong Affinities of Nucleic Acids for IMAC Beads IMAC has previously been used for the isolation and purification of nucleic acids because of the strong affinities of phosphate groups for the IMAC beads [6][7][8]. To further confirm the affinities of nucleic acids for the IMAC beads, six aliquots of each of the nucleic acid samples were loaded onto 0.4 ml IMAC-column at different pH values (pH 1.5, 1.9, 2.8, 3.3, 4.3, and 6.5) and eluted with 3 ml of NH 4 OH (pH 10.5).…”

“…Although many studies have been done to optimize the enrichment of phosphopeptides by IMAC, the effect of nucleic acids in a protein sample on phosphopeptide enrichment has not yet been well clarified. It is known that nucleic acids, which contain abundant phosphate groups with negative charges, have strong affinities for IMAC beads [6][7][8]. Nucleic acids, especially eukaryotic RNAs, are the most common contaminants in protein samples from cells or tissues.…”

DNA nanotechnology: a future perspective

“…These methods may include, but are not limited to, physical separation which may apply the use of metals to chelate partially denatured purine bases and allow elimination of errors [88] or PCR-based approaches such as hairpin PCR, which completely separates genuine mutations from polymerase mis-incorporations. Hairpin PCR operates by converting a DNA sequence to a hairpin following ligation of oligonucleotide caps to DNA ends.…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…The feasibility of using IMAC for the purification of pDNA directly from an alkaline cell lysate was also reported (Tan et al 2007), but its recovery was only possible by introducing a pre-treatment step. The main disadvantage of IMAC is the impossibility to isolate pDNA from a gDNA-containing extract, due to the similar double stranded structure (Cano et al 2005).…”