Jason C. Murphy

Purification of plasmid DNA using selective precipitation by compaction agents

A scaleable method for the liquid-phase separation of plasmid DNA from RNA.

Nucleic acid separations utilizing immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) is widely used for protein purification, e.g., in the isolation of proteins bearing the well‐known hexahistidine affinity tag. We report that IMAC matrixes can also adsorb single‐stranded nucleic acids through metal ion interactions with aromatic base nitrogens and propose that metal affinity technologies may find widespread application in nucleic acid technology. Oligonucleotide duplexes, plasmid, and genomic DNA show low IMAC binding affinity, while RNA and single‐stranded oligonucleotides bind strongly to matrixes such as Cu(II) iminodiacetic acid (IDA) agarose. The affinity of yeast RNA for IDA‐chelated metal ions decreases in the following order: Cu(II), Ni(II), Zn(II), and Co(II). Adsorption isotherms for 20‐mer oligonucleotide homopolymers show that purines are strongly favored over pyrimidines and that double‐stranded duplexes are not bound. IMAC columns have been used to purify plasmid DNA from E. coli alkaline lysates, to purify a ribozyme, to remove primers and imperfect products from PCR reactions, and to separate 20‐mer oligonucleotide duplexes containing centered single‐base mismatches. Potential further applications include SNP scoring, hybridization assays, and the isolation of polyadenylated messenger RNA.

Enhancement of anion-exchange chromatography of DNA using compaction agents

The use of adsorptive chromatography for preparative nucleic acid separations is often limited by low capacity. The possibility that the adsorbent surface area sterically accessible to nucleic acid molecules could be increased by reducing their radius of gyration with compaction agents has been investigated. The equilibrium adsorption capacity of Q Sepharose anion-exchange matrix for plasmid DNA at 600 mM NaCl was enhanced by up to ca. 40% in the presence of 2.5 mM spermine. In addition, compaction agent selectivity has been demonstrated. Spermine, for example, enhances the adsorption of both plasmid and genomic DNA, spermidine enhances binding only of plasmid, and hexamine cobalt enhances only the binding of genomic DNA. Compaction may be generally useful for enhancing adsorptive separations of nucleic acids.

Nucleotide modification at the γ-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase

The mechanism by which HIV-1 reverse transcriptase (HIV-RT) discriminates between the correct and incorrect nucleotide is not clearly understood. Chemically modified nucleotides containing 1-aminonaphthalene-5-sulfonate (ANS) attached to their γ-phosphate were synthesized and used to probe nucleotide selection by this error prone polymerase. Primer extension reactions provide direct evidence that the polymerase is able to incorporate the gamma-modified nucleotides. Forward mutation assays reveal a 6-fold reduction in the mutational frequency with the modified nucleotides, and specific base substitutions are dramatically reduced or eliminated. Molecular modeling illustrates potential interactions between critical residues within the polymerase active site and the modified nucleotides. Our data demonstrate that the fidelity of reverse transcriptase is improved using modified nucleotides, and we suggest that specific modifications to the γ-phosphate may be useful in designing new antiviral therapeutics or, more generally, as a tool for defining the structural role that the polymerase active site has on nucleotide selectivity.

Spermine compaction is an efficient and economical method of producing vaccination-grade DNA

Plasmid DNA inoculations can induce both humoral and cellular immunity, and this technique is now being employed in developing vaccination regimens for a large number of applications. DNA vaccination studies require the preparation of large amounts of purified plasmid DNA with low endotoxin contamination, and the cost burden for multiple injections, multiple animal or large animal studies is significant. We recently reported that selective compaction with spermine can be used to purify large quantities of DNA. We wanted to determine whether this method would produce DNA suitable for vaccination. Endotoxin levels for spermine-compacted DNA were 0.3+/-0.01 endotoxin units (EU)/microg, well within the accepted range (less than 3 EU/microg) for in vivo use. When injected intramuscularly into mice, column-purified and spermine-compacted DNA induced an equivalent antigen-specific CD8+ T-cell response. The labor and time involved in purifying 5 mg of DNA by each method were similar, but the cost of spermine-compacted DNA was only 20% of the cost of column-purified DNA. We conclude that spermine compaction is an efficient and economical method for preparing vaccination-grade DNA.

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

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.

Compaction agent clarification of microbial lysates

Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

Compaction agent protection of nucleic acids during mechanical lysis.

Mechanical lysis is an efficient and widely used method of liberating the contents of microbial cells, but the sensitivity of large nucleic acids to shear damage has prevented the application of mechanical lysis to DNA purification. It is demonstrated that polycationic compaction agents can protect DNA from shear damage and allow chromosomal and plasmid DNA purification by mechanical lysis. In addition to being substantially protected during mechanical lysis, the compacted DNA can be separated with the insoluble cell debris, washed, and selectively resolubilized, yielding a substantially purified DNA product. An additional benefit of this method is that lysate viscosity is greatly reduced, allowing the use of much smaller processing volumes when compared with traditional lysis methods used in nucleic acid purification.

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 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 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.