Purification of nucleic acids and proteins

Metal affinity interactions can be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. A metal affinity precipitation method to separate RNA from linear and plasmid DNA has been described here. A copper charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) was used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility while imidazole chelates copper. RNA was separated from plasmid by precipitation along with the polymer at high salt concentration. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step.; Immobilized metal affinity capillary electrophoresis (IMAGE) was evaluated to study its potential use as a single nucleotide polymorphism detection tool for nucleic acids. m-PEG-IDA-M(II) (where M(II) is either Cu, Zn or Ni) was used in the separation buffer as an affinity modulator of the species with which it interacts. AMP showed an increase in retention time with increasing ligand concentration, particularly more so with m-PEG-IDA-Cu(II) while m-PEG-IDA-Ni(II) showed a higher affinity for GMP. The 20-mer, A20 and the DNA duplex with AA overhang at the 5' end showed increased retention in the presence of chelated copper.; A novel method to design ion exchange adsorbents was examined to test the hypothesis that clustering of charges on ion exchangers might enhance protein binding affinity, or enhance selectivity for proteins displaying charge clusters on their surfaces. CNBr activated agarose-based cation and anion exchangers of identical charge density was prepared using glutamic acid or lysinamide to give a random charge distribution, and pentaglutamic acid or pentalysinamide to create charge clustering. Binding studies with lysozyme, cytochrome c, alpha-lactalbumin and cytochrome b5 showed a general enhancement in protein binding capacity when the same number of charges was offered in a clustered rather than a random distribution. For the clustered charge cation exchange adsorbent, enhancement was selectively higher for cytochrome c, the surface of which displays a distinct charge cluster. The clustered charge anion exchange adsorbent was able to detect minor perturbations that disturb a charge cluster in the protein.