Elimination of contaminantEscherichia coli chromosomal DNA from preparations of P1 artifical chromosome recombinants facilitates directed subcloning

Davidson, Heather; Doherty, Ann; Stevenson, Barbara; Boyd, A. Christopher; Porteous, David J.

doi:10.1002/(sici)1522-2683(19990601)20:7<1469::aid-elps1469>3.0.co;2-w

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2005

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“…The separation of E. coli ‐derived plasmid DNA from contaminating E. coli genomic DNA is of considerable interest on both the laboratory and industrial scales. Genomic DNA contamination can impair shotgun sequencing operations (4, 5), contaminate genomic libraries (6), and is of specific FDA concern in pharmaceutical grade plasmid DNA, with a suggested level below 0.1% per dose (7).…”

Section: Introductionmentioning

confidence: 99%

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

Cano

Murphy

Fox

et al. 2005

Biotechnology Progress

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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.

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