Quantification of Escherichia coli genomic DNA contamination in recombinant protein preparations by polymerase chain reaction and affinity-based collection.
This study describes the development of a novel assay for the quantification of Escherichia coli genomic DNA contamination in recombinant protein samples. The technique is based on PCR amplification and digoxygenin labeling of the genes encoding 5S ribosomal RNA followed by affinity-based collection and detection. Samples containing 1 pg x mL(-1) of extracted E. coli genomic DNA (gDNA) could be measured using this method. Using extracted E. coli gDNA as standards, a 35-cycle PCR reaction exhibited a linear response versus template concentration between 1 pg x mL(-1) and1 ng x mL(-1) genomic DNA even when diluted in a variety of buffering conditions. Comparison of the novel assay with a traditional filter binding and hybridization technique using recombinant protein samples confirmed that the procedure was accurate and sensitive. The assay described in this report is a safer and less expensive alternative to radioactive techniques employed for DNA quantification, utilizing readily available reagents and apparatus.
Gregory
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CA
NeuTec Pharma plc, Infectious Diseases Research Unit, Clinical Sciences Building, Manchester Royal Infirmary, University of Manchester, Oxford Road, Manchester, M13 9WL, United Kingdom. c.gregory@man.ac.uk
Rigg
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GP
Illidge
C M
CM
Matthews
R C
RC
D001483:Base Sequence / Q000235:genetics; D015139:Blotting, Southern / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004076:Digoxigenin / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes / Q000235:genetics; D016678:Genome; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012341:RNA, Ribosomal, 5S / Q000235:genetics
D004269:DNA, Bacterial; D012341:RNA, Ribosomal, 5S; D004076:Digoxigenin