Quantification of Escherichia coli Genomic DNA Contamination in Recombinant Protein Preparations by Polymerase Chain Reaction and Affinity-Based Collection

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...

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Bibliographic Details
Published in:Analytical biochemistry Vol. 296; no. 1; pp. 114 - 121
Main Authors: Gregory, Carl A, Rigg, Gordon P, Illidge, Christopher M, Matthews, Ruth C
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01-09-2001
Subjects:
Base Sequence - genetics
Blotting, Southern - methods
Digoxigenin - analysis
DNA contamination
DNA, Bacterial - analysis
DNA, Bacterial - genetics
E. coli
ELISA
Escherichia coli
Escherichia coli - genetics
Genes - genetics
Genome
Molecular Sequence Data
Nucleic Acid Hybridization - methods
PCR
Polymerase Chain Reaction - methods
recombinant protein
RNA, Ribosomal, 5S - genetics
rRNA 5S
DNA contamination
recombinant protein
E. coli
PCR
ELISA
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Summary: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 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 mL−1 and1 ng 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.
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ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2001.5237