Influence of fragment size on DNA quantitation using DNA-binding proteins and a sensor-based analytical system: applications in the testing of biological products.
A novel immunoassay system which rapidly quantifies picogram levels of total DNA was characterized with respect to the effects of DNA length. Nine chromatographically purified HaeIII restriction fragments of phi X174 were tested. Assay performance was found to be dependent on both the amount and length of DNA present in the sample. DNA fragments longer than 100 base pairs (bp) could be quantitatively detected with this system. Fragments shorter than 100 bp inhibited assay performance and thus could be detected through the use of inhibition studies; however, only qualitative information could be obtained. DNA fragments approximately 10 nucleotides in length had no apparent effect on assay performance. The size of the binding site (number of bases) required for each DNA-binding protein to bind to a nucleic acid fragment is suggested as an explanation for the observed influence of DNA size on assay performance. The total DNA assay was used in conjunction with a Pharmacia FPLC system to characterize the size distribution and amount of DNA in two partially purified biopharmaceutical samples. The results indicate that the majority of residual DNA in these samples is less than 600 bp in length. This technique can be used to rapidly determine the DNA size distribution in an in-process or final product biopharmaceutical sample. This data can then be used in process design and optimization for removal of residual DNA in biological products.
King
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Molecular Devices Corporation, Menlo Park, California 94025.
Panfili
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D000818:Animals; D010584:Bacteriophage phi X 174; D001482:Base Composition / Q000187:drug effects; D001688:Biological Products / Q000737:chemistry* / Q000592:standards; D002417:Cattle; D002845:Chromatography; D004279:DNA, Viral / Q000737:chemistry* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods / Q000592:standards; D009841:Oligonucleotides / Q000737:chemistry
D001688:Biological Products; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009841:Oligonucleotides