A PCR-based semiquantitative assay of DNA impurities in recombinant protein preparations
- 23 Downloads
A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10µg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.
Key wordsDNA determination human recombinant insulin PCR
Unable to display preview. Download preview PDF.
- 1.Farmakopeinaya stat’ya predpriyatiya Institut bioorganicheskoi khimii im. M. M. Shemyakina i Yu.A. Ovchinnikova RAN (Pharmacopoeia Monograph of the Enterprise Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS) FSP42-0452361302. Human insulin, 2002, Moscow.Google Scholar
- 2.Haugland, R.P and Kang, H.C, Chemically Reactive Dipyrrometheneboron Difluoride Dyes, US Patent 4774339.Google Scholar
- 3.Matz, M.V., in Methods Mol. Biol., Hicks, B.W, Ed., Totowa: Humana, 2003, vol. 221, pp. 103–116.Google Scholar
- 4.Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, NY: Cold Spring Harbor Lab., 1989.Google Scholar