Design and Application of rtPCR Method for Analysis of Strain-Producer Residual DNA in Histon H1.3 Substance

Автор: E.V. Kovaleva, A.P. Barannik, Yu.S. Skoblov, E.D. Shibanova, and V.I. Shvets

Страница: 65-75

Design and Application of rtPCR Method for Analysis of Strain-Producer Residual DNA in Histon H1.3 Substance

Biotekhnologiya, 2015, N 2, P. 65-75

UDC 557.112.823

Section:  “Metrology, Standartization, and Control”

E.V. Kovaleva^1,2,*, A.P. Barannik², Yu.S. Skoblov², E.D. Shibanova², and V.I. Shvets¹

¹  The Lomonosov Moscow State University of Fine Chemical Technologies,  119571, Moscow Russia

²  The Shemyakin-and-Ovchinnikov Institute for Bioorganic Chemistry. Russ. Acad. Sci.,  117997, Moscow Russia

e-mail:  katja_kov@mail.ru,  kovaleva@kou.ibch.ru

The application of the real-time PCR quantitative method (rtPCR) with specific primers to the 16S RNA E.coli gene fragment for the detection of residual DNA of E. coli BL21(DE3)/ prhH1.3 strain-producer in the APS of the recombinant human histon H1.3 has been suggested. The sample preparation was optimized, a working standard based on the sonicated total DNA of the strain-producer was designed, and an amplicon for the rtPCR was selected. The specifity of the designed primers was evaluated in comparison with eukaryotic DNA. The efficiency of the method in comparison with that using a commercial kit was analyzed. The designed primers to the 16S RNA gene proved to be by 30 times more sensitive than primers of a reference commercial kit.

Key words:  active pharmaceutical substance,  histon,  real-time PCR,  residual DNA.

25.06.2015, 1535 просмотров.