Absolute and relative QPCR quantification of plasmid copy number in <i>Escherichia coli</i>

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P><P>Real-time QPCR based methods for determination of plasmid copy number in recombinant <I>Escherichia coli</I> cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant <I>E. coli</I> DH5α harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid β-lactamase gene (<I>bla</I>) and for the chromosomal <SMALL>D</SMALL>-1-deoxyxylulose 5-phosphate synthase gene (<I>dxs</I>), respectively. Since both <I>bla</I> and <I>dxs</I> are single-copy genes of pBR322 and <I>E. coli</I> chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of <I>bla</I> to <I>dxs</I>.</P><P>These methods were successfully applied to determine the plasmid copy number of pBR322 of <I>E. coli</I> host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8–3.9% and 4.7–5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within <I>E. coli</I> host cells, 15–20.</P><P>The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant <I>E. coli</I> with a plasmid vector having <I>bla</I> gene.</P>