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Anal Biochem. 2005 Nov 15;346(2):289-94. Epub 2005 Sep 19.

Investigations of the effect of DNA size in transient transfection assay using dual luciferase system.

Author information

1
Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.

Abstract

In transient transfection assays deletions, insertions, or truncations are broadly used to define cis regulatory elements, such as promoters, enhancers, and silencers. This application is based on the assumption that size changes of a test fragment have little or no effect on the apparent activity of the reporter genes. While it is known that unusually large size of a construct, such as PAC DNA, substantially reduces transfection efficiency, the size effect of a DNA fragment ranging from a couple of hundred basepairs to a few thousand basepairs, within which most transient transfection assays are performed, has not been rigorously investigated. Here we report a systematic study on the effect of plasmid size on the measurement of promoter/enhancer activity in transient transfection assay based on the dual luciferase reporter system. By inserting various lengths of stuffer DNA into the luciferase parental plasmids, we found an inverse correlation between the relative luciferase activity and the construct size. Furthermore, this size effect can be only partially corrected by inserting the same size of stuffer DNA into both experimental and control plasmids. The results suggest that the effect of DNA size should be taken into consideration for evaluation of transcriptional elements using transient transfection analysis.

PMID:
16213455
DOI:
10.1016/j.ab.2005.08.029
[Indexed for MEDLINE]

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