Application of Polymerase Chain Reaction in Deletion and Inversion of Internal DNA Fragments
Deletion and inversion of an internal DNA fragment has been wildly used in delineating promoter activity and determining the function of certain cis-elements in the 5’ flanking region of genes (1, 2, 3, 4, 5). These are generally achieved by restriction enzyme digestion and ligation. In most cases, such convenient restriction enzymes are not available. In this chapter, we describe a strategy of deleting or inverting a DNA fragment using a PCR based technique. As an example, an 115 bp element within a 931 bp of the immediate 5’ flanking region of the mouse peripherin/rds gene (cloned in pB Script vector) was deleted. The created DNA fragment can then be used to test for its promoter activity and the functional importance of the deleted element can be evaluated. We also inverted a 158 bp element within a 3304 bp fragment of the same promoter. Similarly, the functional as well as the directional importance of the manipulated fragment can be studied.
KeywordsInternal Fragment Retinal Degenerative Disease Original Fragment Fusion Gene Expression Rhodopsin Regulatory
Unable to display preview. Download preview PDF.
- 2.Higuchi, R. (1990). Recombinant PCR. In: PCR Protocols: a guide to methods and applications, ed. M.A. Innis, D.H. Gelfand, J.J. Sninsky, T.J. White. San Diego: Academic Press, 177-183.Google Scholar
- 3.Shimin chen and Donald J. Zack. Ret 4, a positive Acting Rhodopsin Regulatory Element Identified Using a Bovine Retina in vitro Transcription System. 1996. J. Biol. Chem. 271, No 45: 28549 - 28557Google Scholar