Inter-University Poster Competition 2001/2002 Gallery

A novel PCR-based approach for generating and screening gene expression constructs with progressive deletions using ExoIII nuclease

By FAISAL AL-ALLAF
Imperial College

Generation of progressive deletions in a given DNA construct is a useful approach in order to analyse functional DNA-sequences. The use of ExoIII nuclease followed by single strand specific S1 nuclease is by far the most frequently applied procedure for this goal. Difficulties associated with assessment of the rate of exonuclease cleavage and determination of the desired degree of DNA digestion for transformation and further analyses has made this procedure laborious, expensive, and time consuming. Here, we describe a simple, rapid, and universal PCR-based approach for generating and screening a set of progressive deletions using ExoIII nuclease. This method uses the ligation mix from different time points, of ExoIII treatment, as a template to generate a PCR-product with a primer pair that flanks the digestion site. Thus, the generation of PCR amplicons of variable size indicates the progressive deletion and allows the estimation of the rate of ExoIII mediated cleavage. After transformation, the ligation mix is used again as a positive control for screening and isolation of clones with the desired deletion size. This approach does not only determine which DNA-ligation samples should be used for transformation but also provides a powerful means to screen and select the desired deletion size recombinant(s) from a large number of colonies without any of the DNA preparation steps, required by other analysis methods. We believe that this novel PCR-based method will be a useful tool for research in functional genomics and vector construction. We have used this method to construct various expression vectors containing a bicistronic cassette of LDLR/IRES/EGFP under control of the CMV promoter. These vectors contain variable lengths of the non-coding inter-space sequence, between the stop codon of the LDLRcDNA and the IRES/EGFP cassettes, produced by our sequential deletion method with Exo III treatment. The produced variation in length of the inter-space sequence ranges from 445bp to 34bp. Our results show that the length of the inter-space sequences between the stop codon of the first ORF and the start codon of the IRES element are directly correlated to the expression of the second ORF, EGFP. These data also show that the expression of the EGFP in the constructed vectors is increased by 20 fold when the length of the inter-space sequence is reduced to 34bp.