Megaprimer-Based Methodology for Deletion of a Large Fragment Within a Repetitive Polypyrimidine-Rich DNA

Abstract

Site-directed mutagenesis is often a prerequisite for elucidation of the functional significance of cis- and trans-factors involved in gene regulation. The aim of this study was to delete the primary binding site for heterogeneous nuclear ribonucleoprotein I (hnRNPI) within the inducible nitric oxide synthase (iNOS) 3' untranslated region mRNA. The binding site consists of a 53-nucleotide CU-rich region within a long stretch of polypyrimidines. As a result of primer pair annealing, the repetitive sequence limited the use of several deletion methods based on polymerase chain reaction. Therefore, a megaprimer approach was chosen. The megaprimer was produced by a forward primer outside the polypyrimidine-rich region, and a mutagenic reverse primer annealing to flanking regions of the desired deletion, thereby looping out the target sequence. Subsequently, this megaprimer was used to create the final deletion recombinant. The deletion was verified by sequencing and by ultraviolet cross-linking mouse liver protein extracts with radiolabeled mutant and wild-type RNAs. In conclusion, the megaprimer method offers a solution for generating large internal deletions in repetitive sequences, which facilitates investigations on large repetitive DNA or RNA regions interacting with trans-factors.