Rapid generation of multiple alleles by Recombineering
One of the principal limitations to studying mutational effects in large genes has been the paucity of techniques to rapidly generate multiple alleles. The process is complicated by the absence of unique restriction sites for suitable ‘tailoring’ of large constructs. Selectable disruptive events such as inclusion of a selectable marker, become undesirable while generating subtle alterations in genes. We have designed a rapid and efficient system for generating multiple alleles, allowing insertions between adjacent bases, deletions or single base alterations without final inclusion of selection markers. This technology is an invaluable tool in today’s era of functional genomics and provides a means to functional dissection of genes as well as characterization of regulatory elements.