Systematic design of mouse Vh gene family-specific oligonucleotides

Kabat's database has often been used to design mouse Vh gene-specific 5′ primers. The emphasis was mostly on constructing a universal (degenerate) 5′ primer or 5′ primer set, which would be able to match every mouse Vh gene. We were interested in finding oligonucleotides that could be used as primers or probes that can discriminate between the different Vh gene families. To this end, Kabat's database was reordered into Vh gene families based on more than 80% homology to prototype Vh gene family sequences. Rules were formulated for adequate annealing of putative primers to their respective genes. Putative primers were derived from the consensus sequences of the Vh gene families. A computer program was designed to systematically screen for the most optimal 5′ and 3′ Vh gene family-specific primers. This program enabled us to find a set of framework I-specific 5′ primers, as well as a set of framework III-specific 3′ primers. The found primers were also tested for their use as Vh gene family-specific probes.