v gelatin 0 25 mm each deoxyribonucleoside triphosphate 10 pmoles 5 u2032 primer mix 10 pmoles 3 u2032 primer 1 cdna and 1 unit taq polymerase reactions were incubated at 95 u00b0 c for 5 minutes and then cycled through 94 u00b0 c for 1 minute 55 u00b0 c for 1 minute and 72 u00b0 c for 1 minute after 30 cycles aliquots of each reaction were analysed by electrophoresis on an agarose gel for the heavy chain v region an amplified dna product was only obtained when a primer pool annealing within the start of framework i replaced the signal peptide primer pool the fragments were cloned into dna sequencing vectors the dna sequence was determined and translated to give a deduced amino acid sequence this deduced sequence was verified by reference to the n terminal protein sequence determined experimentally fig2 and 3 shows the dna protein sequence of the mature light and heavy chain v regions of mouse monoclonal 5 44 respectively the murine v region sequences were then cloned into the expression vectors pmrr10 1 and pmrr14 fig7 these are vectors for the expression of light and heavy chain respectively containing dna encoding constant regions of human kappa light chain and human gamma 4 heavy chain the v l region was sub cloned into the expression vector by restriction digest and ligation from the sequencing vector using sfui and bsiwi restriction sites creating plasmid pmrr10 544cl the heavy chain dna was amplified by pcr using a 5 u2032 primer to introduce a signal peptide since this was not obtained in the cloning strategy u2014 a mouse heavy chain antibody leader from a different in house hybridoma termed 162 was employed the 5 u2032 primer.