DNA Sequencing analysis and CDR alignment

DNA sequencing of the ScFv antibody was essentially performed using the chain termination method developed by Sanger et al., 1977 but experiments were performed using either radiolabelled dioxy or dideoxy nucleotides.

Preparation o f M 13 ssDNA from reco m b inant phage

phage suspension was centrifuged at 12,000 g for 10 minutes and the supernatant removed. The pelleted phage were resuspended in 500 pi of Phage Buffer and 1 ml of M l 3 DNA purification resin was added. The DNA/resin mix was poured into a minicolumn, vacuumed until nearly dry and 2 ml of 80% isopropanol added and vacuum dried. The minicolumn was transferred to a sterile tube and 100 pi of HgO added. The tube was immediately centrifuged at 16,000 g for 20 seconds to elute the DNA. The purified ssDNA was analysed by agarose gel as previously described and stored at -20°C until used.

Preparation o f M l 3 dsDNA from reco m b inant E .C oli

The replicative form (RF) of phage DNA which exists as a plasmid in E.coii as dsDNA was purified using a Wizard miniprep kit (Promega). A single colony from an M9 plate was inoculated into 5 ml of 2 x YT-CG and incubated overnight at 30°C with shaking at 250 rpm. 1 ml of the overnight culture was added to 50 ml of 2 X YT-CG and incubated for 4 hours at 30°C with shaking at 250 rpm. The 50 ml culture was processed in 10 ml batches. The cells were pelleted by centrifugation at 1400 g for 10 minutes. The supernatant was removed, the cells resuspended in 400 pi of Resuspension Solution and 400 pi of Cell Lysis Solution added and mixed by inversion and incubated for 3 minutes at 22°C. The lysis reaction was stopped by adding 400 pi of Neutralisation Solution and mixing by inversion. The lysate was cleared by centrifugation at |l0,00(yor 5 minutes and added to a minicolumn containing 1 ml of Purification resin which was vacuum dried. 2 ml of Column Wash Solution was added and the DNA/resin mix was vacuum dried for 30 seconds. The minicolumn was centrifuged at 10,000 g for 2 minutes and transferred to a sterile tube. 50 pi of HgO was added and the tube centrifuged at 10,000 g for 20 seconds to elute the DNA. The purified dsDNA was analysed by agarose gel as previously described and stored at -20°C until used.

P reparation o f DNA sequencing reag ents

The preparation of samples for sequencing using Sequenase 2.0 DNA sequencing kit was used according to the manufacturers instructions using the primer combinations of pCANTAB 5-SI/pCANTAB 5-S3 for the chain and pCANTAB 5-S4/ pCANTAB 5-S6 for the chain (see figure 74 for the sequencing map).

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