One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

Figure 2

Selection of scFv clones and strategy for recovery. (a) The chart reports the relative enrichments, within the indicated selection cycles, for 75 scFv clones. The threshold for inclusion was arbitrary set to a relative representation value of 1 10⁻³ (0.1%). Most clones were maximally enriched at cycle 3. Compared to cycle 4, cycle 3 also showed the highest number of different clones with a relative representation >1 10⁻³ (75 versus 63). Cycle 3 was accordingly selected for recovery of scFv clones. (b) The cartoon describes the strategy, implemented for recovery of scFv clones. The methylated template DNA from cycle 3 sublibrary was copied by Pfu DNA polymerase from overlapping primers (block arrows), corresponding to specific sequences within HCDR3 region of VH. The dashed lines represent the newly synthesized DNA, nonmethylated, since it was generated in vitro. After DpnI digestion, methylated and hemimethylated DNAs are removed, so that the nicked DNA originating from template copy is able to transform competent E. coli cells. The originating colonies thus represent the recovered, bona fide scFv clones. (c) The panel shows the products of the extension reactions, carried out on template from selection cycle 3, with overlapping primers for HCDR3 regions of clones 3_2, 3_5, and 3_67. The upper bands correspond to the supercoiled, methylated template; the lower bands represent the primer-extended, nicked products. SM, size marker; T, template DNA.

(a)

(b)

(c)