1.0 μg of purified targeted dsDNA (for chain shuffling: anti-eGFP scFv clone 44 for VH and anti-eGFP scFv clone G1 for VL; CDR3 mutagenesis: heavy chain framework from anti-eGFP scFv clone 44) was incubated at 37°C for 30 min respectively with 1× lambda exonuclease reaction buffer and 10 U of lambda exonuclease (New England Biolabs, Ipswich, MA, USA) in a total of 50 μL reaction volume. The enzyme was then heat inactivated at 75°C for 10 min. To visualize ssDNA after lambda exonuclease treatment, samples were loaded on a 1% agarose gel with ethidium bromide. ssDNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).Evaluation of ssDNA generation by 6 M urea PAGE
6 M urea polyacrylamide was prepared using 1.875 mL 40% acrylamide/bis-acrylamide (29:1), 0.6 mL 10XTBE, 2.88 g urea, 19.8 μL 30%APS, 2.4 μL TEMED and 3.525 mL dH2O. 15 μL of DNA sample was mixed with 3 μL of 6× loading dye. The mixture was heated at 95°C for 5 min and left to cool before loading. For DNA separation, electrophoresis was carried out at a current of 25mA using mini protean tetra cell (BioRad) for 50 min in 0.5×TBE buffer. Gel was stained with EtBr solution and visualized under UV.Evaluation of ssDNA generation by fluorescence intensity reading
100 ng of DNA in a 10 μL aliquot with 5 μL of iTaq universal SYBR green supermix (BioRad) was used to measure fluorescence using Rotor-Gene 3000 (Corbett Research, Cambridgeshire, UK) at 55°C. Synthetic oligo20 ssDNA (CCGGCCATGGCC(NNK)20GCGGCCGCATAGACTGTT) from Integrated DNA Technologies, USA was used as the ssDNA control, while oligo20 dsDNA was generated by KF filling in of the oligo20 ssDNA as the dsDNA control.Annealing two ssDNA templates
0.4 μg of each digested target ssDNA (for chain shuffling: anti-eGFP scFv clone 44 for VH and anti-eGFP scFv clone G1 for VL; CDR3 mutagenesis: heavy chain framework from anti-eGFP scFv clone 44 and synthetic CDR3 oligonucleotides) in 1×NEB buffer 2 (New England Biolabs) were annealed to create a substrate for KF. Under non-cyclic conditions, the reaction mixture was heated at 95°C for 5 min and left to cool to 30°C with a ramp of 0.1°C/s. The reaction was then left to incubate at 30°C for 5 min. For cycling conditions, the reaction mixture was heated at 95°C for 5 min, and then slowly cooled to 70°C, 60°C, 50°C, 40°C, 30°C, and 25°C with a ramp of 0.1°C/s and every temperature kept constant for 5 min. All temperature cycling was carried out using the MyCycler thermocycler (BioRad).Filling in reaction by Klenow fragment
For filling in ssDNA, 20U KF (New England Biolabs) and 1.5mM dNTPs (Fermentas, Lithuania) were added to a final reaction volume of 50 μL with the annealed ssDNA. The mixture was incubated at 37°C for 4 h and then heat inactivated at 75°C for 20 min. To determine which DNA strand was extended from the annealing site, same amount of reaction sample was loaded on 1% agarose gel with ethidium bromide for visualization and then gel purified with Purification Kit (Qiagen).Cloning for sequencing
The final purified product was digested with appropriate restriction enzymes (New England Biolabs) and ligated to the pIT2 plasmid using T4 DNA ligase and 1× T4 DNA ligase buffer (New England Biolabs) with a final volume of 20 μL according to the manufacturer's protocol. The DNA was prepared using MiniPrep DNA kit from Qiagen according to the manufacturer's protocol. The DNA was sent for sequencing using BigDye Terminator v3.1 cycle sequencing chemistry at 1st Base (Malaysia) using LMB3 Forward primer (CAGGAAACAGCTATGAC) and pIII Reverse primer (GTTAGCGTAACGATCTAA). Sequencing results were analyzed using VBASE2 (43). The sequences of all clones have been deposited by GenBank under the accession no. JX 028841 to JX028874.Determination of library diversity by DiStRO
500 ng dsDNA sample with 10 μL iTaq universal SYBR green supermix (BioRad) in a total volume of 40 μL was prepared and the assay was performed using Rotor-Gene 3000 (Corbett Research) instrument. Reannealing was initiated by denaturation for 2 min at 95°C and subsequent measurements were taked during annealing at 50°C for 180 min taking one measurement per minute. Re-melting was performed starting at 25°C with incremental steps of 0.5°C for 7 s each until the final temperature of 98°C was reached.