5.20.1 Primers and templates on solid support abasic site primer-template
p 1 5´- NH2C6-CGT TGG TCC TGA AGG AGG AT-3´
t 1 5´- AAAACGC TGTAGCA TAG AGT ACATG ACA G TT CX CCT A TC CTC CTT CAG GAC CAA CG-3´ X = stable abasic site analogue
fully matched primer-template
p2 5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC TTA T-3´
t2 5´-AAA GCT CCT TTC TGA ATA TTG AGC TCA TCA GTG AGA AAA CGG CTG CAT ATT GGT GTC AAA GTG TCA CTG AAC TAA AGG CTG ACT TTC CAG ACA AC A TAA GTG TGA TTT AAC ATC TAA AAC-3´
single mismatched primer-template
p3 5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC TTA C-3´
t2 (sequence see above)
distal mismatched primer-template
p4 5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC CTA T-3´
t2 (sequence see above)
triple mismatched primer-template
p5 5´- NH2C6-GTT TTA GAT GTT AAA TCA CA GAA AT-3´
t2 (sequence see above)
surface coverage estimation
p6 5´- NH2C6-CGT TGG TCC TGA AGG AGG AT-biotin-3´
5.20.2 Primers and templates in solution experiments abasic site primer-template
p1´ 5´-CGT TGG TCC TGA AGG AGG AT-3´
t1´ 5´-AAA TCA XCC TAT CCT CCT TCA GGA CCA ACG TAC-3´
X = stable abasic site analogue
fully matched primer-template
p2´ 5´-GTT TTA GAT GTT AAA TCA CAC TTA T-3´
t2´ 5´-CTT TCC AGA CAA CAT AAG TGT GAT TTA ACA TCT AAA AC-3´
single mismatched primer-template
p3´ 5´-GTT TTA GAT GTT AAA TCA CAC TTA C-3´
t2´ (sequence see above)
distal mismatched primer-template
p4´ 5´- GTT TTA GAT GTT AAA TCA CAC CTA T-3´
t2´ (sequence see above)
triple mismatched primer-template
p5´ 5´-GTT TTA GAT GTT AAA TCA CA GAA AT-3´
t2´ (sequence see above)
5.20.3 Overexpression of KlenTaq clones in multiwell format and cell lysate preparation
KlenTaq clones were parallel expressed in 96x deep-well plates containing 1 ml of LB-medium and 100 μg/ml carbenicillin at 37°C. Overexpression was induced via addition of 200 μg/l anhydrotetracycline at OD600~0.8 for 4h. Afterwards, the plates were centrifuged at 4000 x g for 15 min, the supernatants were thrown away and cell pellets were resuspended in 1x KlenTaq reaction buffer containing an additional amount of lysozyme (0.1 mg/ml). The sealed plates were subsequently incubated for 15 min at 37°C and then for 40 min at 75°C. After centrifugation at 4000 x g for 45 min the lysate was directly used for screening.
5.20.4 Spotting of DNA polymerase cell lysate mixtures and screening reactions Immediatly before spotting, the DNA polymerase cell lysates were mixed 1:1 with a KlenTaq reaction mix containing 1xKlenTaq buffer, 0.2 % BSA, 400 μM dATP,dGTP,dCTP, 380 μM dTTP, 20 μM Biotin-11-dUTP and the respective templates in a 96x well plate at 4°C and agitated for 30 sec using a shaker module. The cell lysate mastermix mixtures were centrifuged for 1 min at 4000 x g. Spotting was then performed at ~20°C air temperature and air humidity between 60-70%. The slide tray was also cooled during the whole spotting procedure at 5°C. Cell lysates were spotted in a way that each lysate covered 2 columns and all 5 different primer spot rows with 3 drops per spot resulting in approx. 1.2 nl total spotting volume. The slides were subsequently placed in a humidity chamber consisting of a water filled petri dish at room temperature for ~1 minute until all spots were rehydrated. After incubation, the slides were shortly rinsed twice under gentle agitation in 0.1 x SSC+SDS buffer (15 mM NaCl, 15 mM sodium citrate, pH 7.0, 0.01% SDS) for 5 min and twice with water (5 min each). The slides were then dried under a stream of nitrogen. Afterwards, 70 μl of a streptavidine-alexa fluor® 546 solution 0.4 μg/ml in 1x TBS-T buffer (10 mM Tris-HCl pH 8.0 150 mM NaCl, 0.05% Tween-20) was placed on each slide and directly covered with a square glass cover slips 24 x 60 mm. Incubation was carried out in a humidity chamber (see Figure 21) at room temperature for another 40 min. The cover slip was then removed and the slides were rinsed twice with 1x TBS-T buffer and once with
water. Afterwards, the slides were dried under a stream of nitrogen before readout with a GenePix microarray scanner machine (532 nm channel, 10 μm / pixel resolution) was performed.
5.20.5 Expression and purification of KlenTaq mutants
KlenTaq wt and mutants were purified in batch technique using Ni-NTA sepharose. In detail 100 ml cultures were grown to an OD600 of 1.0 at 37°C following induction with AHT (200 μ g/L) for 3 h. After harvesting cells were lysed for 15 min at 37°C in 5 ml KlenTaq lysis buffer followed by heat denaturation of host proteins at 75°C for 40 min and centrifugation at 20.000 x g for 30 min. Supernatants were incubated with Ni-NTA sepharose slurry. After washing with KlenTaq washing buffer (5 mM and 20 mM imidazol), elution was carried out using washing buffer (200 mM imidazol). After buffer exchange to 2x KlenTaq washing buffer, Tween20, (NH4)2SO4 and glycerol was added to 50 % (see Buffers and solutions, KlenTaq storage buffer). Enzyme purity (Figure 27) and quantity was controlled and determined by SDS-PAGE using an albumin standard dilution curve. Purified enzymes were stored at -20°C.
5.20.6 Primer extension reactions in solution
20 μl of the reaction contained 1x KlenTaq buffer, 100 μM dNTPs, 225 nM template, 150 nM 5´-32P-labeled primer. DNA polymerase concentrations were 1 nM for matched, mismatched and distal mismatched cases and 50 nM for triple-mismatched and abasic site templates. The reaction mixtures without dNTPs were annealed by heating for 2 min at 95°C and cooling to 25°C. Reactions were initiated by addition of dNTPs. After incubation at 51°C for 15 min in cases of match, mismatch and distal mismatch and 1h for the triple-mismatch and abasic site containing reactions, the primer extension was stopped by addition of 40 μl of gel-loading buffer (80%
formamide, 20 mM EDTA). Product mixtures were separated by 12% denaturing PAGE and analysed by phosphorimaging.
5.20.7 Test of the influence of drying and rehydration on the KlenTaq polymerase activity in solution
Solutions of KlenTaq wt ranging from 400, 40, 4, 0.4, 0.04, 0.004 nM in 1x KlenTaq reaction buffer were divided in two 10 μl aliquots, respectively. One aliquot was dried under vacuum using a high-vacuum membrane pump while the other aliquot was stored at room temperature. After 1 h, the dried residue was dissolved in 10 μl water and both aliquots were used for subsequent primer extension reactions using fully matched primer p2´ and template t2´. Reactions (20 μl) contained 1 x KlenTaq buffer, 100 μM dNTPs, 225 nM template, 150 nM 5´-[32P]-labelled primer and 2 μl of the DNA polymerase containing solution. The reactions were stopped after 15 min incubation at 51°C by addition of 40 μl of gel-loading buffer. The products were separated by 12%
denaturing PAGE and analysed by phosphorimaging (see Figure 22).
5.20.8 Spotting and immobilisation of short DNA oligomers
Activation of glass slides and spotting of amino-modified oligonucleotides to glass slides were conducted as previously described (see Section 5.19.2)
5.20.9 Estimation of surface coverage of oligonucleotides bound to the chip surface
To estimate the loading density and the amount of attached DNA primers on the surface, the resulting fluorescence signal of biotinylated immobilised primers with directly spotted streptavidin-alexa fluor®546 conjugate were compared: A DNA oligomer (p6) was immobilised in replicates on a freshly activated aminosilane slide as described above. After incubation with a streptavidine-alexa fluor®546 solution (0.4 μg/ml in 1x TBS-T buffer (10 mM Tris-HCl pH 8.0 150 mM NaCl, 0.05% Tween-20)) the slide was washed twice in 1x TBS-T buffer for 2 min. and in water for 10 sec. Afterwards a dilution series of streptavidine-alexa fluor®546 (1μM – 30nM, 0.4 nl) was spotted directly neighboured to the DNA replicates. Without additional washing the whole slide was scanned using a GenePix microarray scanner machine (532 nm channel, 10 μm / pixel resolution). Under the assumption that binding stoichiometry between
streptavidin conjugate to the immobilised biotinylated DNA strand is 1:1, a linear regression of the fluorescence values obtained from the dilution series made a calculation of the amount of immobilised DNA possible. The spotted amounts of DNA polymerase mutants deriving from the cell lysates were determined by SDS-PAGE.