SUPPLEMENTARY INFORMATION
A High-throughput Screening Method to Reengineer DNA Polymerases for Random
Mutagenesis
Figure S1. Scheme outlining the main steps of SeSaM random mutagenesis method.
Figure S2. Purification of Dpo4. Dpo4 WT and mutants were expressed from E.coli BL21 Gold DE3 lacIq1 and
purified by His-tag affinity chromatography. Crude cell extracts were heat treated to precipitate E.coli host
proteins prior to the chromatography step. Aliquots were collected during purification and analysed by SDSPAGE. Only the purest fractions were taken for dialysis against polymerase storage buffer. M – marker; CFE –
cell free extract; 80°C – heat precipitation of E.coli proteins at 80°C for 10 minutes; FT – flow through fraction
after washing; 1-12 – elution fractions.
Figure S3. Activity values in descending order of Dpo4 catalyzed TvTv mismatch elongation in a 96-well
microtiter plate using optimized Scorpion probe screening system. The measured coefficient of variation was
25%.
Figure S4. Crystal structure of DNA polymerase IV from Sulfolobus solfataricus bound to G:T mispaired DNA
substrate in the presence of an incoming nucleotide (PDB ID 1S97). Altogether, 21 residues were selected for
saturation mutagenesis; 20 residues (Y10, V32, A34, A44, V62, M76, K78, D156, A181,P184, 188, A191,
R242, R247, K275, V289, L293, R331, R332) are shown as sticks and residue R336 represented in balls.
Figure S5. Preparation of a model SeSaM library. DNA modification steps from I to IV of the SeSaM random
mutagenesis method
Table S1. List of primers, DNA probes and other oligos used in the study. (SNN = randomization codon; [FAM] =
6-carboxy fluorescein, fluorescent dye; ~~~~ = Spacer18, hexaethylene glycol; [BHQ] = black hole quencher-3,
quencher molecule; [Biotin] = 5`biotin; ctaagaaagcccg = phosphothioated oligo;)
ID No.
PRIMER NAME
P1
SSM_Fwd
P2
SEQUENCE (5`-->3`)
APPLICATION
CGGTTCCTGGCCTTTTGCTGG
SSM library generation
SSM_RV_Y10
TCAACCTGCGCGTAGAASNNATCAAGTCTACGAACAGC
SSM library generation
P3
SSM_RV_V32
CGAAACGACCGGAGAASNNGCACACAACAACTGGTTT
SSM library generation
P4
SSM_RV_A34
GCTGTCTTCGAAACGACCSNNGAAAACGCACACAACAAC
SSM library generation
P5
SSM_RV_A44
TGCTTCGTAGTTAGCGGTSNNAACCGCACCGCTGT
SSM library generation
P6
SSM_RV_V62
GGCAGGATTTTCTTAGCTTCSNNGATCGGGATACCAGCTT
SSM library generation
P7
SSM_RV_M76
GTTGTTAAACTTCTTTGCGSNNAGGCAGGTAGACGGC
SSM library generation
P8
SSM_RV_K78
GCTCACTTGGTAAACTTCSNNGCGCATAGGCAGGTAG
SSM library generation
P9
SSM_RV_D156
CCGTTCGGTTTAGCCATSNNCGCCGCGATCTTC
SSM library generation
P10
SSM_RV_A181
CCAATGCCTGGAACGTCSNNAATATCCAGTTCGCGG
SSM library generation
P11
SSM_RV_P184
GTGATGTTGCCAATGCCSNNAACGTCAGCAATATCCAG
SSM library generation
P12
SSM_RV_N188
TCAGTTTTTCAGCGGTGATSNNGCCAATGCCTGGAAC
SSM library generation
P13
SSM_RV_A191
CCCAGTTTCTTCAGTTTTTCSNNGTGATGTTGCCAATGC
SSM library generation
P14
SSM_RV_R242
TGCGACCGATGCTCTTSNNCACACGCGTGCGAA
SSM library generation
P15
SSM_RV_R247
ATTACGTTTCATGGTCACAATSNNACCGATGCTCTTGCG
SSM library generation
P16
SSM_RV_K275
GGGATGCGTTTATCCAGSNNATAGTAAGATTCTTCAATAGCG
SSM library generation
P17
SSM_RV_V289
GATGTCCAGATCCTCCGTSNNAGCAACCACGTGGATAG
SSM library generation
P18
SSM_RV_L293
CCGGGACACGATGTCSNNATCCTCCGTAACAGCAAC
SSM library generation
P19
SSM_RV_R331
CACGCCGATACGSNNGATCTTGCGCTC
SSM library generation
P20
SSM_RV_R332
GCGCACGCCGATSNNGCGGATCTTGCG
SSM library generation
P21
SSM_RV_R336
GCCTCGATGAATTTAGAGAASNNCACGCCGATACGGC
SSM library generation
CGCTGAGCAATAACTAGCATAACCGACGATAAATCACAATAGGTTCCTTGT
AGGCTGCCTCACGCCATCACAC
CGCTGAGCAATAACTAGCATAACCGACGATAAATCACAATAGGTTCCTTGT
AGCCTGCCTCACGCCATCACAC
CGCTGAGCAATAACTAGCATAACCGACGATAAATCACAATAGGTTCCTTGT
AGCGTGCCTCACGCCATCACAC
Scorpion probe Assay Primer/Probe
Scorpion probe Assay –
Quencher
Scorpion probe Assay Template Match
Scorpion probe Assay Template 1 Mismatch
Scorpion probe Assay Template 2 Mismatches
P22
6-FAM
[FAM]CGCTGAGCAATAACTAGCATAACCGAC~~~~GTGTGATGGCGTGAG
GCAGC
P23
BHQ-1
GTCGGTTATGCTAGTTATTGCTCAGCG[BHQ1]
P24
P25
P26
Scorpion template
match
Scorpion template 1
mismatch
Scorpion template 2
mismatches
P27
F1
CGACTCACTATAGGGGAATTGTGAGCGGA
SeSaM library generation
P28
R3
CGGGCTTTGTTAGCAGCCGGATCTCAG
SeSaM library generation
P29
SeSaMF1
CACACTACCGCACTCCGTCGCGACTCACTATAGGGGAATTGTGAGCGGA
SeSaM library generation
P30
SeSaMR3
GTGTGATGGCGTGAGGCAGCCGGGCTTTGTTAGCAGCCGGATCTCAG
SeSaM library generation
P31
F1_up
CGCCTGTCACCGACTCACTATAGGGGAATTGTGAGCGGA
SeSaM library generation
P32
R3_dn
GCGGACAGTGCGGGCTTTGTTAGCAGCCGGATCTCAG
SeSaM library generation
P33
Bio_Fwd
[Biotin]CACACTACCGCACTCCGTCG
SeSaM library generation
P34
Bio_Rev
[Biotin]GTGTGATGGCGTGAGGCAGC
SeSaM library generation
P35
SeSaM_Fwd
CACACTACCGCACTCCGTCG
SeSaM library generation
P36
SeSaM_Rev
GTGTGATGGCGTGAGGCAGC
SeSaM library generation
P37
Vector_PTO_Fwd
ctaacaaagcccgAAAGGAAGCTGAGTTG
Cloning of SeSaM libraries
P38
Vector_PTO_Rev
attgtgttcagcGATATCCATGGCCATC
Cloning of SeSaM libraries
P39
Insert_PTO_Fwd
gctgaacacaatCCAGTCGTTATGGTTC
Cloning of SeSaM libraries
P40
Insert_PTO_Rev
cgggctttgttagCAGCCGGATCTCAG
Cloning of SeSaM libraries