site saturation scanning for every lab: supercharging site ... saturation... · scanning for every...
TRANSCRIPT
Site Saturation
Scanning for Every Lab:
Supercharging Site-
Directed Mutagenesis
Holly Hogrefe, Ph.D. R&D Manager
Genomics Solutions Division
Agilent Technologies
11/19/2014 1
11/19/2014
2
New Product Introduction:
QuikChange HT Protein Engineering System
• High-quality libraries, lacking the biases and unwanted mutations
associated with other mutagenesis techniques
• Custom oligonucleotide libraries used to achieve:
• An expanded menu of mutagenesis strategies, not
possible with standard or degenerate oligos:
• Create rationally-designed complex mutant libraries targeting:
• Entire proteins, up to 1000 codons per kit
• Alanine and site-saturation scanning
• Rational combinatorial mutagenesis
Definitions
11/19/2014 3
Page 4 11/19/2014
Random mutagenesis: • Tool for identifying nucleotide or amino acid changes that alter function
• Error-prone PCR conditions used to introduce single bp changes at random
locations in a gene or promoter of interest
• Strategy most commonly used when the location of beneficial changes is unknown
• Advantages over rational (non-random) approaches:
• No prior knowledge of structure required
• Survey large mutational target regions, up to several kbp in size
Agilent Product: GeneMorph Random Mutagenesis kit
• Limitations:
• Screen large libraries to identify useful mutations
• Changes limited to single base substitutions; a large fraction of protein
sequence space is not accessible since most amino acid changes require
2-3 point mutations in the same codon
“With single base mutations, only 5.7 amino acid substitutions on average are accessible from any given
amino acid residue” (Miyazaki and Arnold (99) J Mol Evol 49:716–720)
Page 5 11/19/2014
• Limitations:
• Throughput; 1 mutant per reaction
• Structural information is lacking in many cases
Agilent Products: QuikChange Site-Directed Mutagenesis kits
Site-Directed Mutagenesis: • Process of creating one or more nucleotide or codon changes in a gene or
regulatory sequence
• Rational (non-random) approach that employs DNA polymerase activity to
incorporate mutagenic oligos into wild-type DNA sequences
• Strategy used when location of desired modifications is known; potential target
sites can be identified by analyzing protein structures, amino acid sequence
homologies, or prior mutagenesis data
• Used to determine structure-function relationships in proteins or promoters, identify
post-translational modifications, define protein-protein interactions, modify enzyme
activity, optimize expression, add restriction sites, and correct unwanted mutations
Page 6
The QuikChange Method
• Features a simple 3-step
protocol
• Creates single base changes,
codon replacements, insertions
and deletions with >80%
mutation efficiency
• Employs high-fidelity Pfu
polymerase and linear
amplification to avoid
unintended mutations
• Referenced in tens of
thousands of publications
MIX Denature plasmid and annealing
primers containing desired
mutation X
Gene in plasmid
with mutation
target site
Mutagenic
primers
CYCLE Temperature cycle to extend and
incorporate mutation primers
resulting in nicked circular strands
DIGEST Digest parental DNA template
TRANSFORM Transform the resulting
annealed double-stranded
nicked DNA molecules
After transformation,
XL-1Blue E. coli cell repairs
nicks in plasmid
11/19/2014
Definitions cont’d
Page 7 11/19/2014
Agilent Product: QuikChange Multi Site-Directed Mutagenesis kit*
No. sequences required
No. sites to saturate: 1 2 3 4
Rational design 20 400 8000 160,000
Degenerate codons 64 4096 262,144 16,777,216
Scalability limited by transformation efficiency of E. coli and quality of degenerate codon libraries, as codon
redundancy and amino acid bias increase exponentially as the number of mutation sites is increased;
Site-saturation mutagenesis: • Process of determining the impact of every amino acid replacement at a single codon
• Most practical method uses degenerate oligos (referred to as “codon-saturation”);
oligos synthesized with an NNN codon comprise 64 unique sequences encoding 20
different amino acids at one position
• Site-saturation libraries can be screened to identify the best side chain replacements,
and required structural features (size, charge, etc.) for optimal activity
• Site-saturation with degenerate codons is less-than-ideal: • Bias toward amino acid changes encoded by redundant codons
• Frequency of unwanted termination and rare codons
• Limited scalability or target size:
No. oligos & rxns
4
160,000
1
N = nt position synthesized with equimolar
mixture of 25% AGCT)
Example of Site-Saturation Mutagenesis
Page 8 11/19/2014
McElhinny et al (09) in Molecular Themes in
DNA Replication (Cox L.S., ed) Hogrefe (10) in In Vitro Mutagenesis Protocols (Bramam, J. ed) pg. 271-284
JDF-3 P410L DNA polymerase
(CCT→CTT)
Yield from random selection of 30 QuikChange mutants:
pJDF3
13%
Rare in
EPPCR library
(2 nt changes)
Random mutagenesis: Random mutant
library of Thermococcus sp. JDF-3 Pol B
was expressed in E. coli and screened for
radioactive-ddNTP uptake
Arezi et al (02) J. Mol. Biol. 322:719-729
Definitions cont’d
Page 9 11/19/2014
Alanine (or single mutation) scanning: • Systematic process for evaluating the functionality of a group of amino acids
• A collection of site-directed mutants is constructed, where each mutant
contains an alanine (or other neutral amino acid) substitution at a unique
location; after measuring impact to function, the information is used collectively
to map essential positions
Alanine scanning of the
HIV gp41 loop Jacobs et
al (05) JBC 280:27284-88
30 gp41 alanine mutants
were constructed using
the QuikChange kit with
30 primer pairs; viral entry
was measured using a
Luciferase reporter assay
100%
10%
0%
• Limitations: While powerful, alanine scanning is generally limited to peptides
or small domains due to the cost and amount of labor needed to construct
multiple single-site mutants (one QC reaction and primer pair per codon)
Definitions cont’d
Page 10 11/19/2014
Site-saturation scanning: • Combination of site-saturation and scanning mutagenesis strategies
Agilent Product: QuikChange HT Protein Engineering System
Hypothetical sequence-function map for a 25 amino acid
portion of a protein. The color of each square in the heat
map illustrates the functional consequence of individual
side chain substitutions (single-letter abbreviation) at each
position.
Deep mutational scanning: a new style of protein
science. Fowler & Fields (2014) Nature Methods
11:801-7
“By enabling the impact of mutations to be examined in
an unbiased fashion, deep mutational scanning can
reveal the unexpected”.
• When feasible, provides comprehensive structure-function analysis by surveying
the functional consequences of every side-chain replacement at each location
Significant limitations: Requires a high-throughput cost-effective method for
constructing site-directed mutants, in addition to substantial screening and sequencing
QuikChange HT
Protein Engineering
System
11/19/2014 11
12
+
QuikChange Lightning
Site-Directed Mutagenesis
Agilent’s SurePrint
Technology
Game-Changing Mutagenesis Technology
Quick, accurate, and cost-effective method for constructing
complex rationally-designed mutant protein libraries
QuikChange HT Protein Engineering System
11/19/2014
QuikChange HT Protein Engineering System
Rationally-designed oligo libraries provide:
11/19/2014
13
Constructing oligo libraries with QuikChange offers:
• Fast and easy one-day workflow for in-house library construction
• High incorporation efficiency with minimal polymerase-induced mutations
• Ultra-high transformation efficiencies for maximum library size
• Better quality libraries, more information content
• User-specified codon usage for optimal expression
• No unwanted codons; avoid biases associated with degenerate codons
• Reduced screening requirement
• Comprehensive custom content
• Up to 120,000 user-specified sequences
• Easy-to-use oligo library design tool
• Flexible design options, one or more proteins
• An expanded menu of mutagenesis strategies, not available using standard oligos
• Single-site (Alanine or other) scanning
• Site-saturation scanning
• Targeted combinatorial mutagenesis
QuikChange HT - Workflow
QC HT Step 1: Design mutagenic library
specific to sequence and application with
eArray design software.
X B C X B C X B C X B C
X B C X B C X B C X B C
X B C X B C X B C X B C
X B C X B C X B C X B C
X B C
X B C
X B C
X B C
X B C
X B C
X B C
X B C
X B C
X B C
X B C
X B C
E.coli library
Screening – Sequencing Identify and sequence
selected clones
Nat
gene V
1 V
2
V
3 V
4
V
5 V
6
V
7
V
N
V1,0
01
Amplified OLS
incorporation
Dpn I digestion
Nick repair
(E. coli)
QC HT Step 3: Perform QuikChange
mutagenesis with each oligo set, move onto
mutant screening in less than a day
Site Directed Mutagenesis Incorporate oligo sets separately into plasmid
DNA with QuikChange, followed by Dpn I
enrichment & transformation
PCR & Purify oligo sets 1,2,3...20
SurePrint Inkjet
Oligo Library
Synthesis
QC HT Step 2: Library is synthesized per design. A set of mutant oligos is
designed for each target region and contains identical 5’ and 3’ ends which serve as
PCR primer binding sites. Oligo sets are isolated from the oligo library by PCR.
Oligo library
cleaved
11/19/2014 Page 14
FREE design tool: • Supports codon-based design of mutant protein libraries
• Design one pair of PCR primers and one set of mutagenic oligos (“oligo set”) for
each target region; QuikChange HT kits configured to construct up to 10 or 20
sub-libraries (10 or 20 site kits)
• For each oligo set, selections include: • Mutagenesis strategy
• Oligo length / Target size (17 codons with 100mers; 50 codons with 200mers)
• Codon usage
Design Workflow
11/19/2014
15
eArray Mutagenesis Workspace
QuikChange HT Mutagenesis Strategies
Mutagenic oligos Number unique
per oligo set*
1 mutation type per
codon;
1 codon change per
oligo
17-50
19 mutation types
per codon;
1 codon change per
oligo
323-950
Any of the specified
mutations; 1-4
codon changes per
oligo
Variable
*17-50 codon targets per oligo depending on length (100-200nt);
#Up to 10 or 20 oligo sets per library; 20 oligo sets X 50 codon targets/set = 1000
possible codon targets (~100kDa protein) per QC HT library
Total per library: 120,000#
11/19/2014 Page 16
Mutation (19 separate codon changes)
50AA x 19 mut = 950 oligos 1 QC HT reaction
Oligo
set 2
QuikScan-19: Site-
saturation scanning
systematically replaces each
amino acid in the wild type
mutational region with all 19
other amino acids, resulting
in 19 mutagenic oligos for
each amino acid position.
For deep mutational
scanning.
Target
sequence
Mutation region 1
Mutation (same codon)
Oligo
set 1
QuikScan-1: Separately
replace each amino acid in
the wild-type mutational
region with a selected amino
acid. Ala replacements
(“alanine scanning”) are
commonly used to identify
key functional amino acids. PCR primer annealing sites
Target
sequence
Mutation region 3
mutations
Oligo
set 3
PCR primer binding sites
QuikCombine: Combine
multiple mutations in
groups of 1-4 positions with
define variation at each
site. Create up to 120,000
variants per library, or
combine a few identified
mutations and validate
functional relevance.
Each oligo library can contain up to 120,000 unique sequences
• create 120,000 different combinations of mutations within a single
domain, or…
• target up to 20 regions in the same protein or different proteins
With 20 sets of oligos, 200nt in length, you can target up to
1000 codons per QC HT library.
11/19/2014
17
1
2
3
4
5
QuikChange HT Workflow
1) Order QuikChange HT kit; includes one custom oligo
library and one pair of PCR primers for each oligo set
2) Amplify the oligo sets separately, and purify using the supplied spin cups:
no need to convert amplicon to ssDNA or remove primer binding sites
3-5) Set up one mutagenesis reaction per amplified oligo
set. Follow the standard QuikChange protocol, substituting
PCR amplicon for the pair of mutant oligos
After transformation and overnight growth, mutant libraries
are ready for sub-cloning into a suitable expression strain
(as needed) for screening or downstream analysis
Sub-library 1 Sub-library 2
Test Cases
11/19/2014 18
Single Mutation Scanning
Page 19 11/19/2014
• Before initiating comprehensive site-saturation scanning, we assessed the
mutability of the “fingers domain” of Pfu DNA polymerase
WT FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
450 LMTSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
451 FIMSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
452 FIPMILGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
453 FIPRMMGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
454 FIPSLMRHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
455 FIPSLLMNLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
456 FIPSLLGMLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
457 FIPSLLGQMIEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
458 FIPSLLGHLMEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
459 FIPSLLGHLLMKRQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
460 FIPSLLGHLLEMRQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
461 FIPSLLGHLLEEMKKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
462 FIPSLLGHLLEERMKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
463 FIPSLLGHLLEERQMIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
464 FIPSLLGHLLEERQKMKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
465 FIPSLLGHLLEERQKIMTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
466 FIPSLLGHLLEERQKIKMKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
467 FIPSLLGHLLEERQKIKTMMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
468 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA
469 FIPSLLGHLLEERQKIKTKIMKTQDPIEKILLDYRQKAIKLLANSFYGYYGYA
470 FIPSLLGHLLEERQKIKTKMKMTQDPIEKILLDYRQKAIKLLANSFYGYYGYA
471 FIPSLLGHLLEERQKIKTKMKEMKDPIEKILLDYRQKAIKLLANSFYGYYGYA
472 FIPSLLGHLLEERQKIKTKMKETMNPIEKILLDYRQKAIKLLANSFYGYYGYA
473 FIPSLLGHLLEERQKIKTKMKETQMTIEKILLDYRQKAIKLLANSFYGYYGYA
474 FIPSLLGHLLEERQKIKTKMKETQEMIEKILLDYRQKAIKLLANSFYGYYGYA
475 FIPSLLGHLLEERQKIKTKMKETQDPMKKILLDYRQKAIKLLANSFYGYYGYA
476 FIPSLLGHLLEERQKIKTKMKETQDPIMKILLDYRQKAIKLLANSFYGYYGYA
477 FIPSLLGHLLEERQKIKTKMKETQDPIEMILLDYRQKAIKLLANSFYGYYGYA
478 FIPSLLGHLLEERQKIKTKMKETQDPIEKMILDYRQKAIKLLANSFYGYYGYA
479 FIPSLLGHLLEERQKIKTKMKETQDPIEKIMIDYRQKAIKLLANSFYGYYGYA
480 FIPSLLGHLLEERQKIKTKMKETQDPIEKILMNYRQKAIKLLANSFYGYYGYA
481 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLMNRQKAIKLLANSFYGYYGYA
482 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLEMRQKAIKLLANSFYGYYGYA
483 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLD(-)MKKAIKLLANSFYGYYGYA
484 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRMKAIKLLANSFYGYYGYA
485 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQMTIKLLANSFYGYYGYA
486 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKMIKLLANSFYGYYGYA
487 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAMKLLANSFYGYYGYA
488 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIMILANSFYGYYGYA
489 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKMIANSFYGYYGYA
490 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLMTNSFYGYYGYA
491 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLMNSFYGYYGYA
492 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLAMTFYGYYGYA
493 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLAKMIYGYYGYA
494 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSMNGYYGYA
495 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSLMRYYGYA
496 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSF(-)MNYGYA
497 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGMNGYA
498 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYG(-)MSYA
499 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGY(-)MNA
500 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGMT
QuikScan-1 Library
Pfu fingers domain consists of 2 anti-parallel helices (O-P)
that make numerous contacts with an incoming nucleotide.
Mutations in this domain have been shown to increase
incorporation of modified nucleotides.
1
2
3
4
5
450 500 IPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGY
# active hits*
*library properties: <10% parental DNA background; total size: 1.7x105 cfus
#95% confidence level; takes into account number of unique sequences and oligo synthesis error rate
• Results show the fingers domain is highly mutable; Pfu DNA polymerase
retains some level of detectable activity when mutated at 34 positions in the
fingers domain
• Heat-treated bacterial cultures were prepared from 467# random clones and
screened for DNA polymerase activity
• QuikScan-1 library containing 51 unique sequences was designed to
introduce a Met (ATG) substitution at each codon in the fingers domain
chromophore
-b
arr
el s
tru
ctu
re
E120G
Site-Saturation
Scanning of GFP
Renilla reniformis GFP E120G:
• Previously identified Gly substitution
at position 120 that improves
fluorescence intensity of Rre GFP in
both E. coli and mammalian cells
• Isolated by random mutagenesis of
humanized Rre gfp sequence and
screening the resulting libraries for
increased brightness in E. coli
• Nucleotide substitution (GAG→GGG)
was subsequently incorporated into
Agilent’s Vitality II (hrGFP II)
mammalian expression vectors
• Resides in 5-6 loop, pointing away
from the chromophore
• Enhances solubility and protein
folding Humanized Renilla reniformis GFP mutant expressed in a range of
mammalian cell types by transfection with Vitality II phrGFP II-C vector
11/19/2014 Page 20
Page 21 11/19/2014
GFP Site-Saturation Scan Library Design (expression & screening in E. coli)
Oligo set 1
Strategy: QuikScan-19
Target: 82-231nt / 28-77aa
Oligo length: 200nt
Codon usage: E. coli, R. reniformis
Oligo set 2
Strategy: QuikScan-19
Target: 310-459nt / 104-153aa
Oligo length: 189nt
Codon usage: E. coli, R. reniformis
Oligo set 3
Strategy: QuikScan-19
Target: 541-690nt / 181-230aa
Oligo length: 198nt
Codon usage: E. coli, R. reniformis
Cloning: Incorporated amplified oligo sets into
pMal-GFP-1 using QC HT kit; pMal-GFP-1
vectors contain frameshift mutations in each
target region; oligos were designed to correct
frameshift (1bp insertion) while introducing
desired codon replacement
Screening: After IPTG induction, fluorescent
colonies were visualized using a light source
and appropriate excitation and emission filters.
Clones displaying higher fluorescence intensity
compared to GFP or GFP E120G transformants
were isolated, and plasmid DNA was prepared.
Positives were subject to 1-2 additional rounds
of transformation and screening to verify
isolation of the brightest GFP mutants.
50AA x 19 mut = 950 oligos 50AA x 19 mut = 950 oligos 50AA x 19 mut = 950 oligos
82 231nt 310 459nt 451 690nt
Screening Results: Domain 1
Page 22 11/19/2014
Transformation & Screening
(E. coli) AA:
OS1
28-77*
OS2
104-153
OS3
181-230
Library size (x105) 1.3 2.2 1.4
% fluorescent clones 0.5 5.9 7.7
# colonies screened (x104) 1.3 2.5 1.5
No. clones brighter than wild-type 1 14 10
No. clones brighter than E120G 0 8 7
*70-72aa chromophore
• Isolated only one mutation in domain 1 that confers
increased fluorescence
• Domain 1 appears more sensitive to mutations
(0.5% fluorescent) than domains 2 and 3,
presumably due to proximity of the chromophore
N28G
G *
A
V 2
L
I
M 2
F
P
S 1
T
C 1
N
Q 1
Y
W
D 1 1
E 1 2 1
H 1 1 2
K 1
R 1
1
6
Page 23 11/19/2014
116 120 121 123 124 125 129 131 154
Screening Results: Causal- Brighter than E120G
Comparable to E120G
Putative- (multi-site mutants) Mutations:
Wild-type codon: Key Domain 2
Am
ino
ac
id s
ide
ch
ain
Domain 2
sequence:
5 6 7
Changes to 6 can
modulate fluorescence
G
A
V
L
I
M
F
P 1
S 2
T 1
C
N
Q
Y 1
W
D
E 1
H
K 1
R 1 1
1
6
Page 24 11/19/2014
Screening Results:
G
A
V
L
I
M
F
P
S
T
C
N
Q
Y
W
D 2
E
H
K
R 1
1
6
G
A
V
L
I
M
F
P
S
T
C
N 1
Q
Y
W
D
E
H
K
R 1
1
6
181 184 185 194 210 213 215 225 229 230
9 10 11
Causal- Brighter than E120G
Comparable to E120G
Putative- (multi-site mutants) Mutations:
Wild-type codon: Key Domain 3
Am
ino
ac
id s
ide
ch
ain
Domain 3
sequence:
“hotspot” in 9
Page 25
Comparison of Mutagenesis Techniques
Kit Strategy GFP Target
QuikChange HT QuikScan-19 28-77; 104-153;
181-230 (60%)
5x104
GeneMorph II PCR under low-mid-high mutation
rates; EZClone & pool libraries
1-239 (100%) 5x105
Confirmed, isolated as a single mutation
Observations: Most but not all mutants isolated from QuikScan-19
libraries were single-site mutants; allowing the
identification of 17 causal mutations (11 sites) in one
round of screening
• 10 mutations at 7 sites were brighter than E120G
• 7 mutations at 6 sites were comparable to E120G
• 5 putative sites identified in QuikScan-19 clones
containing secondary mutations; non-specified
mutations were attributed to oligo synthesis errors
(1 per >300nt)
Relative fluorescence
of QuikScan-19
mutants:
> wild-type; = GFP E120G
> GFP 120G
Page 26
Observations cont’d
• In contrast, majority of clones isolated from
EP-PCR libraries were multi-site mutants;
• 4 causal mutations were identified in
single-point mutants; E120G was the
brightest
• 16 putative sites were identified in
multi-site mutants; additional
mutagenesis to distinguish causal from
neutral mutations was not performed
(none were brighter than E120G)
• E120 mutations were common to both
screens (fluorescence emission was
comparable for GFP E120D and E120G)
Key:
> wild-type
= GFP E120G
> GFP 120G
Relative
fluorescence:
QS-19
EP-PCR
Mutagenesis
method:
• 2 of 4 mutations identified by EP-PCR
were located in a region not covered by
site-saturation scanning
Page 27 11/19/2014
Potential Next Steps
Complete structure-function map of Rre GFP???
Improve fluorescence of mammalian hrGFP expression vectors???
OR
What’s the goal?
Page 28 11/19/2014
Potential Next Steps Goal: Complete structure-function map of Rre GFP
• QuikScan-19 on remaining domains; site-saturation scanning of the entire
GFP protein could be accomplished with 5 oligo sets (~4500 sequences)
• Assay and sequence a statistically significant number of random clones to
identify not only the brightest mutants, but mutations that eliminate, reduce
or have no impact on fluorescence
2 oligo
sets
Goal: Improve fluorescence of mammalian hrGFP expression vectors
• QuikCombine to create all combinations of brightest mutations (E. coli)
• Domain 2: N116E, E120D, M121E/Q, V123E,
Y124V/C, R125HDK, K129H, R131HS, V154M
• Domain 3: L184RKSTE, M185Y, F194P, G213N,
G229D
Apply site-saturation scanning & rational
combinatorial mutagenesis to 6 and 9
domains, using codon changes that
optimize mammalian expression
Perform in
parallel with
above using
staggered oligo
sets
• Move to mammalian screening system
Agilent SurePrint
Oligo Library
Synthesis
11/19/2014 29
Long length synthesis is achieved by improved
coupling efficiency & lower depurination rates*
2) Oxidation 3) Deblock
1) Coupling
Repeat n times
Depurination
side reaction
N1
N2
Ni O
O P O
RO O
HO
Injet
Flood
O
O
O
O
P O RO
O
P O RO
O
11/19/2014
30
Optimized synthesis chemistry produces high-
quality long oligos
*Synthesis of High-Quality
Libraries of Long (150mer)
Oligonucleotides by a Novel
Depurination Controlled
Process. LeProust, E. et al
(10) NAR 38: 2522–40
PCR
150mer complex library PCR amplified
oligo set
PCR isolates full-length oligos, generating
sufficient material for QC HT mutagenesis
Page 31 11/19/2014
Long Oligo Advantage
Mutagenize the longest stretch of sequence possible per QC HT reaction
Up to 50 codons with 200mers Up to 17 codons with 100mers
Fidelity of QC HT#
Oligo length 200nt 163nt 112nt
Number clones (bp) sequenced 196 (28.8kb) 198 (21.2kb) 58 (3.5kb)
% correct 63% 70% 81%
% artifacts + wild-type 4% 3% 0%
Number unspecified mutations 84 62 12
Point mutations 4% 8% 0%
Single nt deletions 54% 53% 58%
Deletions of >1nt 42% 25% 34%
Insertion 0% 13% 8%
Oligo error rate 0.29%
(1 per 343nt)
0.29%
(1 per 342)
0.34%
(1 per 294) #PCR errors expected to be <0.2% based on an error rate of 4x10-7MF/bp/d for PfuUltra II DNA polymerase
QC HT Library:
• 93948 unique oligos
• 5 oligo sets (gfp libraries)
• 3 oligos: 112, 163, 200nt
• 2 features per array
Frequency of unspecified mutations is 1 error per ~300 bases; errors are
predominantly deletions (single, multiple)
Trade-off between target length and mutation frequency; longer the oligo, the
greater the likelihood of an unintended synthesis error due to less than
perfect coupling efficiency; simply adjust the number of clones you screen to
take advantage of the benefits of long oligos
Oligo Quality Verified with QC HT Kit Control
• Adapted from standard QuikChange kit control;
processed in parallel with custom oligo sets
• Agilent verifies the quality of each custom oligo library
• 100nt lacZ sequence printed alongside custom oligos
• 10 features per 244K array
• WT lacZ sequence reverts stop codon in pWhiteScript, restoring -gal activity
• Oligo synthesis fidelity verified by scoring percent blue colonies after plating
control QC HT transformations on X-gal/IPTG agar plates
• Researchers can use the QC HT kit control to:
• Confirm oligo library quality
• Troubleshoot issues with protocol (PCR, mutagenesis, transformation) or
reagents (comp cell storage conditions, agar plates)
11/19/2014 Page 32
11/19/2014
Custom
Oligo
Library
Mean %
blue
Std
Dev
1 94.73 2.05
2 92.96 1.91
3 92.00 2.50
4 90.73 3.80
QC HT lacZ kit control
*corresponds to <1 error per 300nt
Quality-controlled oligo libraries
Passing spec: 10l of control transformation
must yield >100 colonies, with at least 80% blue*
Page 33
General
Guidelines for
Screening
QuikChange HT
Libraries
11/19/2014 34
Theoretical Screening Requirements
- Assay sufficient number of clones to ensure that most variants have been sampled
at least once
Variants Probability L1 L2
100 0.95 300 760
100 0.99 460 920
1000 0.95 3000 9880
1000 0.99 4610 11510
10000 0.95 30000 122000
10000 0.99 46000 138000
100000 0.95 300000 1448000
100000 0.99 461000 1611000
If the oligo pool contains V variants, the size of library L required to have probability P of observing a particular variant is given by L1 = -V ln(1-P) The size of library required to have probability P that all variants are represented is L2 = -V ln (-ln(P)/V)
Ala scan (50 variants)
Site saturation scan (950 variants)
- A random sampling of 3-10,000 clones should contain at least the majority of
variants in site-saturation scanning library constructed with 200mers;
- Practical number of clones to screen
- Not limited by transformation efficiency of bacteria (~105-107 transformants per
QuikChange reaction), ensuring fully-representative sub-libraries
- Level of oversampling required is determined by the total library diversity
11/19/2014 Page 35
120K Multi-site (120,000 variants)
Page 36 11/19/2014
General Screening Strategies
Sub-cloning:
• Purify plasmid DNA library and transform suitable
expression host (BL21, etc.)
Low-throughput screen (1-103): Purified protein needed (e.g., specific activity
changes, activity is undetectable in extracts, etc.)
• Affinity protein purification; process 96 colonies per plate
• Measure protein concentration and functional activity to
detect changes in specific activity or expression level
• Sequence change-of-function mutants to identify useful
mutations; optionally, sequence mutants with wild-type
activity for deeper structure-function mapping
Plate appropriate
number of clones
Transfer to 96-
(deep) well plate;
culture & induce
protein expression
Affinity tag
protein
purification
Measure
specific
activity
Sequence to
identify
essential/non-
essential codons
Availability and type of a mutant screening assay
largely determines mutagenesis strategy
• Low throughput → Alanine scan
• Medium throughput → Site-Saturation scan
• High throughput → Multiple site-saturation scanning
libraries, complex combinatorial libraries
Mid-throughput screening
assays (103-106)
Screen recombinant proteins in host cell
background, as extracts (96 well plates)
or filter-lifts (plaques, lysed cells)
Assay activity:
• Directly:
• radiolabelled or colorimetric
substrate (e.g., polymerases, -gal)
• fluorescence (e.g., GFP)
• Indirectly:
• GFP or -gal fusions
• Monitor stability/solubility changes;
expression-level changes in codon
optimization studies
Plate desired
number clones
Incubate with
radio-labeled
substrate
Replica plate
onto filters
Lysis
Isolate clone
from master
plate and
sequence
Identify
beneficial
mutations
Colony-based filter lift
Qualitative
(GoF)
42C 55C
wt wt Mutant polymerase library
Extract screen Example (MMLV RT):
Arezi et al (09) Nucleic Acids
Res., 37: 473-481
Example (T.sp. jdf-3 pol B): Arezi et al (02) J. Mol. Biol. 322:719-729
Selection methods (>106): Viability of recombinant bacteria
under selective metabolic pressure
Binding under stringent conditions
(M13 phage display; e.g., IgG)
Fitness under selective PCR
condition (CSR)
Compartmentalized
Self-Replication
Examples (PCR enzymes):
Hansen et al (11) Nucleic Acids Res. 39:1801-1810;
Arezi et al (14) Frontiers in Microbiology 5: 408
Adapted from
Ghadessy (01)
PNAS 98:
4552-7
Selection
(GoF)
Thermal activity assay:
Colonies were cultured in
deep-well plates overnight.
Cells extracts were prepared
and spotted onto filter paper.
Filters were incubated at
elevated temperature with
primed DNA and radioactive
nucleotides. Mutations that
confer thermal resistance
were identified by DNA
sequencing.
Semi-quantitative
G/LoF
11/19/2014 Page 37
Summary
11/19/2014 38
Evolution of QuikChange Kits
Page 39
1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014
fusion
Accuracy (x105)
PfuUltra Pfu Phusion DV Vent KOD
Pfx Taq
QuikChange
HT Protein
Engineering
System QuikChange
Lightning Site-Directed
Mutagenesis System
QuikChange
Lightning Multi Site-Directed
Mutagenesis System
QuikChange
II & II XL Site-Directed
Mutagenesis System
QuikChange
Multi Site-Directed
Mutagenesis System
QuikChange Site-Directed
Mutagenesis System
Pyrococcus
furiosus isolated
Cloned Pfu
Native Pfu
First of 6
QuikChange
patents issue
PfuTurbo
PfuUltra PfuUltra II
Standard oligos
(1 pair per mutant)
SurePrint Oligo Libraries
(expanded target
regions; up to 50 codons
per reaction)
Standard or
Degenerate oligos
(2-5 per reaction)
Double-to-pentuple
mutants & codon-
saturation libraries
Scanning & targeted-
combinatorial
libraries (up to
122,000 variants)
Parallel improvements in High-Fidelity
PCR & Long-Oligo Synthesis
11/19/2014
QuikChange HT provides fast, efficient, and cost-effective construction of
rationally-designed libraries, enabling comprehensive mutagenesis with
flexible scaling and no prior knowledge of structure-function relationships
11/19/2014
40
• Fast & efficient: 1-day protocol for 3-step cloning with >80% incorporation efficiency
Summary
• Rational design:
• High-quality information content minimizes amount of screening required
• User-specified codon usage eliminates issues with degenerate codon primers;
libraries lack termination codons and biases associated with codon redundancy,
while utilizing host-preferred codons for maximum expression
• Cost-effective:
• In-house method provides lower cost-per-mutant and faster turn-around-times
compared to commercial gene variant libraries
• Use SurePrint inkjet technology to synthesize high-quality oligos up to 200nt in length
• Enables the construction of complex plasmid DNA libraries with targeted mutations
spanning 17-50 amino acids
11/19/2014
41
• Comprehensive; only commercial kit that enables:
• Scanning for deep structure-function mapping with no prior knowledge
• Targeted combinatorial mutagenesis for protein engineering
Key Features cont’d
- Nucleotide-based oligo library design (promoter structure-function studies)
- Kits for incorporating more than one oligo set per QC HT reaction, to
increase library diversity and mutational target size
Future Offerings (potential collaboration and beta-testing opportunities):
• Flexible scaling:
• Easy-to-use mutagenesis design software supports multiple design options
• Construct up to 20 libraries in parallel, targeting one or more proteins
• Choose design strategy that fits experimental and screening requirements, from
alanine scanning to site-saturation scanning and rational combinatorial mutagenesis
Contact info: [email protected]
Ordering Information
42
All Inclusive:
Mutagenic Primer Library + Library Amplification and Cleanup + QuikChange
Lightning cloning (fast protocol) + Competent Cells + Controls
1. QuikChange HT Mutagenesis Library (one tube)
2. QuikChange HT Mutagenesis Sub-Library Primers (2-40 tubes)
3. QuikChange HT Mutagenesis Reagents
4. QuikChange HT DNA Cleanup Kit Room Temperature (bind & wash buffers + spin cups)
5. SoloPack Gold Supercompetent Cells (15 tubes + 1pUC18)
Scope PN Description US List Price
Commercial G5900A QuikChange HT Protein Engineering System 150nt, 10 sites 10,161.30
G5900B QuikChange HT Protein Engineering System 150nt, 20 sites 15,632.77
G5901A QuikChange HT Protein Engineering System 200nt 10 sites 12,391.83
G5901B QuikChange HT Protein Engineering System 200nt 20 sites 19,064.35
Academic/ government
G5902A QuikChange HT Protein Engineering System 150nt-Academic, 10 sites 6,021.51
G5902B QuikChange HT Protein Engineering System 150nt-Academic, 20 sites 9,263.86
G5903A QuikChange HT Protein Engineering System 200nt-Academic, 10 sites 7,343.31
G5903B QuikChange HT Protein Engineering System 200nt-Academic, 20 sites 11,297.39
11/19/2014