gene synthesis using dnaworks dr. david hoover helix systems, scb, cit, nih
TRANSCRIPT
![Page 1: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/1.jpg)
Gene Synthesis using DNAWorks
Dr. David Hoover
Helix Systems, SCB, CIT, NIH
![Page 2: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/2.jpg)
Gene Synthesis
Several methods• ligation - incredibly tedious and inefficient• FokI - sequence dependent (type IIs r.e.)• serial cloning - sequence dependent• assembly or self-priming PCR
![Page 3: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/3.jpg)
Gene Synthesis Methods
Thermodynamically Balanced Conventional
Thermodynamically Balanced Inside-Out
![Page 4: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/4.jpg)
Protein Expression
Protein/Structure Independent Factors:• promoters and upstream elements• translational initiation and termination• mRNA stability• codon bias
Protein/Structure Dependent Factors:• folding and aggregation• proteolysis and degradation• secretion and localization
![Page 5: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/5.jpg)
Codon Bias
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
R:A
GG
R:A
GA
I:A
TA
G:G
GA
P:C
CC
R:C
GA
L:C
TA
R:C
GG
T:A
CA
L:T
TA
S:A
GT
S:T
CA
L:C
TG
S:T
CG
R:C
GC
R:C
GT
A:G
CG
P:C
CG
E. coli
H. sapiens
![Page 6: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/6.jpg)
Synthetic Genes
Benefits:• Codon use optimized for host• Flexibility in subcloning• Ease of complex mutagenesis
Problems:• Time consuming• Complicated• Error-prone
![Page 7: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/7.jpg)
Commercial Sources
Blue Heron Biotechnology (http://www.blueheronbio.com)
DNA 2.0 (http://www.dna20.com/)
Gene Script Corporation (http://www.genscript.com/)
BioNexus Inc. (http://www.genesynthesis.net/)
Entelechon (http://www.entelechon.com/)
GeneArt (http://www.geneart.com/)
Codon Devices (http://www.codondevices.com/)
![Page 8: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/8.jpg)
Commerical Sources
Typical costs:• $0.79 - $3.60 / bp• Complexities?• Intellectual property?
• 800 bp = $1000 (Gene Script)
![Page 9: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/9.jpg)
Genes From Scratch
• oligos ~ $0.20 / nt (NIH discount)• PCR reagents ~ $2 / reaction • sequencing ~ $20 / 600 bp• electrophoresis ~ $3 / gel• labor ~ $20 / hr
GFP, 238 aa, 714 bp, 20 oligos, 1134 nt, 2 reactions, 2 gels, 4 sequences, ~10 hrs = $517
![Page 10: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/10.jpg)
![Page 11: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/11.jpg)
How to design oligosreverse-translate protein into DNA, optimum codon
usage
break into fragments of equal overlap Tm
optimize:• hairpins / mRNA structure• repeats / mispriming• restriction site inclusion / exclusion• length
![Page 12: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/12.jpg)
DNAWorks
http://helixweb.nih.gov/dnaworks/
![Page 13: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/13.jpg)
DNAWorks Output
181 TCTGGTGAAGGCGAGGGTGACGCGACCTACGGTAAACTCACTCTCAAAT agaccact TGCCATTTGAGTGAGAGTTTAAGTAGACGTGG <--- 4 S G E G E G D A T Y G K L T L K F I C T
| | | | | | |
7 ---> 241 ggttccttggccgaccctggttactaccttctcttacggtgttcag TGCCCGTTTGACGGCCAAGGAACCGGCTGG tc <--- 6 T G K L P V P W P T L V T T F S Y G V Q
| | | | | | |
![Page 14: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/14.jpg)
DNAWorks Options
• Job Name• E-mail Address
![Page 15: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/15.jpg)
DNAWorks Options
Codon Frequency Table• E. coli (standard, class II), H. sapiens, C.
elegans, D. melanogaster, M. musculus, P. pastoris, R. norvegicus, S. cerevesiae, X. laevis
• Custom CFT
![Page 16: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/16.jpg)
Gly GGG 599428.00 16.49 0.25 Gly GGA 597986.00 16.45 0.25 Gly GGT 392298.00 10.79 0.16 Gly GGC 814464.00 22.41 0.34 Glu GAG 1441162.00 39.65 0.58 Glu GAA 1043166.00 28.70 0.42 Asp GAT 789799.00 21.73 0.46 Asp GAC 914677.00 25.16 0.54 Val GTG 1028789.00 28.30 0.46 Val GTA 257442.00 7.08 0.12 Val GTT 399567.00 10.99 0.18 Val GTC 528840.00 14.55 0.24 Ala GCG 271820.00 7.48 0.11 Ala GCA 579156.00 15.93 0.23 Ala GCT 672416.00 18.50 0.26 Ala GCC 1018345.00 28.02 0.40 Arg AGG 432954.00 11.91 0.21 Arg AGA 434655.00 11.96 0.21 Ser AGT 441137.00 12.14 0.15 Ser AGC 706723.00 19.44 0.24 Lys AAG 1163126.00 32.00 0.57 Lys AAA 879684.00 24.20 0.43
![Page 17: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/17.jpg)
DNAWorks Options
Parameters• Annealing Temperature• Oligo Length (random)• Codon Frequency Threshold (random, strict,
scored)• Oligonucleotide, Na+/K+, Mg2+ Concentrations• Number of Solutions• TBIO• No gaps in assembly
![Page 18: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/18.jpg)
DNAWorks Options
Balancing act• Fast, simple, cheap?• Slow, complex, expensive? - reliable• Reusable and interchangeable oligos?
![Page 19: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/19.jpg)
DNAWorks Options
Others• Restriction Site Screen (non-degenerate,
degenerate sequences)• Custom Site Screen (mind the format!)• Weights (experimental)
![Page 20: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/20.jpg)
DNAWorks Options
Sequences• protein (X = stop)• nucleotide (can be degenerate)• almost any file format• reverse sequence• fix sequence in gap
![Page 21: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/21.jpg)
DNAWorks Output
Web output• Input for DNAWorks (standalone version)• Header• Initial parameters• Optimization log• Final scores• Final summary
![Page 22: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/22.jpg)
DNAWorks Output
Total output• Sequence blocks• CFT blocks• Pattern block• Trials• Final Summary
![Page 23: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/23.jpg)
DNAWorks Output
Trial outputs• Initial parameters• Final DNA sequence• Assembly• Final scores• Codon report• Histograms• Oligo sequences
![Page 24: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/24.jpg)
Scores / Penalties
• codon usage• length• melting temperature• repeat• pattern• mispriming• AT/GC contents• gapfix
![Page 25: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/25.jpg)
Mutant Run
• Design oligos based on previous set of oligos• Parameters taken from previous run• For single mutation, will output 1 or 2 oligos only
![Page 26: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/26.jpg)
What to look for
Final Summary• Avoid misprimes and repeats• Make sure overlaps are > 12 nt (Short)• Tm range should not be > 3°C (TmRange)
Don't depend entirely on scores• Arbitrary, somewhat dependent on length
![Page 27: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/27.jpg)
Tricks
Choosing codons• random - slower optimization, less constrained• strict - for the fussy• scored - if codon score really matters
Tm, Length ranges, Number of Solutions• To find the "very best" solution• no more than 999
![Page 28: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/28.jpg)
Tricks
Design multi-use and interchangeable oligos• Flanking primers with standard overlaps• Intersperse nucleotide elements between protein
elements• Gap-fix restriction sites• Allow for mutations later on
Random mutagenesis• Nucleotide sequences can be degenerate
![Page 29: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/29.jpg)
Tricks
Thermodynamically Balanced Inside-Out Mode• Multi-step PCR• More controlled, reliable method• Gao X., et al., Nucleic Acids Res 2003
Random oligo lengths• Faster, better optimization• For the not-so-fussy• Probably best for DNA-only genes
![Page 30: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/30.jpg)
Tricks
Set Tm higher• 64°C - 70°C• longer oligos, extra purification ($$$)
![Page 31: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/31.jpg)
Always double check!
Nothing is foolproof• Think carefully about what you need BEFORE
starting work• Always run final sequences through alternate
program (EMBOSS, GCG-Lite)• Make sure oligos are what you intended
![Page 32: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/32.jpg)
PCR
• Mix all oligos and additives• Specific PCR protocols• Analytical gel• Isolate desired products
![Page 33: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/33.jpg)
Assembly ProtocolOligos 1 μl 625 nM each 25 nM each
dNTPs 2 μl 2.5 mM each 0.25 mM each
H2O 19 μl
Buffer 2.5 μl 10X 1X
Pfu pol. 0.5 μl
95°C 2.0 ' 1X
95°C 0.5 '
65>55°(-0.5) 0.5 ' 20X
72°C 0.5 '
72°C 5 ' 1X
4°C hold
![Page 34: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/34.jpg)
Amplification ProtocolPCR mix 2 μl ? ?
dNTPs 8 μl 2.5 mM each 0.2 mM each
3' primer 4 μl 10 μM 400 nM
5' primer 4 μl 10 μM 400 nM
Buffer 10 μl 10X 1X
H2O 70 μl
Pfu pol. 2 μl
95°C 2.0 ' 1X
95°C 0.5 '
62°C 0.5 ' 20X
72°C 0.5 '
72°C 5 ' 1X
4°C hold
![Page 35: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/35.jpg)
![Page 36: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/36.jpg)
Problems
• No product (complete failure)• Wrong size product (mispriming)• Mutations (2 out of 3 correct, 2 errors/kb)
Sequencing is warranted...
![Page 37: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/37.jpg)
Fixes
• Optimize PCR conditions• Break gene synthesis into steps (TBIO)
![Page 38: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/38.jpg)
Errorsp = mutation rate / 1000 nt / duplication (Cline et al., Nucleic Acids Res 24 (1996))
Taq polymerase = 0.008KOD (Novagen) = 0.0027PfuUltra (Stratagene) = 0.00043
The probability of a gene n bp in length having no errors using a polymerase with mutation rate p:
p' = (1 - p)n
Therefore, p' for a 738 bp gene = (1 - 0.00043)738 = 0.728
![Page 39: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/39.jpg)
ErrorsThe number of clones needed to screen to find a correct gene with 95% confidence:
N = log(0.05)/log(1-p')
Thus, log(0.05)/log(1-0.728) = 3 clones need to be sequenced.
From Wu et al., J Biotech 124 (2006)
![Page 40: Gene Synthesis using DNAWorks Dr. David Hoover Helix Systems, SCB, CIT, NIH](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649da05503460f94a8bc20/html5/thumbnails/40.jpg)
Time
• Find protein of interest, design oligos, order oligos
• Run PCR, integrate into sequencing vector, transform
• Pick colony, grow overnight culture
• Miniprep construct, integrate into expression vector, transform
• Pick colony, grow overnight culture
• Run expression growth trials
~ 1 week between concept and initial trial (at best!!)
Can be automated and parallelized (96 well plates?)