DNA全基因合成介绍

DNAWorks Options
Balancing act • Fast, simple, cheap? • Slow, complex, expensive? - reliable • Reusable and interchangeable oligos?
DNAWorks Options
Others • Restriction Site Screen (non-degenerate, degenerate sequences) • Custom Site Screen (mind the format!) • Weights (experimental)
Gene Synthesis Methods
Thermodynamically Balanced Conventional
Thermodynamically Balanced Inside-Out
Protein Expression
Protein/Structure Independent Factors: • promoters and upstream elements • translational initiation and termination • mRNA stability • codon bias
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/)
DNAWorks Options
Sequences • protein (X = stop) • nucleotide (can be degenerate) • almost any file format • reverse sequence • fix sequence in gap
DNAWorks Output
Web output • Input for DNAWorks (standalone version) • Header • Initial parameters • Optimization log • Final scores • Final summary
DNAWorks Output
Total output • Sequence blocks • CFT blocks • Pattern block • Trials • Final Summary
• • • • • • • • codon usage length melting temperature repeat pattern mispriming AT/GC contents gapfix
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
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
Synthetic Genes
Benefits: • Codon use optimized for host • Flexibility in subcloning • Ease of complex mutagenesis
Problems: • Time consuming • Complicated • Error-prone
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
Gene Synthesis using DNAWorks
Dr. David Hoover Helix Systems, SCB, CIT, NIH
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
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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 | | | | | | |
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
599428.00 597986.00 392298.00 814464.00 1441162.00 1043166.00 789799.00 914677.00 1028789.00 257442.00 399567.00 528840.00 271820.00 579156.00 672416.00 1018345.00 432954.00 434655.00 441137.00 706723.00 1163126.00 879684.00
16.49 16.45 10.79 22.41 39.65 28.70 21.73 25.16 28.30 7.08 10.99 14.55 7.48 15.93 18.50 28.02 11.91 11.96 12.14 19.44 32.00 24.20
0.25 0.25 0.16 0.34 0.58 0.42 0.46 0.54 0.46 0.12 0.18 0.24 0.11 0.23 0.26 0.40 0.21 0.21 0.15 0.24 0.57 0.43
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
Protein/Structure Dependent Factors: • folding and aggregation • proteolysis and degradation • secretion and localization
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
Gly Gly Gly Gly Glu Glu Asp Asp Val Val Val Val Ala Ala Ala Ala Arg Arg Ser Ser Lys Lys
GGG GGA GGT GGC GAG GAA GAT GAC GTG GTA GTT GTC GCG GCA GCT GCC AGG AGA AGT AGC AAG Байду номын сангаасAA
DNAWorks Output
Trial outputs • Initial parameters • Final DNA sequence • Assembly • Final scores • Codon report • Histograms • Oligo sequences
Scores / Penalties
Commerical Sources
Typical costs: • $0.79 - $3.60 / bp • Complexities? • Intellectual property?
• 800 bp = $1000 (Gene Script)
Genes From Scratch
• • • • • oligos ~ $0.20 / nt (NIH discount) PCR reagents ~ $2 / reaction sequencing ~ $20 / 600 bp electrophoresis ~ $3 / gel labor ~ $20 / hr
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
70.0%
80.0%
90.0%
0.0%
R:AGG R:AGA I:ATA G:GGA P:CCC R:CGA L:CTA R:CGG T:ACA L:TTA S:AGT S:TCA L:CTG S:TCG R:CGC R:CGT A:GCG P:CCG
E. coli
Codon Bias
H. sapiens
DNAWorks Options
• Job Name • E-mail Address
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
optimize: • hairpins / mRNA structure • repeats / mispriming • restriction site inclusion / exclusion • length
DNAWorks
http://helixweb.nih.gov/dnaworks/
GFP, 238 aa, 714 bp, 20 oligos, 1134 nt, 2 reactions, 2 gels, 4 sequences, ~10 hrs = $517
How to design oligos
reverse-translate protein into DNA, optimum codon usage break into fragments of equal overlap Tm