DNAWorks全基因合成介绍.pptVIP

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 Gene Synthesis Methods 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 Codon Bias Synthetic Genes Benefits: Codon use optimized for host Flexibility in subcloning Ease of complex mutagenesis Problems: Time consuming Complicated Error-prone Commercial Sources Blue Heron Biotechnology () DNA 2.0 (/) Gene Script Corporation (/) BioNexus Inc. (/) Entelechon (/) GeneArt (/) Codon Devices (/) 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 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 optimize: hairpins / mRNA structure repeats / mispriming restriction site inclusion / exclusion length DNAWorks DNAWorks Output 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 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 DNAWorks Options Balancing act Fast, simple, cheap? Slow, complex