Project B
Rational de-novo synthesis of genes from synthetic oligonucleotides
We have constructed a prototype polymerase chain assembly (PCA) workstation which can perform rapid PCA and/or PCR amplification and then separate the full length synthetic dsDNA using tandem immunocapture columns to yield pure synthetic dsDNA (see Figure-1) . Our approach employs rapid gene synthesis by polymerase chain assembly (PCA) in a high speed thermocycler in combination with smart error control strategies to effectively minimize thermal damage [Mamedov et al., J. Biotechnology, 131 (2007) 378-387 and Mamedov et al., Comput Comput Biol. Chem (2008), doi:10.1016/j.compbiolchem.2008.07.021].For example, endothelial protein C receptor (EPCR, 660 bp) and a mutant EPCR-2 (576 bp) that lacked 4 N-linked glycosylation sites were constructed from 35 and 33 oligonucleotides, respectively, and error rates of less than 1 error/kb were obtained. Further, these synthetic genes were cloned and protein expression was tested in the Pichia pastoris expression system [Mamedov et al., J. Biotechnology, 131 (2007) 387-387]. We are using this technology-tool available in our laboratories in the de-novo assembly of chimeric proteins and customized plasmids. I believe the proposed strategy of rapid de novo gene synthesis – when coupled with proteomics.
Figure 1. PCA, PCR, and Affinity Purification of the pUC19 β-lactamaseGene(929 bp). PCA and PCR products. Lanes (M) 100 bp DNA ladder (NEbiolabs); lane 1: PCA product; lane 2: PCR product with unlabeled primers; lane 3: PCR product with labeled primers (A).
Affinity Separation. Lanes (M) 100bp DNA ladder (NEbiolabs); lane 1: 929 bp labeled PCR product ; lane 2: wash sample from BrdU column; lane 3: intermediate elution product from BrdU column; lane 4:wash sample from streptavidin column; lane 5: final elution product from streptavidin column (B).
PCR of Affinity Purification Product. Lanes (M) 100bp DNA ladder (NEbiolabs); lane 1: 929 bp PCR product of the purified product from Figure 1B, lane 5 (C).
Fully Automated Affinity Separation. Lanes (M) 100 bp DNA ladder (NEbiolabs); lane 1: PCR product with labeled primers (sample S); lane 2: Final Elution Product; lane 3: 929 bp PCR of the Affinity Product (D).
TerMaat et al., J.Chromatographic Science, Vol:48, 2010, pp120.
Affinity Separation. Lanes (M) 100bp DNA ladder (NEbiolabs); lane 1: 929 bp labeled PCR product ; lane 2: wash sample from BrdU column; lane 3: intermediate elution product from BrdU column; lane 4:wash sample from streptavidin column; lane 5: final elution product from streptavidin column (B).
PCR of Affinity Purification Product. Lanes (M) 100bp DNA ladder (NEbiolabs); lane 1: 929 bp PCR product of the purified product from Figure 1B, lane 5 (C).
Fully Automated Affinity Separation. Lanes (M) 100 bp DNA ladder (NEbiolabs); lane 1: PCR product with labeled primers (sample S); lane 2: Final Elution Product; lane 3: 929 bp PCR of the Affinity Product (D).
TerMaat et al., J.Chromatographic Science, Vol:48, 2010, pp120.