DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis

Abstract

The availability of sequences of entire genomes has dramatically increased the number of protein targets, many of which will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. The synthetic gene can be optimized for expression and constructed for easy mutational manipulation without regard to the parent genome. Yet design and construction of synthetic genes, especially those coding for large proteins, can be a slow, difficult and confusing process. We have written a computer program that automates the design of oligonucleotides for gene synthesis. Our program requires simple input information, i.e. amino acid sequence of the target protein and melting temperature (needed for the gene assembly) of synthetic oligonucleotides. The program outputs a series of oligonucleotide sequences with codons optimized for expression in an organism of choice. Those oligonucleotides are characterized by highly homogeneous melting temperatures and a minimized tendency for hairpin formation. With the help of this program and a two-step PCR method, we have successfully constructed numerous synthetic genes, ranging from 139 to 1042 bp. The approach presented here simplifies the production of proteins from a wide variety of organisms for genomics-based studies.

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

“…A subsequent PCR using primers 19 and 20 which incorporates two additional glutamates after the ToxA SS and substitutes KDEL for REDLK at the end of DIII was cloned into the Pvu II and Xba I sites of an intermediate vector and then used for a subsequent cloning step. An OmpA SS was substituted for the ToxA SS using primers 21–24 designed using the online DNA Works (Hoover and Lubkowski, 2002) tool with a first round using all four primers and a second round using primers 21 and 24. An Aat II and Xba I fragment was cut out of the cloned PCR 19 and 20 product and substituted for the same fragment from the OmpA SS:TGFα-DII (Δ25 aa):DIb:DIII construct, which results in substituting the KDEL sequence from the other KDEL-containing clone (the product of primers 19 and 20).…”

Analysis of Coiled-Coil Interactions between Core Proteins of the Spindle Pole Body

“…The resulting plasmids were transformed into E. coli BL21 pLysS cells (30). Expression of proteins was induced with 0.4 mM IPTG at OD 600 ) 0.4 for 2 h. Proteins were purified in 8 M urea on a Sephadex G-50 size exclusion column (Pharmacia) and subsequently by reverse-phase HPLC (Vydac C18 column) (39).…”

Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E

“…Using DNAWorks (Hoover and Lubkowski, 2002), Arabidopsis eIF4G 322-1727 , eIF4E1, eIF4E1b, eIF4E1c, and eIF4E1c(long) were designed with codon optimization for expression in Escherichia coli and assembled by overlap PCR of oligonucleotides (Supplemental Figs. S3-S8; Supplemental Tables S3-S7; Horton et al, 1989).…”