DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis.
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.
Hoover
David M
DM
Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, MD 21702, USA.
Lubkowski
Jacek
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D054425:Chemokine CCL27; D019742:Chemokines, CC / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D005796:Genes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000235:genetics; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D019718:Receptors, CXCR4 / Q000235:genetics; D012984:Software; D023083:beta-Defensins / Q000235:genetics
C099677:CCL13 protein, human; C492276:CCL27 protein, human; D054425:Chemokine CCL27; D019742:Chemokines, CC; D018945:Monocyte Chemoattractant Proteins; D009841:Oligonucleotides; D019718:Receptors, CXCR4; D023083:beta-Defensins