|
pubmed:abstractText |
A synthetic gene encoding the hepatitis C virus (HCV) nucleocapsid protein was constructed and expressed in E. coli. To synthesize this gene, we developed a new method that results in the enzymatic synthesis of long polydeoxyribonucleotides from oligodeoxyribonucleotides. The method, designated as the 'Exchangeable Template Reaction' (ETR), uses oligonucleotides as templates for DNA polymerase. A special mechanism was designed to exchange the templates during the polymerase reaction. The mechanism relies on the formation of a single-stranded 3'-protrusion at the 'growing point' of the elongating DNA such that it can be subsequently annealed, in a sequence-specific manner, with the next synthetic oligonucleotide. When annealed to the 3'-protrusion, the added oligonucleotide becomes a template for DNA polymerase, and the protruding 3'-end of the double-stranded DNA is used as the primer. The HCV nucleocapsid gene was assembled with DNA ligase from three fragments synthesized by ETR. The data verify that this method is efficient. The main advantage of ETR is the ability to combine more than two oligonucleotides in one tube together with polymerase and an enzymatic activity that produces a 3'-protrusion (e.g., BstXI) rather than the sequential addition of each component. The data demonstrate that as many as five oligonucleotides can be used simultaneously, resulting in a synthesized DNA fragment of designed sequence. The synthetic gene expressed in E. coli produced a 27 kDa protein that specifically interacted with antibodies from sera obtained from HCV-infected individuals.
|
