Transcriptionally Active Polymerase Chain Reaction (TAP)

摘要

An approach is described for makingtranscriptionally active PCR (TAP) fragments that were used directly in in vitro and in vivo expression experiments. TAP fragments encoding reporter genes were amplified in 1 day using typical PCR methodology and were expressed in cultured cells and in mice at levels comparable with a widely used cytomegalovirus promoter-based plasmid expression vector. Following intramuscular injection, a TAP fragment encoding hepatitis B surface antigen (HBsAg) induced anti-HBsAg antibody titers comparable with those induced by supercoiled plasmid encoding the same antigen. Epitope-tagged TAP fragments were generated and transfected into cells for rapid, high throughput immunocytochemical analysis of the tagged gene products. TAP fragments were also transferred directly into expression vectors by in vivo homologous recombination without conventional cloning, affording a high throughput cloning approach that does not require restriction enzyme digestion, ligations, or thymidine adenine complementation cloning. The methodology has been adapted to a robotic work station enabling the high throughput generation of transcriptionally active genes at the rate of more than 400 different genes per day. This technology offers a practical approach to directly utilize genome sequence data to generate functional proteomes. An approach is described for makingtranscriptionally active PCR (TAP) fragments that were used directly in in vitro and in vivo expression experiments. TAP fragments encoding reporter genes were amplified in 1 day using typical PCR methodology and were expressed in cultured cells and in mice at levels comparable with a widely used cytomegalovirus promoter-based plasmid expression vector. Following intramuscular injection, a TAP fragment encoding hepatitis B surface antigen (HBsAg) induced anti-HBsAg antibody titers comparable with those induced by supercoiled plasmid encoding the same antigen. Epitope-tagged TAP fragments were generated and transfected into cells for rapid, high throughput immunocytochemical analysis of the tagged gene products. TAP fragments were also transferred directly into expression vectors by in vivo homologous recombination without conventional cloning, affording a high throughput cloning approach that does not require restriction enzyme digestion, ligations, or thymidine adenine complementation cloning. The methodology has been adapted to a robotic work station enabling the high throughput generation of transcriptionally active genes at the rate of more than 400 different genes per day. This technology offers a practical approach to directly utilize genome sequence data to generate functional proteomes. transcriptionally active PCR hemagglutinin cytomegalovirus oligonucleotide chloramphenicol acetyltransferase enzyme-linked immunosorbent assay green fluorescent protein Chinese hamster ovary hepatitis B surface antigen Worldwide genome sequencing projects have now yielded complete DNA sequence data for microorganisms, insects, plants, and human and other mammalian genomes. Presently, the exponential increase in genome sequence information has not led to a similar increase in the availability of functional genes or proteins that are encoded by these sequenced genes, and consequently, the biomedical and pharmaceutical research communities have not yet been able to take full advantage of all the sequence data. Today, functional genomics research demands an increasing number of sequenced genes with unknown function to be expressed. By far the most widely used approach to producing transcriptionally active genes is to clone them into a plasmid expression vector, transform and grow bacteria, and purify the plasmids. This tried and true approach has worked well for many years, but it is time- and labor-intensive, particularly when a large number of genes needs to be simultaneously cloned in a transcriptionally active form. Large numbers of PCR fragments can be amplified on the require