There is a growing list of organisms that have been completely sequenced and the entire human genome will be sequenced in the foreseeable future. For example, Borrelia burgdorferi, chlamydia, helicobacter pylori and tuberculosis encode 850, 900, 1,600 and 4,000 genes, respectively. Straightforward, rapid and economical methods are available to obtain the necessary primers and generate pure preparations of each of these genes by PCR, but conventional PCR products are not transcriptionally active so they can not be used directly in functional assays. The typical way to create transcriptionally active genes is to clone the PCR fragments into an expression vector, transform and grow bacteria, and purify the plasmid. Although this process can lead to large quantities of plasmid useful for a variety of different experiments, it would greatly facilitate research and functional screening if the initial PCR product were transcriptionally active. The purpose of this proposal is to develop a practical method for generating transciptionally active PCR fragments, which can be used directly in in vitro transfection assays and in vivo. The method will enable high throughput functional screening of a very large number of genes, on a scale that is not possible today. A kit designed to accomplish this will be developed and made available to scientists.
The resources from this grant will be used to develop a practical method for generating transciptionally active PCR fragments, which can be used directly in in vitro transfection assays and in vivo. This method will give investigators the ability to create very large libraries of 1000s of transcriptionally active genes from the knowledge of their sequence.
