Nucleic Acid Analysis

This chapter describes several of the most common nucleic acid analyses performed in vitro to characterize a cloned DNA segment carrying a gene or genes. Newer polymerases that are used for DNA sequencing include modified T7 phagederived DNA polymerase and a variety of thermostable DNA polymerases such as that from the thermophilic bacterium Thermus aquaticus. The authors have successfully used the kit marketed by U.S. Biochemicals for many years; however, other products may be just as effective. It is strongly recommended that laboratories use kits from U.S. Biochemicals or competing manufacturers for applications in which they need to do manual sequencing and gel electrophoresis. The gel mobility shift assay (also called the gel retardation assay) is based on the differences in the degrees of electrophoretic mobility between nucleic acid fragments and nucleic acid-protein complexes. Although the assay has been used successfully to study binding to RNA, this discussion will be limited to the most common use, the study of binding to dsDNA. The outcome of the assay is the identification of specific DNA targets of the protein of interest. A section reviews ChIP assays from a number of laboratories relevant to prokaryotic systems. Commonly used in vitro techniques described elsewhere in the chapter generally require biochemical purification of a DNA-binding protein of interest and some knowledge concerning the location of its DNA target(s). Once a gene or promoter has been cloned, it is of interest to determine the start site and the size of the RNA transcript.