Nucleotide Sequencing and Hybridizing

Nucleotide sequencing is the analytical chemistry of genetic engineering in general and recombinant techniques in particular. The procedure uses the well-known method of electrophoresis where organic molecules of different size will travel at different rates in a gel matrix under the influence of an electric potential (direct current); larger molecules move more slowly because of their interaction with the gel structure. Thus, DNA chains of varying nucleotide content (complexes of P-S-B) ^* which have been labeled by ³²P insertion at 5′ end will spread on the gel into discrete sequential position. The location of the respective nucleotides is visualized on the gel by staining with ethidium bromide (EtBr) or a fluorescent dye. The EtBr or dye fluoresce under ultraviolet light and a photographic record can be established. Also, when only small amounts of DNA are present, subsequent exposure to an x-ray film gives an autoradiograph of ³²P location which then can be read to ascertain the order of bases.