Sequencing of DNA - determination of the primary structure of the proteins

DNA sequencing permits the rapid determination of sequences of proteins, so allowing the determination of their primary structures.

DNA can be sequenced using:

the "dideoxy" (Sanger) method or

the "chemical" (Maxam and Gilbert) approach

The dideoxy method is currently the most important method for sequencing of DNA. In this method sequencing is carried out using a DNA polymerase to extend a primer along a single-stranded template in the presence of the four dNTPs. The sequencing reaction is terminated in a random fashion by the incorporation of a dNTP analog, dideoxynucleoside triphosphate (ddNTP). ddNTP lacks the -OH group on the 3' carbon of the ribose sugar, and thus if incorporated into a nucleic acid chain during DNA synthesis, terminates synthesis, producing DNA chains of varying length, that all terminate with the same 3' base. There are separated by the high-resolution polyacrylamide gel electrophoresis. By the use of a radiolabel or fluorescent label, it is possible to detect these chains. Radiolabels can be incorporated into the primer used for sequencing (by end - labeling with T4 polynucleotide kinase and [- P] ATP or [- P] ATP) or can be incorporated into the growing DNA chain by the use, for example [- S]dATP or into the ddNTPs [P ddNTPs]. For fluorescent labels, the dyes can be coupled to the primers or can be attached to the ddNTPs.

Radioactive sequencing and primer-labeled fluorescent sequencing requires the use of four separate sequencing reactions, one for each of the ddNTP terminators, and four lanes on the polyacrylamide gel. It is not necessary if 4 different primer dyes are used.

The basis of PCR amplifications can help in process of the sequencing. The popular method of cycle sequencing involves the linear amplification of double- or single-stranded DNA. This amplification is performed in a thermal cycler. Each cycle consists of annealing, extension and thermal denaturation as in a normal PCR, but only a single primer is present in the reaction; this result in a linear amplification. It may be used with radiolabeled and fluorescent dye-labeled primers or radiolabeled or fluorescent dye-labeled dNTPs or ddNTPs. The sequencing reactions can be analyzed by polyacrylamide urea gels with detection on X-ray film or automated fluorescence sequencers. The scheme of the "dideoxy" method is illustrated in Figure below.