SPECIFIC DNA TECHNIQUES
The development of technologies for detailed nucleo-tide sequence determination of DNA molecules has been of immense importance. This knowledge opens the way for very precise DNA modifications, like changing individual nucleotides in order to change an individual amino acid in a protein.
In 1977 two different methods were published for DNA sequencing. The Maxam and Gilbert method is based on chemical degradation of DNA, whereas the Sanger method, also called the chain termination method, uses DNA replication enzymology. The Sanger method is the most popular method and is described here. It uses a DNA polymerase enzyme normally involved in DNA replication. DNA poly-merases are template dependent, meaning that they need a single-stranded DNA molecule which they will copy according to the A-T and G-C base pairing rules, and are primer dependent, meaning that they need a free 30-hydroxyl group of an oligonucleotide as a starting point for the incorporation of deoxyribonu-cleotide triphosphates (dATP, dCTP, dTTP, and dGTP). The primer is a short, chemically synthesized molecule, about 20 nucleotides in length which is complementary and antiparallel to a segment in the single-stranded DNA molecule to be sequenced. Under the right conditions it will hybridize and thus provide a specific starting point for the elongation reaction by the polymerase.
The method depends in essence on the inclusion in the reaction mixture of a so-called dideoxyribonu-cleotide triphosphate (ddNTP). These molecules not only lack the 20 hydroxyl group on the ribose as is normal in DNA, but also the 30 hydroxyl group; hence the name di-deoxy. These ddNTP’s can be incorporated into DNA strands by DNA polymerase. However, since the lacking 30-hydroxyl group is required for DNA elongation, the DNA molecules which have incorporated such a ddNTP are no longer substrate forfurther chain elongation: the chain terminates with a ddNTP and this principle is used for the sequencing reaction. Per reaction four tubes are set up which contain template, primer and the four dNTPs. To the four tubes ddNTP is added. To the first tube ddATP is added, to the second tube ddTTP, to the third tube ddCTP and to the fourth tube ddCTP. The ratio of dNTP versus ddNTP in each tube is chosen in such a way that a small number of templates in each tube will incorporate the specific ddNTP and will no longer be substrates for elongation (chain termination). Therefore in each tube a fraction of the strands will terminate with the specific ddNTP present in that particular tube. The length of the terminated strands is determined by the oligonucleotide primer, which sets a fixed starting point, and the ddNTP incorporated. In the first reaction tube, for example, fragment lengths are determined by the position of the various A nucleotides in the template. After the synthesis reaction the contents of the four individual sequencing tubes are applied to a high resolution polyacrylamide gel electrophoresis system which separates individual elongation products based on their length. Tube 1 reveals the positions of A, tube 2 of C, tube 3 of T and tube 4 of G. The reaction products can be visualized either by autoradiography in case a small aliquot of radioactively labeled dNTP has been incorporated in all reactions (usually alpha-32P-dCTP) or by fluoro-graphy in case a fluorescent group has been chemically added to the sequencing primer during its synthesis. The latter method is especially very well suited for automation. Currently sequencing machines are com-mercially available which, in one run, can sequence over 800 nucleotides. In such machines 20 to 40 runs can be loaded and analyzed simultaneously which tremendously enhances productivity. Needless to say, sequences are handled, analyzed and stored electro-nically. Three interlinked computer sequence data-bases are operational in the world, which are freely accessible via Internet.