Knowledge of the nucleotide sequence of a gene provides important information about its structure, function, and evolutionary relationship to other similar genes in the same or different organisms. Thus, the development in the 1970s of relatively simple methods for sequencing DNA has had a great impact on genetics. Two basic methods for DNA sequencing have been developed: a chemical cleavage method (A. M. Maxam and W. Gilbert, 1977) and an enzymatic method (F. Sanger, 1981). A brief outline of the underlying principles follows.
Sequencing by chemical degradation
This method utilizes base-specific cleavage of DNA by certain chemicals.Four different chemicals are used in four reactions, one for each base. Each reaction produces a set of DNA fragments of different sizes. The sizes of the fragments in a reaction mixture are determined by positions in the DNA of the nucleotide that has been cleaved. A double-stranded or singlestranded fragment of DNA to be sequenced is processed to obtain a single strand labeled with a radioactive isotope at the 5′ end Brown, T.A.: Genomes. Bios Scientific Publ., Oxford, 1999.. This DNA strand is treated with one of the four chemicals for one of the four reactions. Here the reaction at guanine sites(G) by dimethylsulfate (DMS) is shown. Dimethyl sulfate attaches a methyl group to the purine ring of G nucleotides. The amount of DMS used is limited so that on average just one G nucleotide per strand is methylated, not the others (shown here in four different positions of G). When a second chemical, piperidine, is added, the nucleotide purine ring is removed and the DNA molecule is cleaved at the phosphodiester bond just upstream of the site without the base. The overall procedure results in a set of labeled fragments of defined sizes according to the positions of G in the DNA sample being sequenced. Similar reactions are carried out for the other three bases (A, T, and C, not shown). The four reaction mixtures, one for each of the bases, are run in separate lanes of a polyacrylamide gel electrophoresis. Each of the four lanes represents one of the four bases G, A, T, or C. The smallest fragment will migrate the farthest downward, the next a little less far, etc. One can then read the sequence in the direction opposite to migration to obtain the sequence in the 5′ to 3′ direction (here TAGTCGCAGTACCGTA).
Sequencing by chain termination
This method, now much more widely used than the chemical cleavage method, rests on the principle that DNA synthesis is terminated when instead of a normal deoxynucleotide (dATP, dTTP, dGTP, dCTP), a dideoxynucleotide (ddATP, ddTTP, ddGTP, ddCTP) is used. A dideoxynucleotide (ddNTP) is an analogue of the normal dNTP. It differs by lack of a hydroxyl group at the 3′ carbon position. When a dideoxynucleotide is incorporated during DNA synthesis, no bond between its 3′ position and the next nucleotide is possible because the ddNTP lacks the 3′ hydroxyl group. Thus, synthesis of the new chain is terminated at this site. The DNA fragment to be sequenced has to be single-stranded Brown, T.A.: Genomes. Bios Scientific Publ., Oxford, 1999.. DNA synthesis is initiated using a primer and one of the four ddNTPs labeled with 32 P in the phosphate groups or, for automated sequencing, with a fluorophore (see next plate). Here an example of chain termination using ddATP is shown Strachan, T., Read, A.P.: Human Molecular Genetics. 2nd ed. Bios scientific Publishers,. Wherever an adenine (A) occurs in the sequence, the dideoxyadenine triphosphate will cause termination of the new DNA chain being synthesized. This will produce a set of different DNA fragments whose sizes are determined by the positions of the adenine residues occurring in the fragment to be sequenced. Similar reactions are done for the other three nucleotides. The four parallel reactions will yield a set of fragments with defined sizes according to the positions of the nucleotides where the new DNA synthesis has been terminated. The fragments are separated according to size by gel electrophoresis as in the chemical method. The sequence gel is read in the direction from small fragments to large fragments to derive the nucleotide sequence in the 5′ to 3′ direction. An example of an actual sequencing gel is shown between panel A and B.
Références [ + ]
|1, 2.||↑||Brown, T.A.: Genomes. Bios Scientific Publ., Oxford, 1999.|
|3.||↑||Strachan, T., Read, A.P.: Human Molecular Genetics. 2nd ed. Bios scientific Publishers,|