Nucleic acid sequencing reveals the genetic code of a DNA molecule. It may be carried out using one of two methods, each of which results in the production of DNA fragments of various lengths, differing from each other by a single base and from which one can infer the nucleic acid sequence of the molecule. This is accomplished using denaturing polyacrylamide gels. Whereas agarose gels can separate DNA molecules differing in length by 30â€“50 bases, polyacrylamide gels can discriminate among DNA molecules differing in length by a single base. Denaturing gels cause the DNA molecule to become single stranded and remain that way throughout the entire process of electrophoresis. Denaturing gels contain urea and are run at elevated temperatures, both of which promote the separation of the two strands of the DNA molecule.
Again, the DNA must be labeled in order to be visualized. The most common form of labeling is with radioactive isotopes, in particular, 32P, 33P, or 35S. After electrophoresis, the gel is dried and placed next to a sheet of x-ray film in a dark place. During this time the radioactive particles emitted from the isotope in each DNA molecule â€œexposeâ€ the film, and after development, a dark band is seen on the film at the position where the DNA band was located in the gel. This picture, called an autoradiograph, is a mirror image of the position of the DNA bands in the gel.
There are two methods that can be used to sequence DNA molecules. The Maxam-Gilbert method is based on cleavage of DNA at specific sites by chemicals rather than enzymes. However, this method is seldom used anymore; the Sanger method is preferred.
In the Sanger method, the enzymatic synthesis of DNA takes place by the sequential formation of a phosphodiester bond between the free 5' phosphate group of an incoming nucleotide and the 3' OH group of the growing chain. This process takes place throughout the length of the DNA molecule. Dideoxynucleotides lack a 3' OH group, and have a 3' H group instead. In the presence of a dideoxynucleotide, the synthesis of DNA stalls because the diphosphate bond cannot be formed. The chain growth terminates at that point, and the last base on the 3' end of the chain is a dideoxy terminator. This modification of Sangerâ€™s method of DNA sequencing is known as dideoxy termination sequencing.
In the Sanger sequencing technique, four different reaction mixtures are used to sequence a DNA fragment. Each reaction mixture contains the template DNA molecule to be sequenced, radioactively labeled primers, all four deoxynucleotides, DNA polymerase, and a different dideoxy terminator (ddATP, ddCTP, ddGTP, or ddTTP). When one of these terminators is incorporated in the newly synthesized DNA strand, it will stop further synthesis of that strand; the result is that all the strands of various lengths in the reaction mixture end in the same base. The radioactive products are separated by electrophoresis and visualized by autoradiography. Reading from the bottom of the gel (shortest fragments terminated closest to the 5' end) upward reveals the base sequence complementary to that of the template strand.