Sequencing DNA

All DNA sequencing protocols must incorporate a method for making the DNA fragments generated in the reaction "visible"; they must be capable of being detected. For Maxam-Gilbert sequencing a technique called end labeling is used, in which a radioactive atom is added to the ends of the DNA fragment being sequenced. The first step in this process is to use an enzyme, called a restriction endonuclease, to cut the DNA at a specific sequence. If the restriction endonuclease is Hind III, for example, the sequence AAGCTT will be cut.

The ends of the DNA segment made by the restriction endonuclease will have phosphate groups (-PO₃^2-) at its ends. An enzyme called a phosphatase is used to remove the phosphate group. Another enzyme, called a kinase, is then used to add a radioactive phosphate in its place. This reaction will add radioactive atoms onto both ends of the DNA restriction fragment. A second restriction endonuclease is used to make a cut within the end-labeled fragment and gel electrophoresis is then employed to separate the two resulting subfragments from each other. Each subfragment now has one labeled and one unlabeled end. The subfragment whose sequence is to be determined is cut out of the gel to purify it away from the other end-labeled subfragment.

The end-labeled piece of DNA is then divided and the fragments are placed in four separate tubes. They are then treated with different chemicals that weaken and break the bond holding the base to the backbone of the DNA molecule. These chemicals are base specific. In other words, one chemical causes the "C" reaction, in which the bond holding the C base in position is broken. Another chemical breaks the bond holding the G in place (the "G" reaction). Another breaks both G and A bases from the DNA backbone Figure 1. Schematic diagram of autoradiogram showing bands generated by the Maxam-Gilbert method of sequencing DNA. The DNA sequence is read from the bottom of the gel by noting which gel lane contains a band. (the "G+A" reaction), and a fourth breaks the bonds holding the C and T bases in place (the "C+T" reaction).

Each reaction is limited so that each DNA molecule will have only one of its base positions altered. Within the "C" reaction, for example, each DNA molecule will have only one of its C bases weakened and removed. One of the DNA molecules may have the C base closest to the end dislodged. On another DNA molecule, a C that appears 500 bases away from the end may be removed. Every C position in the population of molecules, however, is subject to treatment.

When the first step in the reaction is concluded, another reagent is then used to completely break the DNA strands at the points where the bases have been removed. In this way, a collection of DNA fragments is generated that differ in size according to the position along the strand where the break occurred.

Each reaction is electrophoresed in its own lane on a gel that separates the DNA fragments by their length. A process called autoradiography is then used to detect the separated fragments. In this technique, X-ray sensitive film is placed flat against the gel under conditions of complete darkness. Since the fragments made in the sequencing reactions are end-labeled with radioactive atoms, their emissions will expose the X-ray film at the positions where they are found on the gel. When the X-ray film is developed, bands, like a bar code, reveal the sizes of the fragments generated in each separate reaction.

Each band that has been rendered visible on the X-ray film differs from the one above or the one below it by a single base. The DNA sequence is then read from the bottom of the gel upward. A band found in the "G" reaction lane is read as a G, a band found in the "C" lane is read as a C, a band found in the lane from the "G+A" reaction but with no corresponding band of the same length in the "G" lane is read as an A, and a band in the "C+T" reaction without a corresponding base in the "C" lane is read as a T (see Figure 1).