Sequencing DNA

Because it employs fewer steps and does not require the use of restriction enzymes, the chain termination method of DNA sequencing is used in more laboratories than is the Maxam-Gilbert approach. Chain termination sequencing is a clever variation of the reaction used to replicate DNA, and requires only a handful of components. Four reaction tubes, designated "A," "C," "G," and "T," are prepared and the DNA strand whose sequence is to be determined is added into each tube. This DNA strand is called the template.

Along with the template, a short, single-stranded piece of DNA (called a primer) is added; it attaches specifically to one section of the template and serves as a starting point for synthesis of a new DNA strand. Also added are the four "building block" nucleotides, a buffer (to maintain the proper pH level), the enzyme DNA polymerase, and its cofactor magnesium, all of which are needed to extend the primer into a full-length DNA chain.

Figure 2. Schematic diagram of autoradiogram showing bands generated by the chain termination method of DNA sequencing. A band in any particular gel lane results from the incorporation of a dideoxynucleotide. DNA sequence is read from the bottom of the gel upward.

To make the DNA strands visible, a nucleotide carrying a radioactive phosphorous or sulfur atom is included in the reaction. Also included in each reaction tube is a quantity of a unique dideoxynucleotide, a modified form of a nucleotide that lacks the site at which other bases can attach during chain growth. Thus, when this nucleotide is added to the DNA chain, all further chain growth is terminated. In the "A" tube, a dideoxynucleotide form of the A base, dideoxyadenosine triphosphate, is added. In the "C" tube, dideoxycytidine triphosphate is added. Dideoxyguanosine triphosphate is added to the "G" tube, and dideoxythymidine triphosphate to the "T" tube.

When the reactions are incubated at a temperature suitable for the DNA polymerase, nucleotides that are complementary to the bases on the template are added onto the end of the attached primer. The bases A and T are complementary, as are G and C. Thus, if a T base is on the template strand, DNA polymerase will add the complementary base, A, at the corresponding position in the new, extending strand. If, by chance, the DNA polymerase adds a dideoxynucleotide, chain growth is terminated at that point.

Since dideoxynucleotides are randomly incorporated, each reaction generates a mixture of DNA molecules of variable length, but all are terminated by a dideoxynucleotide. Careful adjustment of reactant concentration will give a set of DNA molecules that terminate at each of the possible positions. The "A" tube, for instance, will contain a mixture of DNA chains, each of which ends in a different "A."

As in Maxam-Gilbert sequencing, each reaction is loaded in its own gel lane and electrophoresal autoradiography is then used to detect the fragments. The base sequence is read directly off the X-ray film from the bottom of the gel upward, noting the lane in which each band appears (see Figure 2). The bands at the bottom of the gel represent the shortest fragments and resulted from termination events closest to the primer. Bands toward the top of the gel represent longer fragments made by termination events farthest from the primer.