The ability to determine the nucleotide sequence of DNA molecules has given a tremendous boost to biological research. Techniques used to sequence DNA follow from our understanding of DNA replication. Consider a solution containing identical single-stranded molecules of DNA, each being used as the template for the synthesis of a complementary daughter strand originating at a short primer sequence. The problem is to determine the nucleotide sequence of the template strand. A brilliant answer to this problem was developed by the English geneticist Frederick Sanger, an achievement rewarded with a share in the Nobel Prize in Chemistry in 1980. (It was his second Nobel; he had also been honored with the award in 1958 for his discovery of a method for determining the sequence of amino acids in a polypeptide chain.)

Recall that a free 3′ hydroxyl group is essential for each step in elongation because that is where the incoming nucleotide is attached (Fig. 12.17a). Making use of this fact, Sanger synthesized dideoxynucleotides, in which the 3′ hydroxyl group on the sugar ring is absent (Fig. 12.17b). Whenever a dideoxynucleotide is incorporated into a growing daughter strand, there is no hydroxyl group to attack the incoming nucleotide, and strand growth is stopped dead in its tracks (Fig. 12.18a). For this reason, a dideoxynucleotide is known as a chain terminator. By including a small amount of each of the chain terminators in a reaction tube along with larger quantities of all four normal nucleotides, a DNA primer, a DNA template, and DNA polymerase, Sanger was able to produce a series of interrupted daughter strands, each terminating at the site at which a dideoxynucleotide was incorporated.

Fig. 12.18b shows how the interrupted daughter strands help us to determine the DNA sequence by the procedure now called Sanger sequencing. In a tube containing dideoxy-A and all the other elements required for many rounds of DNA replication, a strand of DNA is synthesized complementary to the template until, when it reaches a T in the template strand, it incorporates an A. Only a small fraction of the A nucleotides in the sequencing reaction are in the dideoxy form, so only a fraction of the daughter strands incorporate a dideoxy-A at that point, resulting in termination. The rest of the strands incorporate a normal deoxy-A and continue synthesis, although most of these will be stopped at some point farther along the line when a T is reached again. Similarly, in a reaction containing dideoxy-C, DNA fragments will be produced whose sizes correspond to the positions of the Cs, and likewise for dideoxy-T and dideoxy-G.

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Each of the four dideoxynucleotides is chemically labeled with a different fluorescent dye, as indicated by the different colors of A, C, T, and G in Fig. 12.18b, and so all four terminators can be present in a single reaction and still be distinguished. After DNA synthesis is complete, the daughter strands are separated by size with gel electrophoresis. The smallest daughter molecules migrate most quickly and therefore are the first to reach the bottom of the gel, followed by the others in order of increasing size. A fluorescence detector at the bottom of the gel “reads” the colors of the fragments as they exit the gel. What the scientist sees is a trace (or graph) of the fluorescence intensities, such as the one shown in Fig. 12.18c. The differently colored peaks, from left to right, represent the order of fluorescently tagged DNA fragments emerging from the gel. Thus, a trace showing peaks colored green-purple-red-green-purple-purple-blue-green-blue-red corresponds to a daughter strand having the sequence 5′-ACTACCGAGT-3′ (Fig. 12.18c). In the Sanger sequencing method, each sequencing reaction can determine the sequence of about 1000 nucleotides in the template DNA molecule.

Quick Check 3 You have determined that the newly synthesized strand of DNA in your sequencing reaction has the sequence 5′-ACTACCGAGT-3′. What is the sequence of the template strand?