A powerful molecular method for analyzing DNA is a technique known as DNA sequencing, which determines the sequence of bases in DNA. Sequencing allows the genetic information in DNA to be read, providing an enormous amount of information about gene structure and function. In the mid-1970s, Frederick Sanger and his colleagues created the dideoxy sequencing method, based on the elongation of DNA by DNA polymerase; at about the same time, Allan Maxam and Walter Gilbert developed a second method based on chemical degradation of DNA. The Sanger method quickly became the standard procedure for sequencing any purified fragment of DNA.

The Sanger, or dideoxy, method of DNA sequencing is based on replication. The fragment to be sequenced is used as a template to make a series of new DNA molecules. In the process, replication is sometimes terminated when a specific base is encountered, producing DNA strands of different lengths, each of which ends in the same base.

The method relies on the use of a special substrate for DNA synthesis. Normally, DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs), which have an OH group on the 3′-carbon atom (Figure 14.13a). In the Sanger method, a special nucleotide, called a dideoxyribonucleoside triphosphate (ddNTP; Figure 14.13b), is also used as a substrate. The ddNTPs are identical to dNTPs, except that they lack a 3′-OH group. In the course of DNA synthesis, ddNTPs are incorporated into a growing DNA strand. However, after a ddNTP has been incorporated into the DNA strand, no more nucleotides can be added, because there is no 3′-OH group to form a phosphodiester bond with an incoming nucleotide. Thus, ddNTPs terminate DNA synthesis.

Although the sequencing of a single DNA molecule is technically possible, most sequencing procedures in use today require a considerable amount of DNA; any DNA fragment to be sequenced must first be amplified by PCR or by cloning in bacteria. Copies of the target DNA are then isolated and split into four samples (Figure 14.14). Each sample is placed in a different test tube to which the following ingredients are added:

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Either the primer or the dNTPs are radioactively or chemically labeled so that newly produced DNA can be detected.

Within each of the four tubes, the DNA polymerase synthesizes DNA. Let’s consider the reaction in one of the four tubes: the one that received ddATP. Within this tube, each of the single strands of target DNA serves as a template for DNA synthesis. The primer pairs with its complementary sequence at one end of each template strand, providing a 3′-OH group for the initiation of DNA synthesis. DNA polymerase elongates a new strand of DNA from this primer. Wherever DNA polymerase encounters a T on the template strand, it uses, at random, either a dATP or a ddATP to introduce an A into the newly synthesized strand. Because there is more dATP than ddATP in the reaction mixture, dATP is incorporated most often, allowing DNA synthesis to continue. Occasionally, however, ddATP is incorporated into the strand, and synthesis terminates. The incorporation of ddATP into the new strand takes place randomly at different positions in different copies, producing a set of DNA fragments of different lengths (12, 7, and 2 nucleotides long in the example illustrated in Figure 14.14), each ending in a nucleotide that contains adenine.

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Equivalent reactions take place in the other three tubes, except that synthesis is terminated at nucleotides with a different base in each tube. After the completion of the polymerization reactions, all the DNA in the tubes is denatured, and the single-stranded products of each reaction are separated by gel electrophoresis.

The contents of the four tubes are separated side by side on an acrylamide gel (which allows finer separation than agarose) so that DNA strands differing in length by only a single nucleotide can be distinguished. After electrophoresis, the locations, and therefore the sizes, of the DNA strands in the gel are revealed by the radioactive or chemical labels.

Reading the DNA sequence is the simplest and shortest part of the procedure. In Figure 14.14, you can see that the band closest to the bottom of the gel is from the tube that contained the ddGTP reaction, which means that the first nucleotide added was guanine (G). The next band up is from the tube that contained ddATP; so the next nucleotide in the sequence is adenine (A), and so forth. In this way, the sequence is read from the bottom to the top of the gel, with the nucleotides near the bottom corresponding to the 5′ end of the newly synthesized DNA strand and those near the top corresponding to the 3′ end. Keep in mind that the sequence obtained is not that of the target DNA, but that of its complement. To see dideoxy sequencing in action, view Animation 19.3. TRY PROBLEM 26

For many years, DNA sequencing was done largely by hand and was laborious and expensive. Today, sequencing is usually carried out by automated machines that use fluorescent dyes and laser scanners to sequence thousands of base pairs in a few hours (Figure 14.15). The dideoxy reaction is still used, but the ddNTPs used in the reaction are labeled with a fluorescent dye, and a different-colored dye is used for each type of ddNTP. In this case, the four sequencing reactions can take place in the same test tube and can be placed in the same well for electrophoresis. Sequencing machines carry out electrophoresis in gel-containing capillary tubes. The different-sized fragments produced by the sequencing reaction separate within a tube and migrate past a laser beam and detector. As the fragments pass the laser, their fluorescent dyes are activated, and the resulting fluorescence is detected by an optical scanner. Each colored dye emits fluorescence of a characteristic wavelength, which is read by the optical scanner. The information is fed into a computer for interpretation, and the results are printed out as a set of peaks on a graph (see Figure 14.15). Automated sequencing machines may contain up to 96 or more capillary tubes, allowing from 50,000 to 60,000 bp of sequence to be read in a few hours.