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serves as template for DNA synthesis. The primer pairs to 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, by using the target DNA strand as a template. Wherever DNA polymerase encounters a T on the template strand, it uses at random either a dATP or a ddATP to introduce an A in 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 ddA into the new strand occurs randomly at different positions in different copies, producing a set of DNA chains of different length (12, 7, and 2 nucleotides long in the example illustrated in Figure 19.6), each ending in a nucleotide with adenine.

Equivalent reactions take place in the other three tubes. In the tube that received ddCTP, all the chains terminate in a nucleotide with cytosine; in the tube that received ddGTP, all the chains terminate in a nucleotide with guanine; and, in the tube that received ddTTP, all the chains terminate in a nucleotide with thymine. After the completion of the polymerization reactions, all the DNA in the tubes is denatured, and the single-strand products of each reaction are separated by gel electrophoresis.

The contents of the four tubes are separated side by side on an acrylamide gel so that DNA strands differing in length by only a single nucleotide can be distinguished. After electrophoresis, the locations of the DNA strands in the gel are revealed by autoradiography. The shortest strands, which terminated at positions early in the DNA sequence, migrate quickly and end up near the bottom of the gel; longer fragments, which terminated late in the sequence, migrate more slowly and end up near the top of the gel.

Reading the DNA sequence is simple and the shortest part of the procedure. In Figure 19.6, 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 synthesized had 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.

You may have wondered how the primers used in dideoxy sequencing are constructed, because the sequence of the target DNA may not be known ahead of time. The trick is to insert a sequence that will be recognized by the primer into the target DNA. This is often done by first cloning the target DNA in a vector that contains sequences

Primer

Primer site

Vector DNA

Primer site

DNA insert

Vector DNA

Primer

A 19.7 Sites recognized by sequencing primers are added to the target DNA by cloning the DNA in a vector that contains universal sequencing primer sites on either side of the site where the target DNA will be inserted.

recognized by a common primer (called universal sequencing primer sites) on either side of the site where the target DNA will be inserted. The target DNA is then isolated from the vector and will contain universal sequencing primer sites at each end (I Figure 19.7).

Sequencing is often carried out by automated machines that use fluorescent dyes and laser scanners to sequence thousands of base pairs in a few hours (I Figure 19.8). The dideoxy reaction is also used here, but the ddNTPs used in the reaction are labeled with a fluorescent dye, and a different colored dye is used for each type of dideoxynucleotide. For example, a red dye might be used for nucleotides with thymine, a green dye for those with adenine, a black dye for those with guanine, and a blue dye for those with cytosine. In this case, the four sequencing reactions can take place in the same test tube and can be placed in the same well during electrophoresis, given that each ddNTP is distinctively marked. The most recently developed 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 19.8). Automated sequencing machines may contain 96 or more capillary tubes, allowing from 50,000 to 60,000 bp of sequence to be read in a few hours.

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