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FIGURE 8-36 DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (a) DNA polymerases require both a primer (a short oligonucleotide strand), to which nucleotides are added, and a template strand to guide selection of each new nucleotide. In cells, the 3'-hy-

ts with an Incoming deoxynucleoslde n phosphod¡ester bond. (b)The Sanger =oxynucleoslde triphosphate (ddNTP) sis. (The Sanger method Is also known ddNTP Is Inserted In place of a dNTP, the analog Is added, because It lacks ded for the next step, to be sequenced Is used as the tem-and, and a short primer, radloactlvely or fluorescently labeled, Is annealed to It. By addition of small amounts of a single ddNTP, for example ddCTP, to an otherwise reaction system, the synthesized >e prematurely terminated at some lo-normally occurs. Given the excess of that the analog will be Incorporated ; small. However, ddCTP Is present In it each new strand has a high proba-iC at some point during synthesis. The llxture of labeled fragments, each end-sldue In the sequence generates a set jth, such that the different-sized frag-=sls, reveal the location of C residues, ately for each of the four ddNTPs, and tly from an autoradlogram of the gel. migrate faster, the fragments near the icleotlde positions closest to the primer 5 read (In the 5' —> 3' direction) from quence obtained Is that of the strand Ing analyzed.

DNA sequencing is readily automated by a variation of Sanger's sequencing method in which the dideoxynucleotides used for each reaction are labeled with a differently colored fluorescent tag (Fig. 8-37). This technology allows DNA sequences containing thousands of nucleotides to be determined in a few hours. Entire genomes of many organisms have now been sequenced (see Table 1-4), and many very large DNA-sequencing projects are in progress. Perhaps the most ambitious of these is the Human Genome Project, in which researchers have sequenced all 3.2 billion base pairs of the DNA in a human cell (Chapter 9). £ Dideoxy Sequencing of DNA

The Chemical Synthesis of DNA Has Been Automated

Another technology that has paved the way for many biochemical advances is the chemical synthesis of oligonucleotides with any chosen sequence. The chemical methods for synthesizing nucleic acids were developed primarily by H. Gobind Khorana and his colleagues in the 1970s. Refinement and automation of these methods have made possible the rapid and accurate synthesis of DNA strands. The synthesis is carried out with the growing strand attached to a solid support (Fig. 8-38), using principles similar to those used by Merrifield in peptide synthesis (see Fig. 3-29). The efficiency of each addition step is very high, allowing the routine laboratory synthesis of polymers containing 70 or 80 nu-cleotides and, in some laboratories, much longer strands. The availability of relatively inexpensive DNA polymers with predesigned sequences is having a powerful impact on all areas of biochemistry (Chapter 9).

FIGURE 8-37 Strategy for automating DNA sequencing reactions.

Each dideoxynucleotide used in the Sanger method can be linked to a fluorescent molecule that gives all the fragments terminating in that nucleotide a particular color. All four labeled ddNTPs are added to a single tube. The resulting colored DNA fragments are then separated by size in a single electrophoretic gel contained in a capillary tube (a refinement of gel electrophoresis that allows for faster separations). All fragments of a given length migrate through the capillary gel in a single peak, and the color associated with each peak is detected using a laser beam. The DNA sequence is read by determining the sequence of colors in the peaks as they pass the detector. This information is fed directly to a computer, which determines the sequence.

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