C

9.9 Techniques Used in the Polymerase Chain Reaction (PCR) 239

tube, ddGTP to the next, and so on. When the reaction is incubated at an appropriate temperature, the primer will anneal to the template and DNA synthesis will begin. Chain elongation proceeds until a dideoxynucleotide (ddNTP) is incorporated, terminating synthesis at that point. Termination, however, is an infrequent event because of the numerous template molecules and the small amount of the ddNTP relative to its dNTP counterpart. The result is that a tube will contain a set of newly synthesized DNA strands of various lengths. Each of these strands will have been terminated at the point that the specific ddNTP was incorporated into the chain. For example, in the tube that contained ddATP, numerous fragments of different lengths will be obtained, but each fragment will have been terminated at a position that called for the incorporation of the nucleotide base adenine. The sizes of the fragments in a tube indicate the positions of a specific nucleotide base in the synthesized DNA strand.

A special type of gel electrophoresis is used to separate the DNA fragments and determine their relative size. The gel has separate wells arranged side by side. Each well receives the contents of one of the four tubes from the sequencing reaction. The gel conditions (pH, temperature, and gel concentration) denature the DNA and enable separation of fragments that differ by only one nucleotide in length. Autoradiography is then used to detect the relative positions of the newly synthesized fragments. The order that the terminating nucleotides were incorporated can then be determined by "reading" the gel from the bottom up (figure 9.21).

Automated DNA Sequencing

Most automated techniques do not use a radioactive label to detect the newly synthesized DNA, but instead use a fluorescent dye attached directly to the ddNTP. Different dye colors

Figure 9.21 An Autoradiograph of a Sequencing Gel Each dark band represents a specific-sized fragment.The sequence of the newly synthesized strand can be determined by "reading'' the gel from the bottom up.

9.9 Techniques Used in the Polymerase Chain Reaction (PCR) 239

Figure 9.22 Results of Automated DNA Sequencing The order of the colored peaks reflects the nucleotide sequence of the DNA.

are used for each of the four ddNTPs. By having each different ddNTP labeled with a distinct color, the reaction can be done in a single tube and run in one lane of the gel. Again, gel electrophoresis is used to separate the bands. A laser is used to detect the colors of fluorescent bands as they run off the gel, recording their intensity as a peak. The order of the colored peaks reflects the nucleotide sequence of the DNA (figure 9.22).

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