II. DNA sequencing.

III. Polymerase chain reaction.

IV. Genetic diagnosis.

Terms.

Errata Page 302, Table 12.1: Cycle 5 should have 32 copies. Page 303, Figure 12.17 caption: Primers are within the region to be amplified, not flanking it.

I. DNA fragments of different sizes can be separated from each other by means of electrophoresis.

A. Electrophoresis is defined as the separation of charged molecules in an electric field.
1. The phosphate groups of DNA and RNA are negatively charged under most conditions. Therefore, DNA and RNA molecules migrate toward a positive electrical pole.

2. If the electrophoretic medium (a gel, usually made of a complex carbohydrate called agarose) is maintained at a constant pH, the rate of migration of nucleic acid fragments toward the positive pole is dependent only on the size of the fragment, i.e. smaller fragments migrate faster than larger fragments.

B. If sufficient quantity of a small number of fragments is available for analysis, the fragments can be made visible in the gel by staining with a dye that binds to DNA. (See Figure 12.15 in textbook.)

C. More often, the amount of DNA and the large number of fragments require more sensitive and specific methods for identifying the fragments of interest. For this purpose, a Southern blot is used.

1. In a typical Southern blot, DNA is treated with a restriction enzyme to generate fragments. For large pieces of DNA, such as genomic DNA, there may be millions of fragments. In the electrophoresis step, each fragment will migrate at the rate characteristic for its size. Because of the large number of fragments, staining with a general DNA stain produces only a smear.

2. DNA probes that hybridize to a specific DNA sequence can be used to reveal only those fragments that have the specific sequence. In order to use a probe, one must first transfer the fragments from the gel to a membrane to carry out the hybridization. The DNA fragments can be flushed out of the gel onto the membrane by providing a supply of liquid on one side of the gel, placing the membrane on the other side, and placing absorbent materials (paper towels) on the other side of the membrane.

3. When the fragments have been transferred, they are denatured and flooded with the ³²P-labeled probe. After hybridization of the probe to the target fragments is completed, the excess probe is removed and the membrane is placed next to X-ray film. The radiation from the ³²P exposes the film, showing the position of the target fragments. Because of the great sensitivity of the ³²P, very small amounts of DNA can be used.

4. Each combination of DNA, restriction enzymes, and DNA probes produces a different pattern of fragments.

A. In the most common method, nt analogs are added in small quantity during copying of a DNA strand whose sequence is desired. The enzyme DNA polymerase used to copy the strand can add nucleotides only to proper 3'-nucleotides. If an analog has been inserted in the previous step, synthesis stops. Interruption of synthesis by the analogs creates a series of products that terminate at the nt that corresponds to the analog.

B. The mixture of newly formed DNA fragments are separated by size on an electrophoresis gel.

C. Combining the results from different homologs provides the nt sequence.

A. The technique consists of the following steps:
1. Denature the DNA with heat (ca. 90 °C), which breaks the hydrogen bonds and allows the double strands to separate.

2. Lower the temperature to ca. 50 °C in the presence of short DNA primers that are complementary to the boundaries of the DNA to be amplified. This temperature allows double strands to form.

3. Use thermostabile DNA polymerase to extend the primer, using the original DNA strands as templates. This step is carried out at about 70 °C.

B. The steps of this cycle are regulated entirely by temperature. A complete cycle takes 2-3 minutes.

C. Each cycle doubles the amount of DNA, but only the segment that is defined by the primers selected. Thirty cycles would produce 2³⁰ or ca. 1 billion amplification.

A. One can test for specific DNA changes in a gene or one can look for cosegregation of closely linked markers.
1. Specific DNA changes are especially useful if the specific change is known from other evidence. E.g. a mutation such as that in sickle cell anemia or the high frequency of one or a few variant alleles (cystic fibrosis). Contrast that situation with Duchenne muscular dystrophy, where most cases involve new mutations that are individually extremely rare.

2. Rare cells from a fetus escape into the maternal circulation. PCR is so sensitive that DNA sequences that are not present in the mother's genome can be detected and used to predict fetal genotype.

3. If other affected family members are available, cosegregation has the advantage that the specific DNA lesion need not be known.

B. Many late onset diseases can be diagnosed before any expression of the disease.
1. Familial hypercholesterolemia.

2. Familial Alzheimer disease.

C. Preimplantation embryos can also be used for DNA testing. Following in vitro fertilization, one cell is removed at the 8-cell stage, and PCR is used to amplify the DNA.

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