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Genetics 301 Lecture #15
Spring 2003, March 6, 2003

The polymerase chain reaction (details of method discussed last time) allows large amounts of specific DNA sequences to be produced quickly, from even very small amounts of starting material. Examples of applications include DNA testing from crime-scene samples and from ancient DNA. Two types of sequences that are often analyzed are microsatellites (regions containing simple tandem repeat polymorphisms) and sequences of mitochondrial DNA.

DNA sequencing is performed by disrupting normal DNA replication by adding dideoxyribonucleotides into a DNA synthesis mixture. Synthesis is prematurely terminated and reading of band sizes allows the DNA sequence to be read. Dideoxyribonucleotides have also been used to treat HIV infections by disrupting replication of the virus. Automated sequencing using fluorescent dyes for labeling the DNA has allowed large amounts of sequence data to be collected.

Mutations are classified into germline (passed on to progeny) and somatic (occurring in body cells and sometimes associated with cancer). Conditional mutations are those expressed under certain (restrictive) conditions. The wildtype phenotype is seen under permissive conditions. Temperature sensitive mutations are the most widely studied type of conditional mutation. The coat color in the Siamese cat is an example of a temperature-sensitive mutation; pigment is present in cooler body areas (ears, paws) but not in warmer areas where the relevant enzyme is not active.

Spontaneous mutations are those which arise without exposure to a known mutation-causing agent. Induced mutations arise from exposure to such an agent (typically chemicals or radiation).

Base substitution mutations are classified as transisitons (purine to purine, pyrimidine to pyrimidine) or transversions. (purine to pyrimidine, or pyrimidine to purine). Silent mutations are those which either are in a protein—coding region but do not change the amino acid in the protein, or occur outside a protein coding region and will thus generally have no effect on the phenotype. Missense mutations change the amino acid inserted into a protein. Sickle cell anemia is related to a missense mutation leading to valine being substituted in the place of glutamic acid in a protein. This mutation apparently became common in some tropical areas (e.g., Africa) because those heterozygous for the mutation were more resistant to malaria than were homozygous wild type individuals. A nonsense mutation results from substitution of a stop codon for an amino acid. This will result in premature termination of protein synthesis and will generally have a drastic effect on the phenotype.

Induced mutations generally result from chemical or radiation treatments. Chemicals may act by substituting for a base, modifying a base or inserting between bases during replication. The base analog 5-bromodeoyuracil is an example of a compound which substitutes for a base. It normally pairs (in the "keto" isomer form) with A, but can mispair with G when it is present in the "enol" form. (Note: I may have given this backwards in lecture). This will result in a transition mutation. Agents that modify bases include nitrous acid (which removes amino groups, changing them to keto groups, changing pairing) and alkylating agens such as ethyl methanesulfonate and nitrogen mustard. These chemicals add alkyl (e.g., methyl, ethyl) groups to the bases, changing pairng, and can remove purine bases (depurination).

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