cut DNA at specific four–, five–, or six–base-pair sequences. For example, the enzyme EcoR1 (a restriction enzyme derived from the bacterium Escherichia coli) cuts DNA at sites that contain the nucleotide sequence GAATTC. This is a quick and inexpensive way to get sequence information and was widely used in population-genetics studies before the advent of PCR-based sequencing technology. The technique is highly repeatable and robust but has largely been replaced by direct DNA sequencing.


Anonymous Single-Copy Nuclear DNA (Karl et al., 1992)—This technique requires cloning and sequencing fragments of DNA. On the basis of the clones, variation in the nuclear genome can be resolved and characterized. The requirement of cloning (like microsatellites; see Chapter 2) makes this an expensive and labor-intensive approach to initialize, but it is robust and repeatable (Karl and Avise, 1993). In population genetic studies, it is largely replaced by microsatellite methods but has broad applications in phylogeography and phylogenetic studies.3


Minisatellites (Peare and Parker, 1996)—These are the first generation of “DNA fingerprints” and consist of short repeat sequences4 of about 10–60 base pairs that occur in variable copy number, in hundreds of locations in the genome. They are detected with a fluorescent or radioactive probe and can be variable enough to distinguish individuals (Jeffreys, 2005). However, they can be difficult to interpret and have largely been replaced by microsatellites in population-genetics studies.


Random Amplification of Polymorphic DNA (Schroth et al., 1996)—This technique uses PCR primers to amplify short segments of the genome randomly; they are then separated and visualized with gel electrophoresis.5 It has the advantage of not requiring prior knowledge of the genome (sequence data) to design primers. However, it is not widely used in population-genetics studies because of problems with interpretation and repeatability.

3

Phylogeography focuses on the geographic distribution of genetic variation, usually at the level of species and genera. Phylogeographic studies often reveal molecular evolutionary separations below the species level, as is the case with green turtles (Chelonia mydas; Bowen et al., 1992). Phylogenetics is the study of evolutionary history, usually describing relationships among species, genera, and higher taxonomic categories in the format of trees.

4

These are DNA segments that repeat the same sequence multiple times. They are prone to duplication during cell replication and therefore can produce highly variable genetic markers.

5

This is a method of separating DNA fragments by size. The DNA or protein is inserted into a gelatin slab, and an electric current is run through the gelatin to move fragments toward either the positive or negative end.



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