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Technology

There are many methods used for conservation work to identify individuals in key populations, including allozyme analysis, two-dimensional polyacrylamide gel electrophoresis (PAGE), sequence analysis, restriction fragment length polymorphism (RFLP) analysis, PCR-RFLP and microsatellite analysis.

Allozyme analysis, the earliest population genetic measure using a molecular technique involves protein electrophoresis to distinguish allelic forms of an enzyme. The first application of this work to population genetics was by Lewontin and Hubby in 1966 to survey gene-controlled protein variants in natural populations of Drosophila pseudoobscura and provide measures of heterozygosity for those populations (2). This form of analysis is still in use today.

Two-dimensional PAGE combines SDS-PAGE in the first dimension with isoelectric focusing in the second dimension to reveal heterogeneity of charge in proteins and can be used to show genetic polymorphisms in a population.

Sequence analysis can be applied to studies of mitochondrial DNA (mtDNA) diversity. For example, the D-loop of mitochondria is a segment of 500-600 bases in mammalian mitochondria where an opening of the duplex DNA forms and replication initiates. The D-loop is displaced by a short stretch of RNA base-paired to the complementary strand of mitochondrial DNA. Sequence analysis of the D-loop (control region) has revealed that this region is polymorphic in many species. When used with phylogenetic analyses, information about this sequence can be used to trace maternal lineage of an individual.

The mtDNA also exhibits restriction fragment length polymorphisms (RFLP). To determine polymorphisms in restriction fragment length of mtDNA, the mtDNA is first amplified by PCR and then analyzed for RFLPs using a number of restriction enzymes. This combination of PCR and RFLP (PCR-RFLP) has been used to detect genetic polymorphisms in cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) genes, for example.

RFLP analysis has been used for some time in the analysis of polymorphisms of mammalian major histocompatibility complex (MHC) genes and has revealed a large degree of polymorphism, making this region of DNA targeted in assessments of genetic diversity in many animals. Exploiting the diversity in the MHC RFLP and other genetic locations, researchers have made "DNA fingerprints" for different populations. In the DNA fingerprinting technique, DNA from different individuals is digested with restriction enzymes and resolved on an agarose gel. Then, using labeled probes to specific sequences in the polymorphic region, Southern blot analysis is performed to show the bands of interest. In general, the pattern displayed on the gel is specific for a particular individual and can be used to establish family relationships or degree of relatedness between individuals.

Other forms of analyses use PCR-based techniques to reveal polymorphic loci. Microsatellite, or short tandem repeat (STR), loci consist of short repetitive sequence elements 3 to 7 base pairs in length (3-6). These repeats are well distributed throughout the genomes of many species and are a rich source of highly polymorphic markers that may be detected using PCR (7-10). Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and can be distinguished from one another using radioactive, silver stain or fluorescence detection following electrophoretic separation.

The application of genetic techniques to the analysis of population structure and management allows a rigorous analysis of endangered species. Improvements in DNA analysis techniques, as well as theoretical and statistical work adapted from basic science, provide the conservation biologist with a powerful set of tools for potentially saving populations at risk.