238
Sabine S. Loew
mutations during replication (Wilson et al. 1985; Avise et al. 1987; Martin et al.
1992a). Analysis of mtDNA restriction fragments detects sufficient polymorphism to resolve genetic distances among conspecific populations and closely
related species (Tarr and Fleischer 1993; Avise 1994).
O’Brien and associates (1990) combined mtDNA analysis with data on protein
polymorphism to inform management decisions for the Florida Panther. MenottiRaymond and O’Brien (1993) used them to evaluate the history, status, and
management of African Cheetahs. Taberlet and colleagues (1995) used DNA
sequencing and RFLP analysis of mtDNA to delineate the contact zones of two
divergent lineages of Scandinavian Brown Bears, to define conservation units,
and to guide possible translocation efforts.
DNA Sequencing (Maxam and Gilbert 1977, 1980;
Sanger et al. 1977)
DNA sequencing provides the greatest resolution of genetic divergence by actually determining the identity and sequence of all bases within a target region
(typically 500 base pairs long). Consequently, all genetic differences between
samples are detected, instead of only those that result in restriction site changes.
The most commonly used manual sequencing method is called Sanger dideoxy
sequencing. It is based on in vitro synthesis of radioactively labeled singlestranded DNA that is interrupted at either an A, T, C, or G nucleotide. DNA pieces
ending in different nucleotides are run in separate lanes, and their positions are
made visible as individual bands through autoradiography. Each band represents a
particular nucleotide, and consecutive bands are separated by one base pair,
consequently the band pattern reveals the DNA sequence of a molecule. More
recently, automated DNA sequencing with fluorescent dye labels has started to
replace the more cumbersome manual sequence analysis. Nuclear and mtDNA
can serve as templates for DNA sequence comparisons among individuals and
taxa, provided sufficient amounts of purified homologous DNA and suitable
sequencing primers are available. The source DNAs most commonly used for
DNA sequence analysis are discussed below.
Mitochondrial DNA Control Region
This noncoding region contains sequences that control replication and transcription of the mtDNA molecule and is called D-loop region in vertebrates and A+Trich region in insects (Fauron and Wolstenholme 1980; Aquadro and Greenberg
1983). It is the only mtDNA region that consistently contains noncoding DNA in
many taxa, and although evolutionary rates vary within the control region, it is
generally a reliable source for hypervariable mtDNA. This high level of variability has made the control region suitable for sequence comparisons within and
between populations and closely related species (Wilson et al. 1985; Harrison
1989; Thomas et al. 1990; Tarr 1995; Morales et al. 1997). For example, Morin
and co-workers (1992) used D-loop sequence to identify chimpanzee subspecies
Sabine S. Loew
mutations during replication (Wilson et al. 1985; Avise et al. 1987; Martin et al.
1992a). Analysis of mtDNA restriction fragments detects sufficient polymorphism to resolve genetic distances among conspecific populations and closely
related species (Tarr and Fleischer 1993; Avise 1994).
O’Brien and associates (1990) combined mtDNA analysis with data on protein
polymorphism to inform management decisions for the Florida Panther. MenottiRaymond and O’Brien (1993) used them to evaluate the history, status, and
management of African Cheetahs. Taberlet and colleagues (1995) used DNA
sequencing and RFLP analysis of mtDNA to delineate the contact zones of two
divergent lineages of Scandinavian Brown Bears, to define conservation units,
and to guide possible translocation efforts.
DNA Sequencing (Maxam and Gilbert 1977, 1980;
Sanger et al. 1977)
DNA sequencing provides the greatest resolution of genetic divergence by actually determining the identity and sequence of all bases within a target region
(typically 500 base pairs long). Consequently, all genetic differences between
samples are detected, instead of only those that result in restriction site changes.
The most commonly used manual sequencing method is called Sanger dideoxy
sequencing. It is based on in vitro synthesis of radioactively labeled singlestranded DNA that is interrupted at either an A, T, C, or G nucleotide. DNA pieces
ending in different nucleotides are run in separate lanes, and their positions are
made visible as individual bands through autoradiography. Each band represents a
particular nucleotide, and consecutive bands are separated by one base pair,
consequently the band pattern reveals the DNA sequence of a molecule. More
recently, automated DNA sequencing with fluorescent dye labels has started to
replace the more cumbersome manual sequence analysis. Nuclear and mtDNA
can serve as templates for DNA sequence comparisons among individuals and
taxa, provided sufficient amounts of purified homologous DNA and suitable
sequencing primers are available. The source DNAs most commonly used for
DNA sequence analysis are discussed below.
Mitochondrial DNA Control Region
This noncoding region contains sequences that control replication and transcription of the mtDNA molecule and is called D-loop region in vertebrates and A+Trich region in insects (Fauron and Wolstenholme 1980; Aquadro and Greenberg
1983). It is the only mtDNA region that consistently contains noncoding DNA in
many taxa, and although evolutionary rates vary within the control region, it is
generally a reliable source for hypervariable mtDNA. This high level of variability has made the control region suitable for sequence comparisons within and
between populations and closely related species (Wilson et al. 1985; Harrison
1989; Thomas et al. 1990; Tarr 1995; Morales et al. 1997). For example, Morin
and co-workers (1992) used D-loop sequence to identify chimpanzee subspecies