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Sabine S. Loew
distance estimates to resolve phylogenies, especially at intermediate taxonomic
levels (Sibley and Ahlquist 1981). However, it does not provide qualitative data
on character states crucial for many phylogenetic analyses. Few laboratories are
set up for routine DNA-DNA hybridizations. This tool is rarely applied in conservation biology.
Restriction Fragment Length Polymorphism (RFLP)
Restriction enzymes cut double-stranded DNA at specific recognition sites that
consist of short oligonucleotide sequences (typically four, five, and six base
pairs). Restriction of DNA with one or several enzymes results in a range of
fragment sizes that are electrophoretically separated in either agarose or polyacrylamide gels. Complex banding patterns can be detected when either all or
selected fragments are visualized by means of a chemical stain (e.g., ethidium
bromide) or radioactive DNA probes (i.e., short labeled DNA fragments that bind
and hence mark complementary sequences). Furthermore, the development of the
polymerase chain reaction (PCR) allows amplification of homologous locusspecific DNA fragments that can be subjected to restriction fragment analysis.
PCR-based RFLP analysis has the advantages that very little tissue provides
sufficient amounts of DNA and that large copy numbers of each fragment facilitate visualization of the banding patterns. As restriction sites are distributed
throughout nuclear and cytoplasmic DNA, RFLP analysis can be applied to a
variety of DNA regions by using a number of DNA sources, as discussed below.
Single-Copy Nuclear DNA (scnDNA) (Quinn and White
1987; Karl and Avise 1993)
In contrast to repetitive DNA, single copy nuclear DNA (scnDNA) is represented
only once or possibly a few times in a haploid genome. As scnDNA is found in
both coding and noncoding regions, evolutionary rates of change associated with
these sites vary considerably. Genetic variation is detected by hybridizing a locusspecific scnDNA probe to digested whole genomic DNA. Alternatively, scnDNA
can be PCR-amplified at specific loci and digested with restriction enzymes, and
electrophoretically separated fragments are visualized with ethidium bromide
staining. As scnDNA polymorphisms are numerous and their Mendelian inheritance can be established through pedigree analysis, scnDNA provides a wealth of
genetic markers to estimate genetic diversity. Karl and colleagues (1992) showed
the usefulness of scnDNA in a study on the genetic population structure of the
endangered Green Turtle. Previous mtDNA analysis had shown high nest site
fidelity among females and suggested severe limitation of gene flow between
breeding populations (Bowen et al. 1992). Analysis of scnDNA, however, revealed only a moderate degree of genetic substructure, suggesting moderate levels
of male-mediated gene flow between rookeries (Karl et al. 1992).
Sabine S. Loew
distance estimates to resolve phylogenies, especially at intermediate taxonomic
levels (Sibley and Ahlquist 1981). However, it does not provide qualitative data
on character states crucial for many phylogenetic analyses. Few laboratories are
set up for routine DNA-DNA hybridizations. This tool is rarely applied in conservation biology.
Restriction Fragment Length Polymorphism (RFLP)
Restriction enzymes cut double-stranded DNA at specific recognition sites that
consist of short oligonucleotide sequences (typically four, five, and six base
pairs). Restriction of DNA with one or several enzymes results in a range of
fragment sizes that are electrophoretically separated in either agarose or polyacrylamide gels. Complex banding patterns can be detected when either all or
selected fragments are visualized by means of a chemical stain (e.g., ethidium
bromide) or radioactive DNA probes (i.e., short labeled DNA fragments that bind
and hence mark complementary sequences). Furthermore, the development of the
polymerase chain reaction (PCR) allows amplification of homologous locusspecific DNA fragments that can be subjected to restriction fragment analysis.
PCR-based RFLP analysis has the advantages that very little tissue provides
sufficient amounts of DNA and that large copy numbers of each fragment facilitate visualization of the banding patterns. As restriction sites are distributed
throughout nuclear and cytoplasmic DNA, RFLP analysis can be applied to a
variety of DNA regions by using a number of DNA sources, as discussed below.
Single-Copy Nuclear DNA (scnDNA) (Quinn and White
1987; Karl and Avise 1993)
In contrast to repetitive DNA, single copy nuclear DNA (scnDNA) is represented
only once or possibly a few times in a haploid genome. As scnDNA is found in
both coding and noncoding regions, evolutionary rates of change associated with
these sites vary considerably. Genetic variation is detected by hybridizing a locusspecific scnDNA probe to digested whole genomic DNA. Alternatively, scnDNA
can be PCR-amplified at specific loci and digested with restriction enzymes, and
electrophoretically separated fragments are visualized with ethidium bromide
staining. As scnDNA polymorphisms are numerous and their Mendelian inheritance can be established through pedigree analysis, scnDNA provides a wealth of
genetic markers to estimate genetic diversity. Karl and colleagues (1992) showed
the usefulness of scnDNA in a study on the genetic population structure of the
endangered Green Turtle. Previous mtDNA analysis had shown high nest site
fidelity among females and suggested severe limitation of gene flow between
breeding populations (Bowen et al. 1992). Analysis of scnDNA, however, revealed only a moderate degree of genetic substructure, suggesting moderate levels
of male-mediated gene flow between rookeries (Karl et al. 1992).