numbers of repeats. Thus by determining the lengths of these repeated sequences
it is possible to discriminate between different individuals. To achieve this, restrict-
ion enzymes are added that cut the DNA whenever a given sequence of bases
(e.g. GACCAT) occurs, so providing a large numbers of fragments of DNA.
When these fragments are added to a gel with an electric current the shorter frag-
ments move more rapidly. Probes are added that attach (hybridize) to the specif-
ic repeated sequence and make them visible. The different length fragments thus
produce different bands. As chromosomes occur in pairs there will be two bands
for each repeat sequence region and as the tandem repeats occur in a number of
places in the genome the result is a series of bands (the DNA fingerprint) that
is unique to an individual (Jeffreys et al. 1985b; Burke and Bruford 1987). See
Burke (1989a,b), Burke et al. (1991), and Krawczak and Schmidtke (1998) for
methodological details.
Repetitive sequence elements that occur in tandem are known as minisatellite
sequences (typically 10 to 100 bases long) or microsatellite sequences (less than
10 bases). In practice minisatellites are used for DNA fingerprinting as described
above. However, this method requires good quality DNA and can be difficult to
analyze. As a result there is an increasing use of microsatellites and “single locus
probes.” These use exactly the same concept, but analyze just a single region of
tandem repeats so an individual will have just two bands, one from each chro-
mosome (or a single band if homozygous). This method typically incorporates
polymerase chain reaction (PCR), which makes multiple copies of a few stands
of DNA, so allowing the analysis of extremely small samples.
Each individual will have two bands at each microsatellite locus, one inherited
from each parent. By combining many loci it is possible to estimate relatedness
among individuals, for example, Höglund and Shorey (2003) used microsatellites
to determine the frequency of full sib and half sib relationships on a White-bearded
Manakin Manacus manacuslek. This method is routinely used to determine the
parentage of offspring and the frequency and source of dumped eggs.
Relatedness among species and populations. Because mitochondrial DNA
(mt DNA) is passed down the maternal line, it is of no value in establishing pater-
nity. However, sequencing regions of the mitochondrial genome can be used to
investigate phylogenetic relationships among populations of the same species and
among species. Mitochondrial DNA is thought to have a mutation fixation rate
several times greater than nuclear DNA, making it extremely variable and has the
further advantage of not being recombined during meiosis so giving a clear line of
descent. It is easy to work with because it is single copy gene (one allele per
individual) yet has multiple copies in terms of number of molecules per cell. For
example, by comparing sequences of yellow wagtails across the Palearctic it has
226 |Techniques in physiology and genetics