There are many different methods for extracting DNA from tissue, blood, hair, feathers, leaves, roots and other sources. In recent years kits have become widely available and reasonably priced, and as a result the extraction of DNA from many different sample types is often a fairly routine procedure. The amount of starting material can be very small; because a successful PCR reaction can be accomplished with only a tiny amount of DNA, samples as small as a single hair follicle may be adequate (Box 1.2) and therefore lethal sampling of animals is no longer necessary before individuals can be characterized genetically. Examples of non-lethal samples that have been used successfully for DNA analysis include wing tips from butterflies (Rose, Brookes and Mallet, 1994), faecal DNA from elusive species such as red wolves and coyotes (Adams, Kelly and Waits, 2003), single feathers from birds (Morin and Woodruff, 1996) and single scales from fish (Yue and Orban, 2001). Apart from the obvious humane considerations, this has been incredibly useful for conservation studies that require genetic data without reducing the size of an endangered population. When working with small samples, however, particular care must be taken to avoid contamination, because very small amounts of target DNA can be overwhelmed easily by 'foreign' DNA.
From a practical perspective, the storage of samples destined for PCR is relatively easy during field trips; whereas material suitable for allozyme analysis will often require surgery or even sacrifice of the entire animals, and then must be stored in liquid nitrogen or on dry ice until it is brought back to the laboratory, samples for PCR analysis can be removed without dissection and can be stored either as dried specimens or in small vials of 70--95 per cent ethanol or buffer that can be kept at room temperature. One thing to watch for, however, is the problem of degraded DNA that can reduce the efficacy of PCR. The DNA in freshly harvested blood or tissue will remain in good condition provided that it is placed quickly into suitable buffer or ethanol, but improperly stored DNA will degrade rapidly as the DNA molecules become fragmented. The DNA extracted from a non-living sample, such as faecal material or museum specimens, will already be at least partially degraded. If the DNA fragments in a degraded sample are smaller than the size of the desired region, then amplification will be impossible and therefore relatively short DNA sequences should be targeted.
One source of material for PCR, which would have been assigned to the realm of science fiction before the 1980s, is ancient DNA from samples that are thousands of years old. Most fossils do not contain any biological material and therefore do not yield any DNA, but organisms that have been preserved in arid conditions or in sealed environments such as ice or amber may retain DNA fragments that are large enough to amplify using PCR (Landweber, 1999). However, even if some genetic material has been preserved, characterizing ancient DNA is never straightforward because there is typically very little material to work with. This makes amplification problematic, particularly if the degraded DNA fragments are very short. Chemical modifications also may interfere with the PCR reaction (Landweber, 1999).
Even if amplification is possible, the risk of contamination is quite high because foreign DNA such as fungi or bacteria that invaded the organism after death may be more abundant than the target DNA. Furthermore, as is always the case with very small samples of DNA, contamination from modern sources, including humans, can be a problem. In 1994 Woodward and colleagues claimed to have sequenced the DNA from dinosaur bones that were 80 million years old (Woodward, Weyond and Bunnell, 1994), but further investigation showed that the most likely source of this DNA was human contamination (Zischler et al., 1995). Nevertheless, characterization of ancient DNA has been successful on numerous occasions, for example DNA sequences from 3000-year-old moas provided novel insight into the evolution of flightless birds (Cooper et al., 1992), and ancient DNA from Neanderthals lends support to the theory that modern humans originated in Africa relatively recently (Krings et al., 1997).
Other relatively unusual sources of DNA for PCR amplification that are particularly useful in molecular ecology include: faeces, hair and urine, all of which have been used to genotype elusive species such as wolves, lynxes and wombats (Sloane et al., 2000; Valiere and Taberlet, 2000; Pires and Fernandes, 2003); sperm collected from the membranes of birds' eggs, which provides a novel view of some aspects of mating behaviour (Carter, Robertson and Kempenaers, 2000); gut contents from ladybirds and lacewings that were analysed to determine which species of aphids they had consumed (Chen et al., 2000); museum specimens, which may be particularly useful when characterizing widespread species or locally extinct populations (Lodge and Freeland, 2003; Murata et al., 2004); and ancient fungi from glacial ice cores up to 140 000 years old, which can provide data on fungal ecology and evolution (Ma et al., 2000).
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