Background info

DNA JArgon:

DNA: is an acronym for deoxyribonucleic acid. In humans our DNA is comprised of ~3 billion DNA "building blocks" called A, T, C and G (refered to as "bases") which are strung together to form the human genome.

Genome: refers to the entire DNA sequence of A, T, C and G's in an organism (the human genome is ~3 billion bases in length divided around 23 pairs of chromosomes)

DNA sequencing: Is the process by which we determine the order of A, T, C and G's. 

PCR: is an acronym for Polymerase Chain Reaction. It is a process to "photocopy" the DNA. It is critically important in forensics as it allows us to analyses very small starting quantities.

Mitochondrial DNA (mtDNA): Is a small ring of DNA (only 16,000 bases) that is always inherited from your mother. There has been a lot of sequencing work done on mtDNA hence it is often used for species identification purposes. 

DNA tree: Is a way to visualise DNA sequences, rather than comparing similarities and difference by looking at the A, T, C and G code it is common to present this graphically as a tree. The closer things are on the tree the closer the relationships.

Nucleus: Is a compartment in a cell that contains most of the DNA (except mtDNA).

Microsatellites: Also called STR's these are highly variable pieces of repeating DNA (e,g, ATATATATATATAT)  scattered across your genome. When these are characterized they are often able to discriminate individuals.

DNA sexing: Males and females have a different genetic make up for some regions of their DNA, if these differences are characterised it is possible to determine if a sample is male or female   

  

 

Want to know more?

There is an extensive body of work available on the internet that describes in detail some of the techniques and technologies we describe on these pages. Some useful links are:

www.cstl.nist.gov/biotech/strbase/

www.thenakedscientists.com/HTML/articles/article/dalyacolumn8.htm/

www.forensicdna.ca

What is wildlife forensics?

Wildlife forensic biology is concerned with the use of technology, such as molecular biology (DNA profiling and DNA sequencing) to fight against wildlife crime. There are a growing range of modern DNA approaches that can be used in wildlife crime investigations. When these are used DNA_logo_smappropriately they can be highly discriminatory. It is commonly acknowledged that wildlife forensics and its arsenal of tools are under-utilised (and under-funded). However the Australian Wildlife Forensic Services at Curtin University (and a several other laboratories around Australia) are well equipped and placed to carry out forensic and wildlife investigations.  This page introduces some of the biology and the technology underlying forensic DNA typing.  It also gives examples of how this technology is used in establishing whether trade in endangered species complies with the CITES legislation. It also illustrates the broad application of DNA typing that is available at Curtin University.

Our work has been generously supported by The Animal Biology Institute at Murdoch University, The department of environment and conservation (DEC)  the Australian Customs Service and the Department of Environment and Heritage (DEH).  Further information on international wildlife trade is available from the Department of Environment and Heritage (DEH) Community Information Unit on 1800 803 772, or the DEH website at: http://www.deh.gov.au.

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The Convention on International Trade in Endangered Species of wild fauna and flora (CITES) is an international convention to protect the trade of endangered plant and animal species, dating from 1975. CITES became enforceable under Australian law in October 1976. In Australia, CITES initially was enforced under the Customs (Endangered Species) Regulations and then by the Wildlife Protection (Regulation of Exports and Imports) Act 1982. Under amendments effective from 11th of January 2002 the legislative basis for meeting Australia's responsibilities under CITES is now provided by Part 13A of the Environment Protection and Biodiversity Conservation Act 1999 (EPBC Act).

Australian federal and state agencies demonstrate world’s best practice in managing and enforcing national and international treaties for the protection of wildlife.  This is aggressively carried out with inspection and policing at our borders and through educational and a physical presence.  Associated with enforcement has been an increasingly broad range of molecular DNA techniques available that can fulfil a prominent role in wildlife forensic investigations.

The genes, made up of DNA, that define everything about an organism are woven into every individual cell of the organism. A reading of an animal's genetic makeup can, in theory, be taken from a single cell. In practice, geneticists use hair or skin samples, usually obtained by techniques that cause no pain or stress to the animal. In addition, samples can be taken from long-dead animals, which has often helped conservation geneticists plot the history of a species' fragmentation under human-caused influences. Animal cells are made up of essentially four major compounds (fats, salts, proteins and DNA). Once all but the DNA has been digested away, the structure can be analysed relatively easily because it is constructed much like a bar-code, consisting only of four elements (A, G, C and T).

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Examples of forensic investigations;

Food: e.g., caviar from Caspian Sea sturgeon, freshwater and marine turtles;

Pets: many species of exotic amphibians, reptiles and birds;

Testing to distinguish extant elephant and mammoth ivory products

Species identification of cetaceans (dolphin/whale) illegally killed

Species determination of birds from eggs

Timber: rainforest hardwood trees such as mahogany and teak;

Analysis of ‘ivory’ roulette balls to establish whether they are made of ivory

Testing traditional medicine: e.g., rhinoceros horns, bear gallbladders, and various plants in TAM

Animal furs and skins: e.g., the trade in crocodile and alligator skins; and products sold as souvenirs such as figurines made from illegal ivory or marine turtle shells, or jewellery made of coral.

DNA and forensic science


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The discovery that some DNA sequences are unique to an individual has revolutionised the field of forensic science and has enabled the analysis of a whole array of biological materials.  Today, DNA profiling (or STR analysis) is perhaps one of the most reliable method of personal identification available. Modern PCR based technologies have advanced DNA profiling to such an extent that it is DNA profiling can actually be carried out using the minute amount of DNA present on a fingerprint, stamp, hair etc. The aim of DNA analysis is to analyse genetic information found in biological material in a forensic environment and to use the information to infer the source of the sample.


STR analysis is now the most widely used DNA typing procedure as it provides higher discriminating power than RFLP analysis but it also requires minute samples. Short tandem repeats (STRs, e.g. ….CACACACA…) are loci on the chromosome that contain short sequence elements that repeat themselves within the DNA molecule. The repeating sequence is usually 2 - 7 bases in length and the entire strand of an STR is usually small (<400 bp). Since the strands are shorter than those used in RFLP analysis, the technique enables the analysis of DNA samples that have been subjected to extreme decomposition.


During forensic examination, an STR with a known repeat sequence is extracted and separated by electrophoresis in the same way as RFLP analysis. The more STRs that can be characterised, the less chance there is of the DNA of two individuals giving the same results. The effectiveness of this technique has lead to ‘multiplexing’, the extraction and analysis of a combination of different STRs at the same time.


The revolution in DNA research has already made an enormous impact on forensic science. The most important developments have been in the field of human DNA. However, specialised DNA research methods can be powerful tools for investigating wildlife forensics, with new methods continually being developed. Whereas human DNA research is aimed primarily to comparisons between individuals, forensic analysis involving endangered species is used to identify species (is this material from a tiger?), populations (is this ivory from an elephant from Kenya or South Africa?), individuals maternal lineage (is this chick the offspring of this bird?) and individuality (did this bloodstain come from this specific bear?). Moreover, humans are just one of the many species on earth. DNA characteristics used for comparisons between humans are not necessarily useful for comparisons of animals and plants.


What is DNA?


DNA (deoxyribonucleic acid) is a molecule that defines functions, appearance (phenotype) and genetic origin. Each individual (and as such species and population) will have their own unique set of DNA (there are some exceptions - e.g. twins). Plants contain many variable sections of DNA and only a small number of individuals in a plant population are thought to have the same genotype (vegetative clones). DNA is present in most cells, including muscle, skin, blood, sperm, hair roots egg shells, feathers and body tissue. In animal cells, DNA is found in two different parts of the cell: the nucleus (genomic DNA) and in the mitochondria (mitochondrial DNA or mtDNA).


For each young, each parent provides half the genomic DNA (gDNA). This can be used to establish parental relations, and can be made easily if one or both parents are known. Through recombination, siblings inherit various combinations of DNA from the same parents and therefore differ from each other. In contrast, animals generally inherit all their mitochondrial DNA (mtDNA) from the maternal line and so in principle mtDNA is the same in mothers and their offspring. It can be used to determine relationships between species as well as the genetic origin of individuals. Mitochondrial DNA can also be isolated from hairs and bones. Cells contain multiple mitochondria and therefore have multiple copies of mtDNA (up to a few hundred per cell). There is a greater chance of identifying mtDNA than genomic DNA, as there are only two copies of the latter present in each cell. This makes mtDNA particularly useful for identifying partly degraded (e.g. burnt) material. DNA analyses examine just a part of the DNA. The composition of a limited number of selected DNA fragments are compared with reference material.


In sexual reproduction, one chain of each DNA double helix in the nucleus is contributed by each parent. The complementary chains are then synthesised and the helix structure constructed. The long double-helixes are folded in a complicated way to form a chromosome. Humans have 46 chromosomes, 23 from the father and 23 from the mother. Mitochondrial DNA contains a double helix structure from the mother only. The beginning and end of the helix are linked to form a ring, comparable with plasmids in bacteria. The ability to amplify DNA in test systems using the polymerase chain reaction (PCR) constitutes the basis of the current sensitive DNA methods. PCR can be used to obtain multiple copies of certain parts of DNA, allowing sufficient DNA to be obtained for analysis and raising the sensitivity of the method. This means that even minute or partially degraded samples can be analysed after PCR amplification. DNA is the template for the reproduction of genetic material and cellular information. However it is important to consider contamination, where even small amounts of material from ‘other’ sources have the potential to make the results uninterpretable.  There are many ways to minimise this from happening.


The parts of the DNA known as genes contain the information needed to create proteins. This coding DNA makes up only a fraction of the total DNA molecule, most of which (more than 90% in humans) is non-coding and has no known function. About 40% of the non-coding human DNA consists of repeating sequences of bases (repeats). Repeats consisting of a minimum of two and a maximum of ten bases (e.g. CA repeats) are called microsatellites, or Short Tandem Repeats (STRs). This repetitive DNA is found in various places (loci) in the genome, not only in humans but in other species as well.  The lab at Murdoch has a large amount of experience in developing, and using STR markers.


In general, the further apart a species is to another in terms of their evolution, the larger the differences between their DNA will be. Spontaneous mutations in the DNA sequence results in change in DNA. Mutations, particularly in non-coding regions of the DNA are less likely to have a significant effect on the production of proteins, and therefore have less important consequences for the viability of the individual and its ability to reproduce. This explains why the variation in the non-coding DNA is much greater than in the coding DNA. This neutral (or non-coding) DNA is therefore more suitable for comparing individuals and determining parental relations than the coding regions (in which selection acts strongest).


Our wildlife forensics team has experience in the following areas:

-Species identification

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-Population identification

-Individual identification

-Parentage analysis

-Identification of mixed animal tissuegeb

-Sex identification


DNA profiling (or 'DNA fingerprinting')


Genomic DNA is compared by determining the number of repeated units in short tandem repeats (STRs) at several known loci. Developing primers for these types of tests is expensive (~AUD$15,000), but the tests themselves are relatively cheap (~AUD$50/individual - with very high exclusion probability > 1 in a billion).  The PCR amplification uses primers to recognise characteristic lengths of DNA directly preceding or following a repeat. This ensures that only the lengths of DNA that are useful for the test are amplified. In the human method, the number of repeated units in the short tandem repeats is determined. This results in a code of 20 whole numbers that indicate the number of repeats. This code is then compared with the reference DNA sample.


Data from a database of DNA variation (e.g. the frequency of the relevant STRs) for the animal species in question are needed to assign a statistical value to the matches. The number of loci that have to be investigated depends on the animal species. Not every species displays the same variation in DNA, for example on mainland and island kangaroos. In smaller populations in particular, the variation may be very limited and so more loci have to be investigated. DNA genotyping systems are already available for a limited number of large Australian mammals, birds and fishes.


This DNA genotyping method is suitable for identifying individuals, determining parental relations and provenance of a sample (where it came from) and characterising populations. Offspring always share certain characteristics with their parents. If the individuals do share certain characteristics, a genetic relation cannot be ruled out. Determining the probability of a parental relation requires a statistical analysis in which case samples are compared with samples from the population they originate from. This analysis requires more genetic markers than in the identification of individuals, and is therefore more difficult and more expensive.


There are now very sophisticated modelling approaches (e.g. Bayesian and assignment tests) for identifying the origin of individuals. For instance, we have recently used Bayesian modelling to identify genetic structure and assign individuals to their population of origin. We used the findings to investigate the illegal dumping of feral pigs for recreational hunting.


Another application of DNA genotyping is the identification of populations, particularly where it may be important to identify the geographical provenance of a particular individual(s). To determine the genetic profile of populations it is necessary to establish the genotypes of a representative sample of the animals (or plants) in these populations. This information is collated into databases in the same way as for the determination of individual identity. New approaches and statistical analysis (e.g. Assignment tests) of the data can then be carried out to identify the population the case samples most probably come from. Given the small number of genetic databanks, this type of analysis is currently limited to a few species, for which such data is available.


DNA sequencing:

Mitochondrial DNA (mtDNA) is well established and widely used tool for species and to a lesser extent, population identification. DNA sequence analysis is a powerful tool for identifying the source of samples thought to be derived from threatened or endangered species.  Mitochondrial DNA is particularly suitable for determining the animal (or plant) species a material (e.g. powders, ivory, TAM) comes from. It is less suitable for determining whether biological material is from a specific individual. Mitochondrial DNA has numerous advantages, including the fact that it is;

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-generally maternally inherited (thereby reducing the effective population size, and also increasing its sensitivity to genetic drift)

-effectively haploid (a single copy)

-assumed that there is no recombination

-compared with nuclear DNA (see above), a molecule with a rapid rate of base substitution (0.5 - 1% / million years) with is 5 to 10 times the mutation rate at nuclear regions

-located separately in the cytoplasm, away from the nuclear genes

-at high copy number within a cell

-lacks introns and pseudo genes (therefore a relatively simple structure)61789454-4C94-4377-94ED-C1CE7DD5F721


For example, mtDNA contains the genetic code for the protein cytochrome b. It contains the code for a protein of vital importance for the production of energy in the cell. All individuals of the same species have a very specific DNA code for cytochrome b because any variation (mutation) leads to serious health problems in the individual. This piece of DNA is very useful for identifying vertebrate species because it has an intermediate rate of mutation (base substitution). The WGL uses this marker extensively in identifying whales and dolphins that have been taken for illegal fishing. Extensive studies of the cytochrome b gene have shown that the variation between species is much greater than the variation within species.


The sequence of bases in the cytochrome b gene are determined and compared with known sequences in gene databases. In contrast with the method described above for comparing STRs, this method compares sections of DNA that contain the genetic code for the synthesis of an essential protein. This means that the variation between base sequences is smaller than for STRs.


Other characteristic genes have also been identified in mtDNA, such as ‘population markers’ contained in information from the control (or D-loop) region.  These methods can be used for samples of cooked material, partially degraded material or when there is too little material to use less expensive methods. A disadvantage is that DNA sequencing is more labour intensive and expensive than other commonly used methods, such as protein analysis. Almost any other species, but requires that information about the species is accurate, and accessible in GenBank


In our laboratories, species identification has been our core business for many years (associated with conservation projects and ancient DNA projects) and we have a large amount of experience in species identification and are using these methods to identify many species of animals (including mammals, birds, snakes). Examples are listed below.

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 Detailed species identification techniques have been established for the following:

     -Whales and dolphins

     -Commercial kangaroo meat industry

     -Identification of bird species for

     -Snakes/Lizard

     -Pigs

     -Camels

    -Possums

    -Illegal seizure work with CALM/AQIS/Customs etc

    -Identification of birdstrike species (birds colliding with aeroplanes)

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The approach is possible because animals (and plants) inherit the traits determined by mitochondrial DNA (mtDNA) from the maternal line. These characteristics, therefore, do not in principle differ between mother and offspring and are used (because there is no recombination of the DNA during sexual segregation), among other things, to determine genetic relations between species.


 Examples of the use of mitochondrial methods include:

-Species identification of cetaceans used as bait on illegal fishing boats in Australian territorial waters.

-Development of a PCR-RFLP test for commercial kangaroo species, where it is illegal at times to use certain species.

-Identification of taxa involved with birdstrikes on aeroplanes (which costs the aviation industry AUD$3 billion per year)

-Species identification of caviar using a sequence of 413 base pairs. Primers from other methods have been shown to be very successful for this.

-Identifying the presence of material from tigers in Traditional Asian Medicine.

-Identification of bushmeat using GenBank.

-Identification of whale, dolphin and porpoise meat from Japanese and South Korean markets.


DNA gender (sex) determination

Difficulty with sex identification is a frequently encountered problem in various fields of forensics, animal biology and medicine because conventional methods require special skill for sample preparation and identification (e.g decomposition, sexes are monomorphic, no physical evidence or cytological investigation). Recent advances of DNA analysing techniques has considerably hastened up the procedure.


A rapid and straightforward method of sex identification from the DNA in a trace amount of saliva and from bird feathers using PCR is possible. Preparation of DNA using chelex based extraction requires only a short time. To identify particular alleles, samples must be run on high resolution gels along with an "allelic ladder" containing a range of the known alleles of known size. Minute amounts of cells (feather end etc) are collected by centrifugation and heated in a buffered solution containing beads of the resin Chelex. Lysis of the cells and release of DNA is accomplished by a boiling water bath and the alkalinity of the Chelex suspension. PCR is then performed with the DNA-containing supernatant.

Mammal DNA sexing

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We can perform sex identification easily and accurately using PCR of the sex chromosome specific locus for most mammal species. We have experience in identifying primate and cetacean sex. To do this, amplification of X and Y chromosome-specific  DNA (Zf and Sry genes) allows the determination of the sex of many species of mammal, including cetaceans, rodents, marsupials, lacking gender-specific morphological characteristics, for example where there is no skeleton. This process allows one to 

determine the sex of an animal based on a blood or tissue sample.  Control reactions are very important for such exercises.


Bird sexing

Bird sexing is often very difficult due to the fact that more than 50% of bird species are nearly identical to look at. Consequently, it is usually necessary to determine sex by DNA-techniques (or via invasive surgical sexing). Bird sexing is important for profit bird-breeders, conservationist bird-breeding, and evolutionary studies. The genes are located on the sex chromosomes of nearly all birds, and is divided into the W and Z gene, representing corresponding sex chromosome location.


Unlike in mammals, in birds the females are the heterogametic sex having a W and a Z sex chromosome. Males have two Z sex chromosomes. Since females are heterogametic, they will be the only ones who possess the W gene. However, both males and females possess the Z gene. This is the basis of gene amplification and sex determination for this experiment. The primers used for this experiment amplify homologous regions of the genes by binding to conserved exons and amplifying across an intron. These introns differ in length, with W gene (in females) being a few bases larger than the Z gene in most species.


Morphological identification of animal hair

All mammalian hair predominantly consist of the protein keratin, which due to its chemical structure is very durable, this durability is responsible for the survival and preservation of hair. Hairs are essentially comprised of three main compartments; the outer protective cuticle which are arranged like tiles on a roof, the cortex (which forms the bulk of the hair) and the medulla. The image below shows a generalised diagram of a hair shaft which shows an example of; (A) cortex with pigment granules; (B) cuticle scales; and (C) mudulla. 

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The morphological identification of animal hairs is based on the fundamental aspects of microscopy, biology and zoology. The purpose of this type of examination is to attribute the animal source of an unknown hair sample to a particular taxon (animal group) based on well-defined, genetically based features that are characteristic to that group. For example, the image below shows two different examples of medulla pattern configuration, cuticls scale patterns and cross-sectional morphology characteristics of hair from Otariidae - Australian fur seal (A-C) and Leporidae - European rabbit (D-F). 

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