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The majority of cells making up the human body are diploid cells carrying identical DNA, with the exception of haploid gametes (egg and sperm) and red blood cells (which have no nucleus). Several types of biological evidence are commonly used in forensic science for the purpose of DNA analysis, including blood, saliva, semen, skin, urine and hair, though some are more useful than others. The use of biological evidence in DNA and genetic analysis varies, with areas of study including blood typing, gender determination based on chromosome analysis (karyotyping), DNA profiling and, more recently, forensic DNA phenotyping. Since the advent of DNA profiling in the 1980s, it has been successfully utilised in criminal cases, disaster victim identification and paternity testing to name a few. However despite their merits, DNA fingerprints are not ideally used as the sole piece of evidence in a case, and in certain countries, such as the United Kingdom, DNA fingerprints must be presented in conjunction with other evidence.

DNA Structure and Function
It is vital to understand the structure and function of DNA and how this relates to DNA analysis in forensic science. DNA, deoxyribonucleic acid, is a molecule arranged into a double-helix, its structure first described by James Watson and Francis Crick in 1953. It is composed of nucleotide trisphosphate molecules, referred to as the ‘building blocks’ of DNA. These molecules consist of a trisphosphate group, a deoxyribose sugar and one of four nitrogenous bases. The four bases involved in a DNA molecule are adenine and guanine (purines) and thymine and cytosine (pyrimidines). These bases bond to the deoxyribose sugar and one of the other bases to form base pairs, with adenine and thymine bonding through two hydrogen bonds, and guanine and cytosine bonding with three hydrogen bonds.

DNA is essentially the molecule that holds all genetic information and ‘instructions’ for an organism. The human genome is composed of over 3 billion base pairs of information organised into 23 chromosomes. Genes are the regions of DNA that encode and regulate protein synthesis, though this involves just 1.5% of the entire genome. A significant amount of the human genome, approximately 75%, consists of extragenic DNA, which contains regions that do not actually contain known gene sequences. About 50% of extragenic DNA is made up of something called repetitive DNA, which is of particular use in forensic DNA analysis. Repetitive DNA is further sub-divided into tandem repeats (including satellite DNA, microsatellites and minisatellites) and interspersed repeats (SINE, LINE, LTR and Transposon). Tandem repeat DNA and the variation between them (polymorphisms) is the focus of many DNA profiling techniques. It is due to the number and location of these polymorphisms that every individual has unique DNA which produces a distinctive band pattern when analysed.

It is through the extensive study of the genome that DNA fingerprinting has been produced as a useful and reliable technique in forensic science.

Sources of DNA Evidence & DNA Extraction
In terms of forensic DNA analysis, there is a variety of possible sources of DNA evidence. The more useful sources include blood, semen, vaginal fluid, nasal secretions and hair with roots. It is theoretically possible to obtain DNA from evidence such as urine, faeces and dead skin cells, though this is often classed as a poor source due to the lack of intact cells and high levels of contaminants preventing successful analysis. Such samples will be collected depending on the type of sample (see crime scenes page for more details of evidence collection and preservation).
Prior to analysis, it will be necessary to extract DNA from the sample. This is generally achieved through the following simplified steps.

• The sample cells are lysed (broken down) in a buffer solution.
• Denatured proteins and fats are pelleted through centrifugation.
• The cleared lysate is then passed through a column, often containing a positively charged medium that binds to the DNA.
• Contaminating proteins, fats and salts are then removed through several washes.
• The DNA is recovered in a buffer solution/water.
• The amount of DNA is often then quantified using spectrophotometric techniques.
• Various methods of extraction have been devised for different types of sample.

DNA samples for comparison are generally collected from suspects using buccal swabs, in which a sterile swab is scraped along the inside of the cheek to collect epithelial cells to use in producing a DNA fingerprint. Sir Alec Jeffreys is often described as the father of DNA fingerprinting.

Single Nucleotide Polymorphisms
The use of DNA analysis in forensic science is based on a variety of techniques focusing on polymorphisms, which essentially refers to variation in sequences. Different sequences are studied in different techniques, including single nucleotide polymorphisms, minisatellites (variable number tandem repeats), microsatellites (short tandem repeats) and mitochondrial DNA, each different with regards to length and repetition.
Single Nucleotide Polymorphisms (SNPs) are the simplest and most common type of genetic variation, composing around 90% of genetic variation in humans. They occur during meiosis when DNA is replicated, with each SNP representing a difference in a single nucleotide. For example, a SNP may replace cytosine with a thymine in a stretch of DNA, and this will not change the length of the DNA. There are about 10 million SNPs in the human genome, with one found at every 100-300 base pair. These can act as biological markers.

In the analysis of SNPs, which can consist of up the four alleles, the specific base present in the SNP must be established. This is achieved using DNA sequencing techniques. However DNA sequencing is not generally used in forensic science except in the analysis of mitochondrial DNA.

Dideoxy Method
Also known as the Sanger Method, this form of DNA sequencing was developed by Fred Sanger in 1975. A labelled primer is utilised to initiate the synthesis of DNA. Four dideoxy nucleotides are added and randomly arrest synthesis. Fragments are produced which are subsequently separated using electrophoresis or, in more modern automated systems, capillary electrophoresis. Specialised software is then used to convert the band patterns into a DNA sequence. Initially unstable, hazardous radioactive tags were used, soon replaced by fluorescence dyes. However the varying mobility of these different coloured dyes meant that the order of bands did not necessarily accurately represent the order of the nucleotide sequence. The dyes would also sometimes behave unpredictably, making them unreliable and not particularly ideal for use in forensic cases. If conditions were unsuitable for the DNA template or the dideoxy nucleotides, the reaction would be unsuccessful. The computer software itself was problematic, with peaks often overlapping, making it difficult to establish the correct order of nucleotides.

Minisatellites or Variable Number Tandem Repeats (VNTRs)
Variable Number Tandem Repeats (VNTRs) are highly polymorphic sequences in the exon region of DNA repeated at various points along the chromosome, 4-40 times. They are about 20-100 base pairs in length, though their specific lengths are not strictly defined. These tandem repeats are inherited from both parents, therefore no one will have the same VNTRs as either of their parents. The number of repeats at the locus affects its position after electrophoresis and the length of the DNA after the chromosome has been cut with a restriction enzyme. Minisatellites were the first type of polymorphism to be used in a criminal investigation in the case of British murderer and rapist Colin Pitchfork.

However VNTRs are not generally used in forensics, as this technique is often a more expensive process and takes longer due to VNTRs having a greater length than, say, STRs.

Microsatellites or Short Tandem Repeats (STR)
Short Tandem Repeats (STRs) are regions of the genome composed of approximately 1-5 bases and repeated up to 17 times. STR markers will either be simple (identical length repeats), compound (two or more adjacent repeats) or complex (several different length repeats). They are found on 22 autosomal chromosomes as well as both X and Y sex chromosomes, though those on the Y chromosome differ less due to lack of recombination. Only a select number of STR markers are used in forensic DNA profiling (10 in the UK and 13 in the US).They express a high degree of polymorphism, making them of particular use to the forensic scientist. The variability in STRs is caused by the inaccuracy of DNA polymerase in copying the region. As STR regions are non-coding, there is no selective pressure against the high mutation rate, resulting in high variation between different people.

Though there have been thousands of short tandem repeats found in the human genome, only a small number are utilised in forensic DNA analysis. STRs used in forensic science tend to be tetra- and penta-nucleotide repeats, as they are both robust, suffer less environmental degradation, and provide a high degree of error free data. STR loci are ideal for use in forensic science for a number of reasons. They represent discrete alleles that are distinguishable from one another, they show a great power of discrimination, only a small amount of sample is required due to the short length of STRs, PCR amplification is robust and multiple PCR can be used, and there are low levels of artefact formation during amplification. An early use of microsatellites is in the identification of Auschwitz camp doctor Josef Mengele.

Restriction Fragment Length Polymorphisms (RFLPs)
Restriction Fragment Length Polymorphisms (RFLPs) were used in the first technique developed to analyse variable lengths of DNA fragments produced through DNA digestion. It exploits variations in DNA sequences due to the differing locations of restriction enzyme sites. The method uses restriction endonucleases to ‘digest’ the DNA by cutting it at specific sequence patterns. The resulting restriction fragments are then separated using gel electrophoresis and transferred to a membrane using the Southern Blot technique. After the separated DNA fragments are transferred, probe hybridisation is used to detect the fragments.

However DNA analysis with RFLP required relatively large amounts of DNA and degraded samples could not be analysed with accuracy. More effective, faster and cheaper DNA profiling techniques have seen been developed, so RFLP is generally no longer used in forensic science.

Polymerase Chain Reaction (PCR)
The amount of DNA evidence obtained during the investigation of a crime is often very small, thus for successful DNA profiling some form of amplification is ideal. Polymerase Chain Reaction (PCR) is a technique which allows for the exponential amplification of DNA fragments to lengths of approximately 10,000 base pairs. This means that, theoretically, a single copy of a DNA fragment could be amplified to millions of copies in just a few hours. PCR is particularly beneficial in the amplification of minute amounts or degraded samples.

A successful PCR reaction requires a number of vital primary components. Oligonucleotide primers which are complementary to the DNA target and mark the target to be amplified, with two primers being used. The base sequence of one primer binds to one side of the target whilst the other primer binds to the other side of the target, with the DNA between the primers being amplified. Fluorescent tags are often added to the primers to visualise amplified DNA in electrophoresis. DNA polymerase enzyme allows the DNA strand to be copied by adding nucleotides to the 3’ end of the primers. Other components required include a reaction buffer with MgCl to ensure ideal conditions for the functioning of the DNA polymerase enzyme, deoxyribonucleotides to build the DNA molecule, and template DNA. Modern PCR uses thermostable DNA polymerases. Most commonly used is the Taq polymerase, which has largely replaced the previously used E.coli-derived polymerase. This was isolated from Thermus aquaticus, which is an organism capable of surviving in temperatures over 70oC. However Taq polymerase lacks the ability to proof read. VENT polymerase is from Thermococcus litoralis, which can survive in temperatures over 100oC.

The PCR cycle consists of three primary steps: denaturation, annealing and extension. The process is generally conducted in a small, plastic centrifuge tube with the temperature carefully controlled using a thermal cycler.

• Denaturation: The sample is heated to 94-95oC for about 30 seconds. This separates the double-stranded DNA by breaking hydrogen bonds, allowing primers access.
• Annealing: The samples is kept at 50-65oC, depending on the primer sequence, to allow hydrogen bonds to form between the primers and the complementary DNA sequence.
• Extension: Also known as the elongation stage. The sample is heated to 72oC for a duration depending on the length of the DNA strand to be amplified and the speed of the polymerase enzyme (Taq polymerase) which builds up the strand. Deoxynucleotide triphosphates are added to the 3’ end of the primer.

Each PCR cycle can take only 5 minutes. This procedure can then be repeated as necessary until the original sequence has been amplified a sufficient amount of time, with the amount being doubled with each cycle. Following PCR, the products are separated using electrophoresis.

Unfortunately PCR is not suitable in the analysis of longer strands of DNA, and so cannot be used with earlier techniques such as RFLP. It must be taken into consideration that certain compounds can inhibit PCR reactions, often substances associated with the stages of extracting and purifying the DNA. Such substances include proteinase K (which degrades the polymerase enzyme), ionic detergents and gel loading dyes. Similarly, certain substances present in blood can inhibit PCR, such as haemoglobin and heparin.

Various alterations have been made to improve the PCR method. Multiplex Polymerase Chain Reaction involves the amplification of numerous DNA sequences in a single reaction through the use of primers that produce non-overlapping allele sizes, allowing numerous regions of a sample to be tested simultaneously.

PCR Errors
Various factors can contribute to errors and inaccuracies in data produced by the polymerase chain reaction technique. PCR is often carried out using DNA polymerases such as Taq DNA polymerase, which does not have the ability to ‘proof read’, resulting in errors in amplification. The greater the amplification, the more likely it is that such errors will occur. Mispriming is also a potential problem, with products being formed from non-target sites. Excessive primer dimers may be formed, which are by-products of PCR produced when one primer is annealed to another causing primer extension. This may all result in unexpected variability in PCR success across a series of samples or previously successful conditions failing.

Electrophoresis
As mentioned, during DNA analysis the individual fragments of DNA can be separated using electrophoresis to produce the distinct ‘DNA fingerprint’. Electrophoresis is essentially a method of separating molecules by their size through the application of an electric field, causing molecules to migrate at a rate and distance dependent on their size. In gel electrophoresis, a porous gel matrix is used, often consisting of agarose gel for simple work or polyacrylamide gel for more specific procedures. The gel is often floating in a buffer solution to ensure the pH level is maintained and the applied electric current is conducted. Samples to be analysed are placed in small wells at the top of the gel using pipettes. A control sample and a standard/marker sample will often be run simultaneously. As the electric current is applied, the negatively charged DNA fragments begin moving through the gel towards the positively charged anode. The gel essentially acts as a type of molecular sieve, allowing smaller molecules to travel faster than larger fragments. Following electrophoresis, it may be necessary to visualise these bands using radioactive or fluorescent probes or dyes. Electrophoresis not only separates DNA but also allows for the fragments to be measured, often expressed in base pairs. Measuring the length of these fragments can ultimately allow the number of repeats to be determined and thus the genotype at that locus.

Earlier techniques used flat-bed gel electrophoresis, though the faster and automated capillary electrophoresis is now used more often. In this technique, abbreviated to CE, the gel is held in a fine capillary tube through which the fluorescently-labelled DNA passes through much as in gel electrophoresis, often with an added DNA size standard. Positioned along the capillary is a laser beam which causes the DNA to fluoresce as it passes. This can then be detected and fed directly to a computer system in the form of an electropherogram. Unfortunately capillary electrophoresis is not able to separate more than one sample at a time, though a genetic analyser can be used to separate a series of samples one after the other.

Blotting Techniques
Following gel electrophoresis, probes are generally used to detect specific molecules. However because probes cannot be directly applied to the gel, blotting methods were initially utilised. There are a number of common blotting techniques: Southern blot, Western Blot, Northern Blot, Eastern Blot and Southwestern Blot.

• Southern Blot – Used in DNA analysis. DNA is extracted, separated with electrophoresis and transferred to the membrane. Labelled probes are added, which hybridise to specific sequences and identify them.
• Western Blot – Used to detect proteins. SDS-PAGE used to separate proteins, which are transferred to membrane. Specific antibodies are then added, followed by a substrate to visualise bands.
• Northern Blot – Used to study gene expression. Similar to the Western blot, except RNA is being analysed.
• Eastern Blot – Used to study proteins. Considered to be an extension of the Western blot.
• Southwestern Blot – Combines features of the Southern and Western blot. Used for the rapid characterisation of DNA binding proteins and their sites.

Low Copy Number (LCN) DNA Analysis
Low Copy Number DNA Analysis, referred to as LCN, is a technique developed by the UK’s Forensic Science Service in an attempt to increase the sensitivity of DNA profiling methods. Samples containing small amounts for badly degraded DNA often leads to problems such as poor quality fingerprints or even completely negative results. This technique reduced these issues.

Developed in 1999,LCN is essentially an extension of the Second Generation Multiplex Plus (SGM+) technique, and is generally used when the previous technique has failed or has produced weak results. Improved sensitivity is achieved through an increased number of PCR cycles, with standard techniques generally using 28 cycles but LCN using 34. This could ultimately allow for DNA profiles to be successfully obtained from minute amounts of sample and even from single cells (see below).

However the increased sensitivity of this DNA profiling technique brings about amplified concerns over issues of ease of contamination and amplification of these contaminants, mixed profiles being produced and wrongful accusations. With techniques such as LCN, it is now more important than ever that investigators wear suitable protective clothing and follow strict anti-contamination procedures, and controls are used in analyses.

In 2007, LCN came under great scrutiny through the case of R vs Hoey, which led to the use of this technique being temporarily banned until a thorough review could be conducted. Sean Hoey was tried for involvement in the Omagh bombing in 1998, charged with numerous counts of murder in addition to terrorism and explosives charges. The DNA evidence used against him was based on the relatively new LCN technique which, at the time, was only being used in British courts. However due to the lack of data and known error rates regarding the technique, serious concerns were raised. Hoey was subsequently found innocent.

Single Cell DNA Fingerprinting
Closely linked to LCN analysis is single cell DNA profiling. Dr Ian Findlay and his colleagues at the Australian Genome Research Facility first reported the successful development of a DNA fingerprint from a single cell in 1997. 226 buccal cells from four individuals were isolated using micromanipulation, amplified using the UK Forensic Science Service’s Second Generation Multiplex (SGM) assay to increase sensitivity. Six STR markers and amelogenin for sex-typing were amplified. The results from single-cell analysis were compared with known DNA profiles. Whereas a full and correct profile was only obtained in 50% of the single-cell tests, 91% of tests exhibited some form of result.

Previous DNA fingerprinting methods often required hundreds of cells in order to obtain a profile. The single cell is obtained by swabbing the material and identifying the cell to be analysed using microscopy prior to analysis. This technique is particularly fast, taking a matter of hours, and has a specificity of about 1 in ten billion.

Single-cell DNA profiling is particularly useful in rape cases, as DNA in sperm cells is highly conserved due to it being so compacted in the protein head. There is also potential for the technique in use in documents. Human DNA can be placed in documents such as Government bonds, wills and security documents, to track their flow. However the main issue with this particular use is that close relatives may handle the documents, particularly when dealing with documents such as wills, and so the technique may not be appropriate. Single-cell DNA analysis is also ideal in IVF procedures, in which single cells can be analysed for genetic defects.

However there are some major concerns with this method. As an increased number of PCR cycles are required to amplify the DNA, this brings about problems of allele drop-in, where additional alleles are added to the sample. Allele drop-out is a similar problem with increasing amplification samples. When starting with a single cell or small amount of sample, any contaminants already present in the sample will be amplified. Similarly, any PCR inhibitors present will be more concentrated, causing amplification problems. One of the primary concerns is the ease of contamination by cells from other individuals. This could result in samples being contaminated and rendered useless or, worse still in the case of forensics, innocent individuals being wrongly accused. More work is required to validate the technique, particularly to use it in forensic science.

The work in single-cell DNA analysis led to the Forensic Science Service in the UK developing low-copy number DNA analysis.

Mitochondrial DNA (mtDNA)
Mitochondrial DNA is a circular molecule of DNA 16,569 base pairs in size, first referred to as the Anderson sequence, obtained from the mitochondrion organelle found within cells. This organelle is involved in the production of cell energy. There can be anywhere between 100 and 1000 mitochondria within a cell, each one containing numerous copies of the mitochondrial genome. This sequence is entirely functional and highly conserved, so there is very little variation between individuals. However there is a 1000 base pair long non-coding D-loop, known as the control region, which contains two hypervariable regions referred to as HV1 and HV2. The variations within these regions are generally single nucleotide polymorphisms (SNPs). SNPs do not alter the length of the mtDNA, and it is these regions that are focused on in the forensic analysis of mitochondrial DNA. Mitochondrial DNA is often subject to a relatively high rate of mutation due to its lack of DNA reparation, causing variation between individuals. The variation within this small portion is itself not especially significant, with HV1 and HV2 differing by 1-3% between non-related individuals.

In the analysis of mitochondrial DNA, the DNA is extracted and the HV1 and HV2 regions amplified using PCR. The base pair sequence of these regions is then established through DNA sequencing (see DNA sequencing section). This is then compared with the Cambridge Reference Sequence and differences noted. Other samples can then also be analysed an comparisons made to establish potential similarities. Mitochondrial DNA is generally used when other methods such as STR analysis have failed. This is often in the case of badly degraded bodies, in cases of disaster or accidents where an individual is too badly damaged to identify, and sometimes in taxonomy to determine species using the cytochrome b gene.

The most significant advantage of the use of mitochondrial DNA is the possibility of analysing even highly degraded samples. If a specimen is severely decomposed to the point that it is not possible to successfully extract a DNA profile using nuclear DNA, it may be possible through mitochondrial DNA. Additionally, only a very small sample size is required.

However the use of mtDNA does have its disadvantages. As mitochondrial DNA is only maternally inherited, this cannot form a full DNA fingerprint of the individual, thus this technique is only beneficial if the DNA profiles of maternal relatives are available, such as the individuals mother or biological siblings. Because of this, mtDNA is significantly less discriminatory than, for example, Short Tandem Repeats. Detecting sequence differences is also relatively time consuming and expensive.

Forensic DNA Phenotyping
Attempts have been made to utilise DNA analysis in the identification of phenotypic characteristics such as skin colour, hair colour and eye colour in a study known as forensic DNA phenotyping (FDP) or phenotypic profiling. This is generally achieved using single nucleotide polymorphisms (SNPs) rather than STRs. SNPs have a lower mutation rate and so are more likely to become fixed in a population, thus they are often found to be population-specific. It may be possible to estimate ethnic origin based on the presence of rare SNPs or STRs linked to particular population groups, though this theory becomes problematic with individuals of mixed ancestry.

Most work on phenotype SNPs has focused on pigmentation, and SNPs in a number of pigmentation genes have been associated with various human hair, skin and eye colour phenotypes.

Advances are already being made in this area of study. The Forensic Science Service has developed an SNP typing assay involving mutations in the human melanocortin 1 receptor gene (MC1R), which is associated with red hair. Specific alleles along this gene are associated with red hair, thus an individual inheriting this allele from each parent results in a high likelihood of that individual having red hair. Establishing that an individual is likely to have red hair is limited in forensic science, as red hair is not particularly common, though it is more common to certain populations.

Some research has been carried out into the genetics of eye colour, namely relating to the OCA2 gene on chromosome 15, which is also involved in the pigmentation of both skin and hair. IrisPlex is a recent test developed which allows for the accurate prediction of blue and brown eye colour.

Studies have been conducted examining the frequency of specific short tandem repeat alleles in groups of varying geographic ancestry. This research has resulted in the possibility of likely ancestry being established due to certain alleles being more likely in specific groups. However this particular use of DNA analysis is not infallible and can only be used as an estimation. The Forensic Science Service has developed an ethnic inference test, which provides the likely origin of a DNA sample from a number of groups (white European, Afro-Caribbean, Middle Eastern, South-East Asian and Indian Sub-continental).

Some countries and states are implementing specific legislation relating to the use of phenotypic DNA analysis. Many jurisdiction presently only allow the analysis of non-coding DNA, though the Netherlands currently allows forensic phenotyping under certain regulations. The phenotypic use of SNPs have also been used in non-forensic analysis, such as in determining the ethnicity, hair and skin colour of ancient remains.

However there are a number of problems and concerns with this approach. It is unlikely that a few chosen SNPs will provide a foolproof ‘picture’ of the sample’s source due to the complexity of multigenic traits as well as external factors such as environment and aging. Even if the technique was perfected for use in forensic science, phenotypes such as hair or eye colour can easily be masked through dyes and coloured contact lenses, limiting its forensic use. There are also privacy concerns relating to the possible traits determined, though it has conversely been argued that visible traits such as hair and eye colour cannot be considered private. Though should further advances be made to the extent that genes were located for traits such as aggression and predispositions to certain diseases, more serious concerns would be raised over the sensitivity of such information.

Researchers anticipate that further advances could allow for additional details to be ascertained, such as the likelihood that the individual smokes, along with the possibility for genes for the likes of handedness, aggression and homosexuality.

Y Chromosome Analysis
Much of modern DNA profiling is based on the analysis of short tandem repeats found on autosomes. However one particular branch of DNA analysis focuses on the amelogenin marker, the only marker on the sex chromosome, useful in the analysis of the Y chromosome. The Y chromosome, generally found only in males, is a small chromosome which, unlike other genes, is only altered through the infrequent occurrence of mutation. However similar to mitochondrial DNA (which is maternally inherited), the combination of alleles in this instance is theoretically identical between father and son, assuming mutation does not occur. Furthermore, Y chromosome analysis discrimination is comparatively low. Y chromosome analysis is particularly useful in cases of sexual assault and rape in which mixed DNA profiles may be encountered. Numerous systems have been developed to analyse some of the STRs present on this chromosome, such as Applied Biosystems’ Yfiler.

DNA Databases
Numerous countries have produced computerised databases containing DNA profiles to aid in the comparison of DNA fingerprints and the identification of suspects and victims. The first Government DNA database was established in the United Kingdom in April 1995, known as the National DNA Database (NDNAD). As of 2011, there were over 5.5 million profiles of individuals in the system. Similarly, the FBI in the US formed their own DNA database, the Combined DNA Index System (CODIS), in 1994, though it was not implemented in all states until 1998. Staff members involved in the handling and analysis of evidence will often also submit their DNA profiles to the database in the case of accidental contamination. There is the possibility for DNA databases to be shared between countries, however some countries focus on different loci in DNA fingerprinting.

DNA databases are not only used to make direct matches between the DNA fingerprints of one person, but to also conduct familial searching. This involves the search for genetic near-matches between a victim/suspect and a member of their family whose DNA profile is stored. This technique is based on the principle that related individuals are likely to express similarities in their DNA profiles. The first prominent use of familial searching was in the case of British serial killer Joseph Kappen, who was seized after his son’s DNA profile was obtained and then linked to him.

The production of DNA databases has allowed for the faster apprehension of suspects through comparing new crime scene samples to those already stored in the database, providing links between criminals and other crimes. It has also been widely used in cold cases, in some instances proving the guilt of an individual decades after they committed the crime. Conversely, wrongly imprisoned individuals have been exonerated through the advent of new DNA analysis techniques and databases. There is also the potential benefit of identifying bodies that have been too badly damaged or decomposed to identify, provided the individual’s DNA profile is stored.

However despite the advantages of such databanks, there has been significant controversy relating to the subject. Standard practice in many locations is to take a DNA sample of an individual when arrested, but this raises concerns over whether their DNA should be retained if they are then found innocent. There are also worries over innocent people being identified as matches or partial matches to DNA found at a crime scene even if they were not involved in the crime but had innocently attended the scene at some other point in time. This particular problem is becoming increasingly concerning as DNA fingerprinting techniques become more sensitive. There are also privacy concerns over the availability of sensitive genetic information, such as susceptibility to certain diseases and familial relationships. A more administrative disadvantage of such databases relates to the need for a facility that is both large enough to store such data but also has adequate security, a combination that can prove extremely expensive.

DNA Profile Interpretation
The primary purpose of forensic DNA profiling is to obtain a DNA ‘fingerprint’ from a biological sample and compare this to profiles obtained from DNA from a crime scene, an individual or profiles stored on a database. The process of modern DNA profiling includes statistically determining the chance that two people will share the same profile by establishing how common a genotype or collection of genotypes is within a population. The more loci studied and the greater heterozygosity of these loci, the smaller the chance two people will share a profile. However such figures can only ever be estimations and do not take certain factors into consideration, such as biological relatives. DNA evidence tends to be presented in terms of a random match probability, rather than definitively stating whether two profiles match or not.

Jeffreys, A J. Thein, S L. Wilson, V. Individual-Specific Fingerprints of Human DNA. Nature. 1985, 316)6023), 76-79.

Watson, J D. Crick, F H. A Structure for DNA. Nature. 1953, 171, 737-738.

Findlay, I. Taylor, A. Quirke, P. et al. DNA Fingerprinting from Single Cells. Nature. 1997, 389(6651), 555-556.

Jackson, A. R. W, Jackson, J. M., 2011. Forensic Science. Essex: Pearson Education Limited.

White, P. C., 2004. Crime Scene to Court: The Essentials of Forensic Science. Cambridge: The Royal Society of Chemistry.

Often found at the scenes of violent crimes, the analysis of bloodstains can provide vital clues as to the occurrence of events. Though bloodstain pattern analysis (BPA) can be a subjective area of study at times and often reliant on the experience of the investigator, the idea that blood will obey certain laws of physics enables the examination of blood at an incident scene and on items of evidence to offer at least an insight into what was likely to have occurred.

The successful interpretation of bloodstain patterns may provide clues as to the nature of the offence, the possible sequence of events, any disturbance to the scene that may have occurred, and even the position of individuals and objects during the incident. It may prove beneficial in refuting or corroborating eyewitness accounts.

Types
The appearance of a bloodstain can depend on a number of factors, including the velocity at which it was travelling, distance travelled, the amount of blood, the angle of impact, and the type of target onto which it lands.

Single Drop
These bloodstains typically refer to blood drops that have fallen vertically, whether it be from an injured person or another object, and landed onto another surface. As a blood drop falls perpendicular to a surface it maintains a spherical form until impacting. The size and appearance of this stain will depend on a number of factors. The volume of a single drop of blood will vary depending on the quantity of blood present and the surface area available from which the drop is falling. As would be expected, a larger surface area would allow for larger drop of blood to form before falling. The height from which the blood falls will affect the size of the stain, with greater heights tending to result in larger bloodstains. Furthermore, the target surface itself will cause an effect, with absorbent surfaces usually producing smaller stains than non-absorbent targets. The nature of the target can alter the appearance of the stain. For instance, a rough target surface can result in increased distortion to the stain and even satellite stains, which are additional stains radiating outwards. A drop of blood falling into an existing bloodstain will result in a drip pattern.

Impact Spatter
This type of bloodstain is the result of a forceful impact between an object and wet blood, causing the blood to break into smaller droplets. A greater force will typically produce smaller droplets, with the density of blood drops decreasing moving further away from the initial blood source. The study of impact spatter may provide insight into the relative position of individuals and objects during an incident and the nature of the incident.

Cast-Off Stain
Cast-off bloodstains occur when centrifugal force causes blood drops to fall from a bloodied object in motion. Similarly, cessation cast-off patterns may result from the sudden deceleration of an object. In this instance, the blood flung from a blood-stained object, such as a weapon, may produce characteristic patterns of numerous individual blood drops forming a curved or straight line. If an object is repeatedly moved, each subsequent swing will result in less cast-off as less blood remains on the object. Bloodstains produced in this fashion can be particularly difficult to interpret as there is a great deal of possible variation in patterns produced. However depending on the nature of the motion of the bloodied object, cast-off blood will at least produce relatively linear stains.

Transfer Bloodstains
Transfer or contact stains result when a bloodied surface comes into contact with another surface, transferring blood to that secondary target. The study of this type of bloodstain can prove particularly beneficial in establishing a sequence of events at the incident scene and tracing the movement of objects or individuals. In some cases it may even be possible to establish what object the transfer stain was likely to be caused by, for instance if a particular pattern is produced that can be traced to a blood-bearing object. Similarly, such bloodstains may be left by the hands of an individual, thus opening the possibility of fingerprint evidence.

Projected Pattern/Arterial Damage Stain
This type of bloodstain results from the discharge of pressurised blood onto a target surface, for instance the ejection of blood from a punctured artery. Areas of the body in which wounding may cause arterial bloodstains include the carotid artery, the radial artery in the wrist, the femoral artery in the inner thigh, the brachial artery in the arm, temporal regions of the head, and the aorta (though damage to the aorta is less likely due to increased protection of the chest cavity). Blood is expelled from the artery as the heart continues to pump and, as the blood travels, it breaks up into smaller individual droplets. Bloodstains produced will usually represent the beating of the heart as blood is expelled in periodic spurts. The resulting bloodstains can vary depending on a variety of factors, including whether the victim was stationary or moving as blood was being ejected, where on the body the injury occurred and the extent of the wound. If a wound is smaller in size, naturally smaller blood drops will be produced, which can subsequently be expelled further from the injury site than larger blood drops.

Pool Stains
Pooling bloodstains refer to the accumulation of blood on a particular surface, generally from prolonged bleeding from a wound or accumulation of arterial blood. If a body is not present at the incident scene, depending on the quantity of blood present, it may even be possible to roughly estimate whether the victim is likely to be dead or alive based on how much blood they have lost.

Insect Stains
These are bloodstains resulting from insect activity. The presence of insects such as flies at an incident scene, particularly one involving blood, is not uncommon (see the forensic entomology page). Flies may feed on blood and tissues at the scene and then, following regurgitation or excretion, produce small circular stains known as flyspeck. This minute stain could be mistaken for alternative bloodstains, such as expirated blood. Furthermore, small additional stains may be caused by insects walking through a stain, thus spreading the blood.

Expiration Stains
Often associated with injury to the respiratory tract, this type of bloodstain is caused by blood being coughed or otherwise expelled from the mouth. The stains will often be slightly diluted in appearance due to the additional presence of saliva or mucous. When blood is expirated from the mouth, it will often produce a pattern of small, round stains that could be likened to a fine mist.

Examination of Bloodstain Patterns
Various factors must be taken into account in order to successfully interpret a bloodstain. The surface onto which the blood is found may have had an effect on the behaviour and appearance of the stain. For instance, a bloodstain pattern may appear different if landing on an absorbent surface such as fabric as oppose to tile or plastic. Studying the state of the bloodstain may be able to shed light onto how much time has passed since the blood was shed, as over time blood will naturally coagulate (the process by which liquid blood turns into a gelatinous substance through various clotting factors). Furthermore, the extent of drying or coagulation will depend on the quantity of blood present – for instance a single drop will dry significantly faster than a large pool of blood. During this process of coagulation serum stains may be formed, which occur when the serum (liquid portion of the blood) separates.

Bloodstains at an incident scene may not always be visible to the naked eye, either due to low amounts of blood present or an individual cleaning in attempts to remove signs of bloodshed. Despite the use of cleaning reagents or even attempting to cover the stains with paint, detectable traces will generally remain, which can be visualised using various chemicals or specialised light. Although blood will not fluoresce under UV light like some bodily fluids, it will significantly darken, thus enhancing its visibility. Furthermore, certain chemical reagents can be used to visualise latent bloodstains. These tests, such as luminol and phenolphthalein, generally work by reacting with a constituent of blood to produce some kind of chemiluminescence. However it should always be remembered that these chemical reagent tests are often presumptive, meaning that they can only indicate that the stain is possibly blood. In reality, other substances may react with the reagent in the same way.

A lack of a bloodstain can be just as revealing. The absence of blood in a continuous bloodstain is known as a void, and may suggest that something or someone was present in that area when the bloodstain was caused. This could indicate an object present at the time of the incident has been removed from the scene, or an individual (or even multiple individuals) were present in specific locations when blood was shed.

It can easily be incorrectly assumed that blood found at an incident scene belongs to a victim, however it must be taken into account that some bloodstains may have resulted from the perpetrator being injured at some point. Either way, the information available from the presence of bloodstains is not limited to bloodstain pattern analysis, but also DNA analysis. See the DNA analysis page for more information.

Point of Origin – Directionality and Angle of Impact
In the reconstruction of an incident scene involving bloodstains, it is often beneficial to establish the point of origin of bloodstains, based on directionality and angle of impact. The examination of certain bloodstains may allow for the determination of the direction of travel of blood as it impacted the target. Whereas a drop landing perpendicular to a surface (depending on the type of surface) will tend to produce a more circular pattern, those landing at an angle will result in an elongated stain. The tapered end of this stain will generally point in the direction in which the droplet was travelling. Small amounts of blood may break away from the parent stain entirely – these are known as satellite stains.

Although it may be possible to estimate area of origin purely through visual observation of bloodstain patterns, in some instances trigonometry may be utilised to determine a more precise point of origin. Depending on the type of bloodstain pattern, it may be possible to establish the angle at which a blood droplet hit a target, referred to as the angle of impact. By measuring the ratio of the width of the bloodstain to the length, it can be possible to calculate the angle of impact. If the angle of impact of multiple bloodstains is established, it may be possible to determine the area of convergence (the point where lines of travel from multiple stains meet) through stringing techniques and establish the area of origin.

Documentation and Collection
Documentation of bloodstain evidence will most typically be carried out using photography, including photographs of the wider scene along with close-up images of particular bloodstains. A ruler or other form of scale may be placed in the photograph in order to give perspective as to the size of a bloodstain. Sketches and even videos may also be utilised for further documentation. Collection of bloodstain evidence can be a complex matter, as the evidence will not likely be confined to a small object that can be easily removed from the scene. After rigorous documentation of the evidence, ideally the bloodstains themselves will be collected. This can involve simply removing objects from the scene or, more problematically, sections of carpet or large pieces of furniture. Evidence removed should be packaged in such a way that the stains are not altered or damaged. Collection of blood evidence for the purpose of DNA profiling will generally be conducted using a swab.

 

Jackson, A. R. W, Jackson, J. M., 2011. Forensic Science. Essex: Pearson Education Limited.

Scientific Working Group on Bloodstain Pattern Analysis. [online] Available at: [http://www.swgstain.org]

White, P. C., 2004. Crime Scene to Court: The Essentials of Forensic Science. Cambridge: The Royal Society of Chemistry.

Forensic anthropology refers to a specialised branch of physical anthropology particularly applied to medico-legal matters. When dealing with a set of human remains, a primary fact to ascertain is the identity of the individual and how they may have died, which is understandably not straightforward if all that remains of a body is the skeleton.

Through the study of bones, an array of information can be ascertained regarding the remains including, but by no means limited to, age, gender, ethnicity, cause of death, and even indications of lifestyle such as where a person might have lived. The adult human skeleton consists of some 206 individual bones, with there being even more in the skeleton of a child, whose bones have not undergone certain fusion processes yet, and many of these bones may prove useful to the anthropologist.

Bones develop from cells known as osteoblasts, first beginning as soft cartilage before the bone hardens through the introduction of various minerals, a process known as ossification. Bones can be divided into a number of classes; short, long, flat, sesamoid and irregular bones (Gunn, A, 2009). Short bones, such as the carpal bones within the wrist, tend to be as wide as they are long. Long bones are, as the name suggests, longer in length and also tend to be slightly curved, for example the femur. Flat bones, such as the ribs and breastbone, could be described as being fairly flat and plate-like. Sesamoid bones refer to small bones embedded in a tendon, often found in joints, such as in the knees and wrists. Finally, irregular bones refer to a certain class of bone that do not belong in the other categories, such as the bones composing the spine.

Species Determination
It is initially essential to establish whether recovered bones are of human or animal origin. Whereas the answer to the question may be obvious when a full set of skeletonised remains are present, a great deal more expertise is needed when only a few or even a single bone is found. The general shape, size and structure of the bones may be sufficient to determine likely species, and methods for distinguishing between human and non-human remains have also been established based on microscopic differences in bone structure (Urbanova, P et al, 2005). If the bones are relatively recent they may contain the proteins required to carry out serological tests to establish the species.

Sex
Establishing the sex of skeletonised human remains is not generally too difficult, as there are a number of morphological differences between the skeletons of males and females. If the remains have not reached the latter stages of decomposition, some indicators of sex may still be present in the softer tissues. For instance, the prostate gland in males and the uterus in females do not decay until later than other soft tissues. Should the bones be all that remains, perhaps the most significant indicator of sex is the pelvis. In a female the pelvis presents a U-shaped sub pubic arch, as oppose to the V-shape found in a male pelvis. As would be expected, the female’s pelvis is also generally more spacious to allow for childrearing, with a wider sub-pubic angle and sciatic notch. Furthermore, examination of a female’s pelvis may even indicate whether or not she has previously given birth, offering a further detail for identification purposes. Through examination of the skull it may also be possible to determine the likely sex of the remains, with the skull of a male tending to display a larger, squarer and more pronounced jaw, a more prominent supraorbital ridge (brow), and more rectangular eye sockets (orbits). Though not an infallible means of sex determination, the general size of bones can provide some indication as to whether the remains belong to a male or female. As the muscles in a male tend to be larger and better developed, the bones are generally larger and to an extent more robust than those of a female. However it should be noted that establishing sex based on the human skeleton is often challenging when dealing with the remains of pre-pubescent children, as certain indicators, such as the widening of the hips in a female, may not have occurred until puberty. Furthermore, the natural sex of an individual may not be consistent with the gender of that individual (for instance, a person designated female at birth may be living as a male), hindering the identification process.

Ethnicity
Establishing the ethnicity of an individual is generally carried out by studying the skull, which is typically classed as belonging to a Caucasoid (or Caucasion), Negroid or Mongoloid. The cranium itself is typically long, narrow and high in Caucasians, similar in Negroids but lower, and more rounded in individuals of Mongoloid ancestry. The size of the nasal opening may be used as an indicator for ethnicity, with the nasal cavity of a Caucasian being narrower and higher in comparison to the broader opening belonging to an individual of Negroid origin, and Monogloids sitting somewhat in between. The eye orbits can also provide clues as to the possible ethnicity, with individuals of Caucasian ancestry tending to have sloped orbits, as oppose to Negroids who generally possess more rectangular eye orbits and people of Mongoloid ancestry with rounder orbits. The mastoid process, which refers to a particular part of the skull just behind the ear, tends to appear as a wider projection in Negroids whereas this is often more pointed and narrow in Caucasian individuals. The teeth may also prove beneficial to a certain extent, with individuals of a Mongoloid ethnicity tending to have upper incisors that could be described as ‘shovel-shaped’ with a curved inner surface, as oppose to the more flat surface found in the teeth of Negroids and Caucasians. An additional observation to note relating to teeth, Caucasians tend to have smaller teeth and more overcrowding, commonly resulting in impacting third molars that must be removed. However the determination of ethnicity is certainly not straightforward, with the occurrence of ‘racial hybridity’ being a problem in establishing ethnicity of an unknown individual. This is the result of breeding between different racial groups, resulting in individuals possessing features that are typical of two or more racial groups.

Age
Once a body has decayed to such an extent that only bones remain, the estimation of age may not be as obvious as if soft tissues were present. Fortunately, there are many indicators in the skeleton that can be used to establish the likely age of the victim at the time of death. As previously mentioned, the skeleton of a younger individual is composed of significantly more than the 206 bones found in an adult. This is because as the human skeleton develops, a process known as ossification occurs in certain areas, which is essentially the fusion of bones. The appearance of certain ossification centres can estimate the age of a younger individual, though this becomes decreasingly beneficial as an individual ages. In the human skull there is a series of zigzag lines known as sutures which separate the plates of the skull. Over time these sutures harden and become less distinct. The epiphyseal fusion of bones (the fusion of the shaft of a bone to the end of the bone) can equally act as an age indicator. In addition to this, the study of tooth eruption can also be a useful indicator of the age of an individual, though from approximately the mid-twenties this is not necessarily valuable. Once an individual has reached a particular point in adulthood, generally around the mid-20s, accurately determining age becomes difficult, because tooth eruption has generally reached a stage of completion as has the fusion of bones. Depending on the age of the individual, there may be some signs of age-related conditions such as osteoporosis or arthritis, though once again there are inaccuracies to take into account as not all older people will develop these conditions and furthermore some younger people may be susceptible to them.

Age of Remains
Establishing the age of a set of remains, as in how long ago the individual died, is often a difficult task. With more recent sets of remains, there may still be some tissues present on the body to help pinpoint the age of the body. Certain soft tissues and ligaments can last for up to 5 years, so the presence of these may at least be able to narrow down time since death to the last few years.

Typically, isotope analysis can prove to be particularly beneficial in establishing the likely age of remains. Isotopes are atoms of the same element, and with the same chemical properties, but differing in the number of neutrons within the nucleus (and thus have a slightly different atomic mass). Among the most common elements to be studied in isotope analysis are carbon, nitrogen, oxygen, strontium and hydrogen. This branch of study, which can be focussed upon unstable or stable isotopes, is based on the principle that many elements within the body exist as various isotopes, many of which are taken into the body by eating, for instance. Bones and teeth are usually subjected to isotopic analysis in cases of dating skeletonised human remains, though if present hair and nails may also be used.

The isotopic analysis of stable isotopes, such as carbon-14, is beneficial in estimating time since death. Whilst an organism is living it is constantly taking in carbon, for instance through the ingestion of other organisms containing the isotope. However upon death, it ceases acquiring new ¹⁴C and so levels of ¹⁴C begin to decline. It is thus possible to establish a likely time period since death by measuring the quantities of ¹⁴C present in the remains. Despite the benefits of this technique, it is not as accurate as would be ideal, and establishing time since death using isotope analysis can only allow for an estimation. ¹⁴C dating is also only ideal for older remains (older than at least 100 years), as the technique is simply not accurate for younger remains. Due to this fact, the study of the ¹⁴C isotope is not necessarily of the greatest benefit to forensic science as other isotopes may be.

For example, the analysis of the lead-210 isotope (²¹⁰Pb) is perhaps of more use to the forensic scientist due to its shorter half-life of 22.3 years. As with the ¹⁴C isotope, ²¹⁰Pb is absorbed by the body through the ingestion of food and, upon death and thus the halt of food intake, the isotope will begin to decay. By measuring the amount of this isotope in a set of remains in comparison to the levels of the isotope seen in the body of a living person, it is possible to determine the likely time elapsed since death. Similarly, research is being conducted into the analysis of a polonium isotope (²¹⁰Po), which has a significantly shorter half-life of only 138 days and thus has the potential to pinpoint time since death with greater accuracy.

However it must be taken into account that exposure to certain conditions can affect the isotopes present in a sample and the ratios of these isotopes. Furthermore, isotopes are ubiquitous in the environment thus it is entirely possible for detected isotopes in a sample to actually be the result of contamination.

Lifestyle
As previously discussed, the study of isotopes making up a set of remains can be used to provide various forms of information, such as the likely age of the remains. Another particularly beneficial use of isotopic analysis is to determine the possible geographical origin of remains (known as the provenance) through the study of stable isotopes, often through the use of stable isotope ratio mass spectrometry (SIRMS). The study of various elements such as hydrogen, carbon, oxygen, nitrogen and strontium can theoretically be used to trace an individual across various locations throughout their life.

Different geographical locations are known to have different, often distinct isotope ratios. The varying isotope ratios in different areas occurs due to a process known as isotopic fractionation, in which certain isotopes become enriched over others. The elements studied in this instance exist in different isotopic forms – for instance, strontium exists as four stable isotopes; ⁸⁴Sr, ⁸⁶Sr, ⁸⁷Sr and ⁸⁸Sr. Because their ratios vary between different geographical locations, thus people and animals will ingest food and water from those locations, the ratio of isotopes measured in a sample can act as a kind of ‘fingerprint’, indicating the likely location in which a person had lived. Furthermore, because isotopes are taken into the body at different points throughout an individual’s life, isotope ratios can also infer certain changes in a person’s location throughout his or her life.

Stature
In order to estimate the height of an individual from the complete skeleton, direct measurements can be taken and thus height determination is usually relatively straight forward, provided the presence of tissue prior to decomposition is taken into account. When dealing with an incomplete skeleton or only certain individual bones, estimating the height of an individual may still be possible. By measuring the length of the long bones, ideally the femur, fibula and tibia in the leg, it is possible to estimate height based on stature tables. These tables take into account the race, sex and age of the individual along with the measurement of the long bone. Determination of an individual’s stature based on a skeleton or bones will only ever be an estimation, as it is not always possible to accurately take into account the changes soft tissue would have had on the individual’s height in life.

Injuries and Cause of Death
By X-raying skeletal remains, it may be possible to obtain information that could lead to both establishing a cause of death and even identifying an individual. There may be evidence of injuries obtained earlier in life that have left noticeable markings on the bones, for instance fractures and breakages or even the presence of artificial bones. Furthermore, there may be evidence of bone disease such as osteoporosis. Studying the teeth of the remains may provide important clues, particularly if the individual had any distinguishing dental features or dental work such as fillings carried out. All of this information may be compared to the medical records of known individuals to aid in confirming or disputing the identity of the skeletonised remains. Dental records in particular often prove beneficial in identifying an individual who cannot be identified by any other means, providing they have had dental work carried out and have some dental records stored somewhere.

DNA
Unsurprisingly, bone contains very little nuclear DNA, thus typical DNA profiling methods are not likely to be possible. However bone does contain mitochondrial DNA, which typically persists for longer than nuclear DNA. Mitochondrial DNA is solely inherited from the maternal bloodline, thus it does not contain any genetic information from the individual’s father. If mitochondrial DNA can be successfully extracted and analysed, it may be possible to compare it with living maternal relatives to aid in identification.

Facial Reconstruction
Provided the skull is in a reasonable state, it may be possible to reconstruct the face of an individual based on the skull by various available methods to aid investigation of an individual. The process of photosuperimposition is fairly rudimentary. A photograph of the skull is taken and a photograph of an individual in life overlaid to determine whether the features of the individual match those of the skull. This technique is not always ideal, as the photograph of the individual must be altered in size to match that of the skull, which may not always be accurately possible. A little more complex, facial reconstruction essentially involves rebuilding the likely facial features of an individual using a cast of the skull as a baseline. The technique generally utilises facial markers placed in specific locations on the skull and modelling clay, which is intricately applied to the surface of the skull to the required depths to simulate facial tissue, smoothed and coloured to resemble skin. Further additions such as the nose, ears and lips will then be constructed. Following this additional features such as prosthetic hair and eyes can be added in attempts to reconstruct the facial features of the individual. Of course when dealing with an entirely unidentified victim certain presumptions must be made, such as the colour of a person’s hair and eyes or the presence of facial hair. Even the depth of tissues on an individual’s face can only be estimated to a certain extent, as the underlying bone structure will not indicate this. The technique of facial reconstruction may also be carried out using sophisticated computer software if available. Upon completion of any forms of facial reconstruction, the image produced can be distributed as necessary to aid identification.

Decomposition
Please see the human decomposition page for more details.

 

Beard, B. L. et al. Strontium isotope composition of skeletal material can determine the birth place and geographic mobility of humans and animals. J Forensic Sci. 45(2000), pp1049-61.

Gunn, A (2009). Essential Forensic Biology. Oxford: John Wiley & Sons.

Forensic Magazine. Tracing Unidentified Skeletons Using Stable Isotopes. [online][Accessed 13 Jan 2015] Available: http://www.forensicmag.com/articles/2007/01/tracing-unidentified-skeletons-using-stable-isotopes

The University of Western Ontario Journal of Anthropology. Racial Identification in the Skull and Teeth. [online][Accessed 20 Feb 2015] Available:http://ir.lib.uwo.ca/cgi/viewcontent.cgi?article=1137&context=totem

Urbanova, P et al. Distinguishing between human and non-human bones: histometric method for forensic anthropology. Anthropologie. 43(2005), pp. 77-85.