The Physical and Biological Aspects of Forensic Science

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BIOLOGY:
Development of Forensic Science:
Forensic science refers to the application of science to the law or criminal investigations. That is, scientific knowledge and technology is applied to the creation and enforcement of laws. Famous pioneers in the field of forensic science include persons such as Leone Lattes (1887 – 1954) and Edmond Locard (1877 – 1966). Lattes developed a procedure which enabled the determination of blood group in dried bloodstains, while Locard postulated Locard’s Exchange Principle. This principle states that once items come in contact with each other, some exchange will occur and a trace will be left behind.

The Crime Scene:
The crime scene refers to the location at which a crime has occurred. It must be managed properly in order to maximize the potential for evidence which would help to solve the crime. This management includes cordoning off the area and ensuring that the appropriate specialists, such as forensic scientists or pathologists, are called. In addition, a log book must be kept at the crime scene which indicates the persons who have entered and left the scene. This helps to differentiate between traces left behind by investigators or other persons entering the scene and evidence left behind as a result of the crime itself. All evidence collected must be properly labelled, documented and recorded and photos or sketches of the crime scene should be made. Documentation of evidence is essential as it is useless to analyze a piece of evidence and then not be able to identify its origin. The photos or sketches are useful as they can be used for reference after the crime scene has been cleared. The chain of custody begins at the crime scene at the point of collection. Physical evidence is any object which establishes that a crime has been committed or which links the crime to a victim or perpetrator. There are several types of crime scenes and correspondingly several types of physical evidence. The types of crimes include sexual assault, murder, drug raids, arson, fraud and robbery. The types of evidence collected in relation to these crimes include hair, semen, blood, fibres, fingerprints, narcotics, empty gasoline bottles, documents and tools.

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Bloodstain Pattern Analysis:
Bloodstain pattern analysis refers to the examination of shapes, locations and patterns of bloodstains in an attempt to provide an explanation of the physical events which caused the stains. This analysis is based on the premise that the patterns are characteristic of the forces that created them. There are several classifications of bloodstain patterns. The first type is passive, and this refers to for example a drop of blood. These types of bloodstains occurred without any external force, except gravity, acting on them. The second type is transfer bloodstain patterns. These occur when a wet, bloody surface comes in contact with a secondary surface, and may leave a resulting imprint of the object. This group can be further subdivided into contact bleeding, swipe/smear, wipe and smudge, each of which usually occur due to different forms of contact and can be used to corroborate or contradict statements given by the accused and the alleged victim. The third type is cast-off stains which result from a released or thrown blood-bearing object in motion. The fourth type is arterial spurt or gush, which results from blood exiting the body under pressure from an artery. The final type is a projected pattern, which occurs when the blood source is impacted by some external force greater than gravity. This force may be either internal or external and the pattern seen depends on the amount of force which was exerted. Medium velocity forces of 5 to 25 feet/sec usually result in preponderant stain sizes of 1 – 4 mm, whereas high velocity forces such as approximately 100 feet/sec would result in preponderant stain size usually of less than or equal to 1 mm. High velocity forces occur for example in the case of gunshots. Altered patterns may also be seen which are physically or physiologically changed. These changes may arise from blood clots, diluted stains (such as if someone was trying to clean up the blood), insect activity or as a result of obstruction by an object.

Four P’s of Evidence Handling:
These factors are important in order to maintain the integrity and value of the evidence collected. These factors are:
Protection = avoid contamination of the evidence
Procurement = ensure that the collected evidence coincides with the documentation
Package = packaging methods should be chosen so as to ensure that testing is not impeded or prevented
Preserve = ensure that the evidence is properly stored

Blood:
Preliminary examination for blood first involves screening using the Kastle Myer test. This is based on the property of haemoglobin which enables it to accelerate the oxidation of phenolphthalein, in a peroxidise-like manner, such that there is a colour change from colourless to pink. In cases where the Kastle Myer test gives a negative result, but the presence of blood is thought to be likely the luminol test is employed. This test is not the first choice test since it degrades the DNA present, but it is very sensitive and can detect tiny traces of blood up to 300,000 times diluted. The luminol reacts with haemoglobin to produce molecules which emit light.
After a positive preliminary test confirmation testing must be done. This involves determining the species of origin of the blood in question, which can be done using the precipitin test. The precipitin test involves an immuno-electrophoresis reaction using an antigen-antibody reaction. The antibody may be an anti-human serum or may be for other animals which could possibly have been present. In a positive reaction a precipitate is produced.

Weapons:
Weapons found at a crime scene must be handled in the same manner as all forms of evidence collected. Some weapons may have biological evidence such as bloodstains which can be analyzed while others are sent straight to the Ballistics Department. Common weapons include guns, knives, cutlasses, hammers and blunt objects such as metal pipes.

Semen:
Evidence which is thought to have semen present must first go through preliminary examination. The first test which is done is usually the acid phosphatase test. Acid phosphatase is an enzyme which is found in high concentration in semen but is also found in low concentration in vaginal secretions. The test is based on sodium alpha-napthyl phosphate and fast blue dye, which turns purple in the presence of acid phosphatase.

Swab and Smear:
Swabs and smears are used to facilitate microscopic examination, often especially in cases of sexual assault. In most cases the doctor who examines the victim takes swabs of specific areas including vaginal, anal, penile and oral/buccal, depending on the case. For some cases the oral swab can be used for comparison. Swabs are usually examined for the presence of spermatozoa. On the other hand smears can be examined for the presence of spermatozoa, red blood cells, white blood cells, Trichomonas vaginalis and N. gonococci.

Other Body Fluids:
Other body fluids can be tested as evidence and these include sweat, urine (based upon the presence of creatine), saliva or faecal tests. The urine test is based on identifying the presence of creatine through the use of pictric acid and sodium hydroxide which forms a brown-orange colour when positive.

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Hair and Fibres:
Hair samples can be examined and used to give an indication of the race, type of animal and whether natural or synthetic. In cases where the root is present on the hair then DNA analysis can also be carried out.
Fibre samples are analyzed to see if they are natural or synthetic (and the class of synthetic fibre), used for colour comparison and used for texture comparison with statements given.

DNA:
DNA contains all the genetic information of the cell and is made up of genes and alleles, which are variations of genes. The DNA is tightly coiled into chromosomes which vary in number from one organism to another. DNA can be used for several applications including forensic investigations, paternity testing, identification of missing persons or catastrophe victims and lineage studies. Humans have 22 pairs of autosomal chromosomes and one pair of sex chromosomes. DNA also contains short tandem repeats (STR) which vary in length and number from one person to another. These STR markers are therefore useful in DNA analysis and currently eight STR markers, each with a variety of sizes or alleles, are being used for analysis. For example, TPOX is an STR found on chromosome 5 and its repeat unit is AATG and this unit can be repeated between 6-13 times. The STR may be repeated six times o one homologous chromosome and eight times on the (one from each parent) but it is still the same gene on both chromosomes, only the STR size is different. In this example the genotype for TPOX would be “8,6”. When the DNA is separated by gel electrophoresis the larger sizes move slower on the gel. Sources of biological evidence include blood, semen, urine, hair, fingernails, tissue, saliva, teeth and organs. The processes involved in DNA analysis are extraction, quantification, PCR and STR typing. DNA extraction can be done using several methods.

DNA Extraction:
Firstly, the Chelex (inorganic) method can be used. It involves the use of PBS (phosphate buffer solution) to free the cells from the sample. The sample is then boiled in a solution containing chelating resin beads that bind metal ions such as magnesium which causes the cells to lyse and release the DNA. This is not a very efficient method as it doesn’t produce much DNA. The second method is the organic method which uses phenol chloroform iso-amyl (PCI). PCI precipitates proteins, fats and extraneous materials thus leaving only the DNA in solution. Alcohol is then used to clean and precipitate the DNA, thereby increasing its purity. Thirdly, the DNA IQ (Promega) method can be used. A lysis buffer containing a salt (guanidine thiocyanate – GTC) is used which causes the DNA to stick to the paramagnetic resin. Following this a wash is done with low salt or alcohol based wash buffer and the DNA is isolated. Finally, the Maxwell 16 method can be used which uses pre-dispensed reagent cartridges and has moderate throughput. The inorganic and organic methods are older and the DNA IQ and Maxwell 16 methods have now been developed for several reasons. The older methods involved procedures such as soaking samples, which results in inefficient DNA recovery and may cause significant loss of DNA. The older methods also can introduce amplification inhibitors and in addition the chemical PCI is hazardous. The newer methods allow for DNA extraction without these problems.

DNA Quantification:
DNA quantification is used to determine the amount of amplifiable DNA. 0.1 to 0.3 ng of DNA is required to get accurate results in the analysis. A kit called the Plexor HY from Promega is used with Corbett Rotor Gene 6000 Real-Time PCR. During the PCR, as DNA is amplified the fluorescence decreases. The reporter used is iso-dC and the free nucleotide dabcyl-iso-dGTP binds to the reporter at the end of amplification. This is known as fluorescence quenching.

PCR:
PCR is used to copy target STRs through thermal cycling. The components used for PCR are nuclease-free water, STR buffer containing dNTPs and Mg2+, primers and Taq DNA polymerase. The primers and Taq DNA polymerase enable stringent copying of only STR sequences. The primers are tagged and have an affinity for the markers. They are tagged by two fluorophores, namely tetramethylrhodamine (TMR) which is red and fluoroscein (FL) which is green. Four of the eight STRs are complementary to the primers with TMR, while the other four are complementary to the primers with FL. These tags aid in band visualization after electrophoresis.

STR Typing:
STR typing is mainly done using two methods. Firstly, polyacrylamide gel electrophoresis method can be used. In this method the sample must be denatured and mixed with a loading dye (often bromophenol blue) which keeps the sample in the well by making it heavier. The samples can then be loaded and the electrophoresis is carried out followed by scanning and examination of the gel. In this method another fluorophore called carboxy -X- rhodamine (CXR) is used to tag the internal lane standard and is added to each well. The FMBIO machine is then used which recognizes the different wavelengths of the fluorophore tags and converts this data into digital data and visualizes the colours. It is important to use these fluorophores since some of the STRs will be the same size and therefore will move the same distance on the gel. Without the colour difference it would not be possible to identify them as separate STRs. The four which are tagged green are of varying sizes and so they will not overlap with each other. The same is true for the four which are tagged red. After the visualization is done the genotype must be derived from the images. This is done by identifying the two alleles of each of the eight STRs and the resulting data would be as follows, for example for CSF – 8,6.
The second method used is the ABI Genetic Analyzer 3100. In this method the amplified DNA fragments are loaded into the machine which has 16 silica capillaries which enable the separation of the fragments by electrophoresis.

Y-STRs
The Y chromosome is found only in males and is therefore passed on from father to son. Due to this fact the Y chromosome can offer particular information which is useful to several applications including genetic genealogy studies. Analysis of Y-STRs is especially useful in sexual assault cases which do not have clear results. Since only the Y-STRs will be tested for, the female victim’s profile will not interfere with the analysis. Y-STRs are smaller than the other STRs and will remain in tact even in highly degraded samples and 9 core Y-STRs are used in a reference database. The advantages of using these are that it makes it easier to exclude suspects and easier mixture analysis. However, the disadvantage to using this method is that it is not easy to include someone since the Y chromosome is the same in each generation.

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