Degradation of Blood Sourced Dna on Knives

Degradation of Blood Sourced Dna on Knives Persistence of DNA: An examination of degradation of blood sourced DNA on knives, by household substances and the forensic implications A  review of relevant and current peer-reviewed literature Contents (Jump to) Incidence of homicides Knife crime Forensic presumptive tests Phenolphthalein and Leucomalachite False positives Luminol Luminol variations False positives DNA qPCR Singleplex Multiplex RFLP STRs LCN Advancing techniques Forensic Markers General Household Cleaners Bleaches Detergent All-purpose cleaner Anti-Bacterial disinfectant Similar studies Research project outline References Figures Figure 1. Incidence of homicide victims and accused 2003-2013 Tables Table 1.  Homicide figures and methods of killing from 2003-2013 Table 2.  Homicide methods in Lothian and Borders 2006-11 Abbreviations CODIS – Combined DNA index system DNA – Deoxyribonucleic acid LCN Low copy number LMG – Leucomalachite green PCR – Polymerase chain reaction qPCR – Real time Polymerase chain reaction RFLP –Restriction fragment length polymorphisns SGM – Second generation multiplex STR – Short tandem repeat Often after an assault with a weapon, i.e. a stabbing or murder, attempts are made to clean and or dispose of the weapon used. The most readily available weapon is a knife, whether the attack is pre-planned or ‘spur of the moment’, knifes are easily available to buy and are also, part and parcel with every household i.e. kitchen. Incidence of homicides Incidences of homicide in Scotland are at the lowest point for ten years, having dropped from 109 in 2003 to 62 in 2013, as seen in Figure 1. Fig 1. Incidence of homicide victims and accused 2003-2013 (ScotGov, 2013) Table 1. Homicide figures and methods of killing from 2003-2013 (ScotGov, 2013) Table 1 shows that homicide using a sharp instrument is the most common method, at the highest point in 2010-11 was 61% and at the lowest point in 2005-06 was 35.79%. Knife crime Table 2 shows that more than half (58.2%) of the murders committed in Lothian and borders between 2006 and 2011 used a kitchen knife. Table 2. Homicide methods in Lothian and Borders 2006-11, Adapted from (Kidd, Hughes and Crichton, 2013) Forensic presumptive tests Forensic presumptive tests can be used at scenes of crimes for various reasons. They can test to see what a substance might be i.e. drugs, blood. There are various reagents available for use in the presumptive testing for the presence of blood at a crime scene, using varied types of reactions. Phenolphthalein and Leucomalachite Phenolphthalein is the main reagent used in the Kastle-Meyer presumptive test for blood. This test is mainly used one unidentifiable stains, therefore this is used upon visible (patent) samples. The Kastle-Meyer test is a catalytic method, the phenolphthalein will cause an alkaline solution to turn pink after its oxidation by peroxide when blood is present. According to (Johnston et al., 2008), it will detect blood as dilute as 1 part in 10,000. False positives There are several false positives for the test and these include according to (Virkler and Lednev, 2009) chemical oxidants and fruit and may also include vegetable peroxidases. (Garofano et al., 2006), show that the Kastle-Meyer, Phenolphthalein, test is not as sensitive to blood as Luminol, which is stated as detectable to 1 part in 10,000,000. Leucomalachite Green or LMG is another widely used catalytic method to presumptive test for blood. It works by the same principle as Kastle-Meyer with the exception of the stain being gently rubbed with filter paper containing the reagent. After no colour has developed hydrogen peroxide is again added as in Kastle-Meyer and a green colour change in this case is in indicative of the presence of Blood. Johnston et al, also state that Leucomalachite green has a sensitivity similar to that of Phenolphthalein, 1 part in 10,000. Luminol Luminol is a forensic presumptive test for latent blood, i.e. blood which cannot be seen. Crime scenes are often cleaned afterwards by the perpetrator in an attempt to hide any evidence of what had occurred. Luminol allows crime scene investigators to see the full picture at a cleaned crime scene. Webb et al, 2006, State that Luminol is known as the most sensitive of the presumptive tests currently used at crime scenes. Luminol variations According to (Patel and Hopwood, 2013) There are two more commonly used Luminol formulations. These formulations were developed by Grodsky in 1951, Luminol I, and Weber in 1961, Luminol II. Luminol I or the Grodsky formulation uses a base of sodium carbonate and sodium perborate as the oxidising agent. However Luminol II or the Weber formulation uses a base of sodium hydroxide and hydrogen peroxide as its oxidising agent. Commercial luminol products have become more readily available in the recent years and brands such as BlueStar and BlueStar Magnum and Lumiscene, have come onto the market. These products offer the advantage of being easier to prepare than the more traditional formulations. However according to (Quinones et al., 2007) The Grodsky formulation of Luminol I can have a detrimental effect on the ability to subsequently perform DNA analysis in comparison to the Weber and BlueStar alternatives. False positives Luminol’s sensitivity may be an advantage at finding the smallest drop of blood, but it is also its downfall. DNA DNA is without doubt the greatest scientific discovery of forensic use to date. PCR Akane et al., 1994 investigated the role haem from blood played in interfering with DNA and in inhibiting the polymerase chain reaction. This early study suggested that a haem-blood protein complex caused inhibition issues to the polymerase chain reaction and forwarded the investigations into a more reliable PCR technique. qPCR qPCR or real time polymerase chain reaction, RFLP RFLP or Restriction Fragment Length Polymorphisms STRs STRs or Short tandem repeats, are genetic markers which were first discovered in the early 1990’s. (Edwards et al., 1992) Showed that STRs were an effective tool when used for human identity testing. They showed that there were enough variation through alleles for individuals to be positively identified. STR profiling works by comparing sizes of the sample DNA STRs with standardized databased allelic ladders. New alleles are still being discovered with variations in size which may not be found in commercially available ladders. LCN (Gill et al., 2000) explain that LCN or low copy number, in a PCR amplification technique which can be applied to DNA samples with as little as Kloosterman Kersbergen, 2003, explain that ‘28 + 6’ improved efficacy of DNA sample genotyping. Their 34 cycle PCR technique instead of the normal 28 PCR cycles offered an alternative approach to genotyping forensic DNA samples, which are perhaps low quality or degraded. Low copy number analysis of DNA is not without problems. The most common issues with LCN are allelic drop-out, heterozygote imbalance, stutter peaks/products and unexpected allelic peaks. Allelic drop-out Stutter products Forensic Markers In the US a system known as CODIS is used for DNA profiling. CODIS uses 13 loci and Amelogenin. The loci used are CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. The DNA profiling system used in the UK is SGM Plus (SGM+). This system looks at 11 different loci on different chromosomes (independent inheritance) with a large number of alleles. As well as looking at Amelogenin, the sex marker, the SGM+ includes the markers D2S1338 and D19S433 along with eight CODIS overlapping loci FGA, TH01, VWA, D3S1358, D8S1179, D16S539, D18S51, and D21S11. (Cotton et al., 2000) Validated the most recent 11 loci STR DNA analysis method for use in standard forensic casework. The technique was also validated for use in casework involving > 1ng of DNA, i.e. LCN. HUMVWFA31/A (vWA) HUMTHO1 (THO1) HUMFIBRA (FGA) General Household Cleaners If you have committed a crime, disposing or destroying of the evidence is more than likely your next step. More often than not this is problem faced by forensic scientists, that a deliberate attempt to remove any of the biological material i.e. evidence, blood, using a variety of cleaning materials. Most people store their cleaning supplies in their kitchen or bathroom, so general household cleaners are to be examined. Every household will contain basic cleaning materials such as bleaches, detergents, disinfectants and perhaps multi-cleaners. Cleaning materials not only have the ability to potentially cause contamination to of any of the surviving evidence but also to degrade any DNA which may still be present on the evidence. Degradation of the DNA will make it difficult to produce and gain a profile which could be used to link the weapon to the crime. Bleaches Bleaches can cause many problems at crime scenes, thanks to the American television show Crime Scene Investigation (CSI Las Vegas) most people will know that bleach renders the forensic presumptive blood tests pretty much useless. According to (Harris et al., 2006), Out of all the cleaning products on the market bleach also has the most harmful effect on the quality of DNA available to obtain a profile. They also state that bleach seemed to cause continued degradation of the DNA over time. Common brands of household bleaches include Domestos, Harpic, Mr Muscle, Cif and Supermarket own brands. Detergent Common brands of household detergents include Fairy, Imperial leather, Carex, Daz and Bold. Anti-Bacterial disinfectant Common brands of household anti-bacterial disinfectant include Dettol, Savlon, TCP and supermarket own brands. Similar studies Research project outline This study aims to investigate whether the knife substrate has any effect on the quantity of DNA retained on the weapon after an assault; whether deliberate attempts to remove any biological material, using a variety of household cleaning materials, affect the quality and quantity of DNA that is recoverable and if DNA is recovered from a weapon, whether it was initially detected by presumptive blood tests. The knife substrates used here were, 18† Kitchen knife and 20† Serrated kitchen knife. The household cleaning materials were as follows, Bleach (Domestos), Detergent (Fairy) and Anti-Bacterial disinfectant (Dettol). Blood samples are to be applied to the blade of each knife, it should be allowed to air dry and then cleaned until no blood or residues thereof are visible. Each knife should then again be allowed to air dry and be swabbed using a double swab technique. Each cleaning material is applied to the two different knife substrates and the pairs are numbered 3-18, with knife set 1 2 being control, the initial DNA for comparison should be swabbed before the control knifes are dried and washed with warm water only. Cleaning materials should be used according to the manufacturers’ guidelines. PCR will be performed on the extracted samples using The control samples will be used to examine which was the largest contributing factor in compromising the quality and/or quantity of the sample gained. References The Scottish Government, (2013) Statistical Bulletin, Homicide in Scotland 2012-13, {Online} Available: https://alpha.scotland.gov.uk/government/uploads/system/uploads/attachment_data/file/417/Homicide_in_Scotland_2012-13_statistical_bulletin.pdf [Accessed: 02/10/2014] Cotton, E. A., Allsop, R. F., Guest, J. L., Frazier, R. R., Koumi, P., Callow, I. P., Seager, A., and Sparkes, R. L. (2000) Validation of the AMPFlSTR SGM plus system for use in forensic casework. Forensic Sci Int. Vol.112(2-3), pp.151-61. Edwards, A., Hammond, H. A., Jin, L., Caskey, C. T., and Chakraborty, R. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics. Vol.12(2), pp.241-53. Garofano, L., Pizzamiglio, M., Marino, A., Brighenti, A., and Romani, F. (2006) A comparative study of the sensitivity and specifity of luminal and fluorescein on diluted and aged bloodstains and subsequent STRs typing. International Congress Series. Vol.1288(0), pp.657-659. Gill, P., Whitaker, J., Flaxman, C., Brown, N., and Buckleton, J. (2000) An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Sci Int. Vol.112(1), pp.17-40. Harris, K. A., Thacker, C. R., Ballard, D., and Court, D. S. (2006) The effect of cleaning agents on the DNA analysis of blood stains deposited on different substrates. International Congress Series. Vol.1288(0), pp.589-591. Johnston, E., Ames, C. E., Dagnall, K. E., Foster, J., and Daniel, B. E. (2008) Comparison of presumptive blood test kits including hexagon OBTI. J Forensic Sci. Vol.53(3), pp.687-9. Kidd, S., Hughes, N., and Crichton, J. (2013) Kitchen knives and homicide: A systematic study of people charged with murder in the Lothian and Borders region of Scotland. Med Sci Law. Vol.54(3), pp.167-173. Patel, G., and Hopwood, A. (2013) An evaluation of luminol formulations and their effect on DNA profiling. Int J Legal Med. Vol.127(4), pp.723-9. Quinones, I., Sheppard, D., Harbison, S., and Elliot, D. (2007) Comparative Analysis of Luminol Formulations. Canadian Society of Forensic Science Journal. Vol.40(2), pp.53-63. Virkler, K., and Lednev, I. K. (2009) Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Science International. Vol.188(1–3), pp.1-17.

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