INVESTIGATION OF THE EFFECT OF TIME AND SURFACE TYPE ON AMPLICON CONTAMINATION OF DNA PROFILES

Transcription

1 INVESTIGATION OF THE EFFECT OF TIME AND SURFACE TYPE ON AMPLICON CONTAMINATION OF DNA PROFILES Arwa Al Hanbali (BSc Agr, GradDip App Mol Biol) Centre for Forensic Science University of Western Australia This thesis is presented in partial fulfilment of the requirements for the Master of Forensic Science 2010

2 DECLARATION I declare that the research presented in this 48-point thesis, as part of the 96- point Master degree in Forensic Science, at the University of Western Australia, is my own work. The results of the work have not been submitted for assessment, in full or part, within any other tertiary institute, except where due acknowledgement has been made in the text... Arwa H. Al Hanbali Winthrop Professor Ian Dadour (Supervisor) Dr David Berryman (Secondary Supervisor) 2

3 ACKNOWLEDGMENTS I would like to thank Dr David Berryman and Professor Ian Dadour, for their endless guidance over the past year. I would like to extend my gratitude to Professor Mike Jones, for supporting me and offering invaluable advice when I needed it. Many thanks goes to my mother for her support and endless words of encouragement from the time that I decided to go back to study, and to my deceased father, who has always been my role model and guiding light towards achieving my goal. My utmost appreciation also goes to my beloved family and friends in Perth; without their support and constant help in taking care of my children, I would not have been able to finish my study. Especially to my husband Zaid for supporting me and my children, Rabea, Rasha, Rania and Rami, without their faith and frequent reassurance, this thesis could not have been completed. 3

4 ABSTRACT Forensic analysis of the Deoxyribonucleic Acid (DNA) obtained from biological evidence, such as blood or cellular samples, commonly known as DNA profiling or DNA typing, has become an important tool for individual identification in the criminal justice system. However, the disturbing possibility of deliberate contamination of the DNA evidence at the crime scene still exists. Accidental DNA contamination is widely researched and many preventative procedures have been put in place to eliminate this form of DNA contamination. On the other hand, deliberate DNA contamination of the DNA evidence at the crime scene has not been discussed in much detail. The deliberate contamination of the DNA either by planting a person s DNA at a crime scene or by contaminated the forensic evidence samples using another person s DNA amplicons still exists. Both can produce misleading DNA profiles. The current study shows that the deliberate contamination of the biological samples with amplicons under simulated crime scene conditions resulted in an alteration to the DNA profile of the original genomic DNA. Not only do immediate additions of PCR amplicons to samples alter the genomic DNA profiles, amplicons remain present in the genomic DNA profiles even after a period of up to seven weeks under the conditions used in this study. However, the alteration of the genomic DNA with amplicons contamination is not obvious after a period of five weeks. Amplicons alleles were still present but there was a drop-off of fluorescence intensity of the alleles and these peaks could be dismissed as stutter peaks. The study also showed that the surface type can influence the recovery of biological samples; however, in all cases the recovery of contaminant amplicons was observed from the swabbed surfaces. Previous studies show that the use of substrate controls in DNA analysis has limited value (Gill, 1996) in contrast, this study suggest that swabs an area adjacent to a sample or stain at the crime scene, may be useful in detecting the presence of amplicon contamination at crime scenes. Finally this study has verified that a hair washing step before DNA extraction can eliminate amplicon contamination of hair samples. 4

5 TABLE OF CONTENTS DECLARATION...2 ACKNOWLEDGMENTS...3 ABSTRACT...4 TABLE OF CONTENTS...5 TABLE OF FIGURES...8 LIST OF TABLES...10 CHAPTER 1: LITERATURE REVIEW BACKGROUND DNA Profiling in Forensic Science Basic DNA Structure and Definitions DNA Profiling in the Criminal Justice System History of Forensic DNA Profiling Technology METHODOLOGY OF DNA PROFILING WITH STR MARKERS Sample Collection and DNA Extraction DNA Quantification Polymerase Chain Reaction (PCR) Amplification Polymerase Chain Reaction (PCR) Advantages and Limitations Forensic DNA Amplification Kits AmpFlSTR Profiler Plus DNA Profiling Kit Separation and Detection of PCR Product (STR Alleles) THE STR MARKERS USED FOR DNA PROFILING STR Markers Commonly Use in DNA Profiling Issues Related to STR Analysis Issues and Challenges Related to Mixtures of STR DNA CONTAMINATION Accidental Contamination Deliberate Contamination PROJECT AIMS...39 CHAPTER 2: MATERIALS AND METHODS Sample Collection and Preparation The Characteristic of Genomic Blood and Hair DNA Profiles DNA Extraction and Quantification Amplification Conditions Detection of DNA after PCR Amplification Preparation of Contaminant Amplicons Re-amplification of Diluted Contaminant Amplicons Effect of time on the Presence of Amplicons in Blood and Hair DNA Profiles after Direct Contamination The Addition of 10-3 and 10-4 K562 Amplicons to Blood and Hair Samples 46 5

6 2.5 Effect of time on Presence of Sprayed Amplicons in Blood and Hair DNA Profiles under Simulated Crime Scene Conditions Preparing the Crime Scene Model Spraying Amplicons onto Crime Scene Samples Contamination of Bloodstain Samples Deposited on Different Surfaces Spraying 10-3 K562 Amplicons on Blood Samples Contaminated Blood DNA Profiles Using Identifiler CHAPTER 3: RESULTS The Characteristics of Biological Sample Profiles Genomic K562 and 9947A Standard DNA Profiles The Characteristics of the Genomic Blood and Hair DNA Profiles The Characteristics of the Re-amplified DNA Amplicons Examine the Effect and Presence of PCR Amplicons on the Blood and Hair DNA Profiles after Direct Contamination The Effect of K562 DNA Amplicons on Blood DNA Profile The Survival of K562 DNA Amplicons in Contaminated Blood DNA Profile The Effect of K562 DNA Amplicons on the Hair DNA Profile The Survival of K562 DNA Amplicons in Contaminated Hair DNA Profile Effect of Sprayed PCR Amplicons on Blood and Hair Samples under Simulated Crime Scene Conditions The Effect of Spraying 9947A and K562 Amplicons on Blood DNA Profile The Survival of 9947A and K562 Amplicons in Contaminated Blood DNA Profile The Effect of Spraying 9947A and K562 Amplicons on Hair DNA Profile The survival of 9947A and K562 Amplicons in contaminated Hair DNA Profile The Influence of Different Surface Types on the Contamination of Blood DNA Profile...82 CHAPTER 4: DISCUSSION Introduction Characterisation of Biological and Standard K562 and 9947A DNA Amplicons Profiles The Effect of PCR Amplicons Applied Directly on the Biological Samples The Effect of Amplicons Sprayed Directly on the Biological Samples under Simulated Crime Conditions The Presence of Amplicons in the Biological DNA Profiles The Influence of Different Surface Texture on amplicon Contamination of Bloodstain samples under Simulated Crime Conditions CHAPTER 5: CONCLUSION

7 CHAPTER 6: FUTURE STUDIES...93 CHAPTER 7: REFERENCES...95 i. APPENDICES ii. ABBREVIATINS iii. DEFINITIONS

8 TABLE OF FIGURES Figure 1: The structure of DNA double helix (Source: National Library of Medicine) Figure 2: Comparison between the DNA marker methods used for DNA profiling Figure 3: Overview of DNA profiling protocol (Source: Butler, 2005) Figure 4: General procedure for isolation of DNA using Qiagen anion-exchange resin column Figure 5: The qpcr amplification plot. The Cycle at which the plot crosses the threshold known as the Ct value Figure 6: The PCR amplification steps Figure 7: Schematic diagram shows the fluorescent dye label color and relative PCR product size ranges for various STR loci found in The AmpFlSTR Profiler Plus Figure 8: GeneMapper Software plot of the AmpFlSTR Profiler Plus allelic ladder Figure 9: Schematic diagram illustrate the differences between the VNTR and the STR markers Figure 10: Misleading DNA profile caused by pull-up peaks Artifacts Figure 11: Misleading DNA profile caused by stutter, blob and noise artifacts peaks Figure 12: Interpretation of DNA profile containing possible mixtures of STR. 34 Figure 13: A Comparison between the two different STR allele peak patterns obtained from single source and mixed sources of heterozygous samples Figure 14: Timeline illustrating the potential of DNA transfer before, during or after a crime Figure 15: Preparation of the biological samples used in this study Figure 16: Crime Scene Model Preparation. Refer to Appendix D for the preparation steps Figure 17: Set-up of the Simulated Crime Scene Model. The crime scene setup included the positive and negative controls Figure 18: Seven different materials were used to examine the recovering ability of the blood DNA profile and amplicon contamination Figure 19: Genomic profiles of the standard K562 and 9947A DNA Figure 20: Genomic Blood and Hair DNA Profiles Figure 21: Electropherograms Showing the K562 Amplicon DNA Profiles Figure 22: Gel Electrophoresis show image of PCR product Figure 23: Electropherogram Shows the Genomic Blood DNA Alteration after 2 Weeks of 10-4 K562 Amplicons Contamination Figure 24: Electropherogram Shows the Contaminated Genomic Blood DNA Profile after Ten Weeks of Contamination with 10-4 K562 Amplicons Figure 25: The Impact of 10-4 K562 Amplicon Contamination on Blood DNA Profile Figure 26: Electropherogram Shows the Contaminated Genomic Hair DNA Profile after One Week of 10-4 K562 Contaminations Figure 27: Electropherogram Shows the Contaminated Genomic Hair DNA Profile after Five Weeks of 10-4 K562 Contaminations Figure 28: Electropherograms Show the Results of Spraying the PCR Amplicons onto Bloodstain Samples

9 Figure 29: The Contaminated Genomic Blood DNA Profiles with 10-3 K562 Amplicons under Different Time Periods Figure 30: The Contaminated Blood DNA Profiles with 10-3 K562 Amplicons after Five and Seven Weeks Figure 31: The Comparison between the Pre and Post-contaminated Blood DNA Profiles with A Amplicon after Seven Weeks Figure 32: Electropherograms Showed the Comparison between the contaminated Hair DNA Profiles with and without the washing step Figure 33: The Contaminated Genomic Hair DNA Profile Extracted from Unwashed Hair Sample Figure 34: The Contaminated Genomic Hair DNA Profile with A Extracted from Washed hair sample Figure 35: Electropherograms Show the Comparison between Contaminated Blood DNA Profiles with 10-3 K562 amplicons Swabbed from Different Surfaces Figure 36: Summary of research outcomes assessed in this project

10 LIST OF TABLES Table 1: AmpFlSTR Profiler Plus ID Loci and Allele Information Table 2: Labels of the biological samples extracted after each time period Table 3: Set of samples extracted from the first trail time period after direct contamination Table 4: Set of samples extracted from the second trail time periods after direct contamination Table 5: Set of Samples Extracted After Each Time Period under the Simulated Crime Scene Conditions Table 6: Labels of the Bloodstain Samples Table 7: Genomic K562 and 9947A DNA Profiles Table 8: Genomic blood and hair DNA profiles Table 9: The RFU Values of the Contaminated Genomic Blood DNA with 10-4 K562 Amplicons after Two and Ten Weeks Table 10: A Summary of Amplicon and Contaminated Blood DNA Profiles and their Respective RFU Values Table 11: The Comparison between Genomic Hair DNA Profile Before and After A and/ or 10-3 K562 Amplicon Contamination under the Simulated Crime Scene Conditions, and their RFU Values

11 CHAPTER 1: LITERATURE REVIEW 1.1 BACKGROUND DNA Profiling in Forensic Science Forensic Science is the application of scientific knowledge to interpret information gathered from physical evidence to solve legal problems at the court of law (Butler, 2005). Forensic Science covers a wide range of fields such as anthropology, biology, chemistry, engineering, genetics, medicine, pathology, phonetics, psychiatry, and toxicology, all of which assess physical evidence that may be used to solve a crime. The types of physical evidence that are typical elements of a crime include biological evidence, trace evidence, impression evidence (such as fingerprints, footwear impressions, and tyre tracks) (Rudin & Inman, 2002). Among the many new tools that science has provided for examining forensic evidence is the analysis of Deoxyribonucleic Acid (DNA). Forensic DNA analysis, also called DNA profiling, is a molecular method used to characterize DNA obtained from biological samples to identify people and other organisms based on differences in the sequence found in their DNA (Butler, 2005). The most common forms of DNA evidence come from blood or cellular samples such as saliva, semen, skin and hair, and can be obtained from crime scenes, suspects, victims and convicted offenders. The application of DNA profiling covers a broad range of disciplines, including human and wildlife forensic science, diagnostic medicine, and animal and plant sciences. In the forensic field, DNA profiling is used to identify people who might be related to each other, to link a suspect to a crime, as well as to exonerate the innocent. In addition, it can be used to identify victims of crimes and disasters and for paternity testing. 11

12 1.1.2 Basic DNA Structure and Definitions Deoxyribonucleic acid (DNA) is the biological blueprint of life. DNA is located in the nucleus of each living cell, and consists of two strands wound around each other in a double helix to resemble a twisted ladder (Figure 1). It was first described by Francis Crick and James D. Watson in 1953 (Rudin & Inman, 2002). Figure 1: The structure of DNA double helix (Source: National Library of Medicine). DNA strands are made from repeating nucleotides, each nucleotide consisting of a sugar (deoxyribose) and phosphate backbone, cross linked with two types of nucleic acid bases referred to as purines (adenine and guanine) and pyrimidines (cytosine and thymine) (Siegel et al, 2000). The two nucleotide chains are held together by hydrogen bonds between the bases of each strand, where guanine pairs with cytosine and adenine pairs with thymine. The backbone of each strand is formed by the alternating phosphate and deoxyribose sugar units that are connected by phosphodiester linkages (James, 2005). The majority of the genetic information located in the nucleus is in the form of chromosomes. Every species has a characteristic number of chromosomes; for instance, humans have 23 pairs of chromosomes with one copy inherited from the mother and one from the father. This DNA is referred to as a nuclear or 12

13 genomic DNA and it consists of 22 pairs of autosomes and one pair of sex chromosomes (label as X and Y locus). The X- and Y- loci are used to distinguish between male and female; where female DNA distinguish as (X, X), and male DNA as (X, Y) (Sullivan et al., 1993). Each nucleated cell in the living organism contains a complete copy of the DNA with the exception of the red blood cell. The DNA usually consists of coding and non-coding regions of DNA sequences. The non-coding regions often consist of repeat units, and are found either within genes as introns or between genes with no known function (Yu Jun et al., 2002). These repeats may contain several hundred to several thousand base pairs (bp) regions and are often referred to as satellite DNA (Ellegren, 2004). Other repeats that are highly polymorphic and may contain bp are often referred to as minisatellite or variant number tandem repeats (VNTR) (Tautz, 1993; Chambers & MacAvoy, 2000). In the other hand, the repeats that are consist of di-, tri- and tetrancleotide tandem repeats, and are often referred to as Short Tandem Repeats (STR) normally have less than bp (Hulten et al., 1995; Carey and Mitnik 2002) (Refer to Figure 9). These repeats occur in all individuals and each individual has a unique combination or pattern of such repeated sequences - except for that obtained from identical twins - which often differ between individuals and act as a potential targets in forensic DNA analysis for criminal investigations (Hulten et al., 1995; Jeffreys et al., 1985; Blamire, 2005). The locations of such repeats on the particular chromosome usually know as loci or locus DNA Profiling in the Criminal Justice System DNA profiling has become the gold standard tool for individual identification in criminal and paternity cases within the criminal justice system in Australia, as well as in most jurisdictions around the world. The first court case that involved the use of DNA evidence was in 1986, and resulted in the conviction of Colin Pitchfork, (Lincoln, 1997). In this case, the restriction fragment length polymorphism (RFLP) technique was used to help in the resolution of a double homicide case and established a link of the same individual to two murders 13

14 (Butler, 2005). In Australia the first criminal case to use DNA profiling was in the Australian Capital Territory (ACT) Court of Appeal in 1989 when Desmond Applebee was convicted of three counts of sexual assault after the DNA collected from a sample of his blood matched DNA extracted from blood and semen on the victim's clothes (Gans & Urbas, 2002; Coelli, 1989). When DNA profiling was first introduced into the criminal justice system, there were many concerns regarding the validity of the scientific methodology behind the laboratory test (Rudin & Inman, 2002), laboratory accreditation and quality control, sample collection procedures and chain of evidence management (Gans & Urbas, 2002). These concerns led to the development of international standards and quality control regulations, and accreditation of forensic laboratories to provide scientific validity of DNA profiling. This permitted the results of DNA tests to be admitted as evidence in criminal proceedings (Carey & Mitnik, 2002; Jobling & Gill, 2004; Australian Institute of Criminology 1990). Even with these regulations, there are still some important issues with the use of DNA profiling in courts. One issue that is still a major concern is the possibility of accidental and deliberate contamination of forensic samples (Frumkin, et al., 2009; Berryman, 2003; Thompson, 2006) History of Forensic DNA Profiling Technology The ABO typing used in the early days were based on serology tool used to determine differences amongst individuals was discovered by Karl Landsteiner in 1960 (Jobling & Gill, 2004). The basis for those blood types was a set of proteins on the surface of red blood cells, they come in two varieties: A and B. Despite being relatively quick to carry out, it is not informative enough to enable identification of individuals (Jobling & Gill, 2004). In 1980, Botstein and his colleagues discovered a region of DNA that consists of short sequences repeated many times, with the number of repeats varying among individuals known as variable number tandem repeats (VNTR) (Carey & Mitnik, 2002). Botstein used these VNTRs combined with the restriction fragment length polymorphism (RFLP) technique to construct a human gene map (Carey & Mitnik, 2002). 14

15 The repeat regions of VNTR s can take different size repeat units, and may contain base-pairs regions (Ellegren, 2004; Tautz, 1993; Chambers & MacAvoy, 2000). These sequences are usually identified using restriction fragment length polymorphism (RFLP) analysis. RFLP is a method that involves the use of restriction enzymes to cut the regions of DNA surrounding the VNTRs and it results in different amounts and sizes of restriction fragments for different individuals (Rudin & Inman, 2002). The RFLP technique can determine the variation in the length of a defined DNA fragment through the hybridisation of probes to Southern blots of DNA digested with a restriction enzyme revealing a unique blotting pattern characteristic to a specific genotype at a specific locus (Jobling & Gill, 2004). In 1984, while searching for disease markers in DNA, Jefferys (Jeffreys et al., 1985) found that certain VNTR s within the human genome do not code for any known function. They occur in all individuals and each individual has a unique combination or pattern of such repeated sequences (Jeffreys et al., 1985). Jefferys was the first to use the RFLP technique to examine the VNTR regions of the DNA strand use for human identification. The technique was commonly referred to as DNA fingerprinting. Currently, this technique is known as DNA profiling to prevent any confusion with fingerprints of actual fingers. Although, to undertake DNA profiling using RFLP requires a large amount of DNA, time and expert labor, it still maintains the highest degree of discrimination (Butler, 2005). DNA profiling has come a long way since Jeffreys discovery 20 years ago. New and improved techniques have been developed to perform DNA profiling with regards to sample processing, speed and the sensitivity of assays (Figure 2). In particular, the introduction of Polymerase Chain Reaction (PCR) analysis (Refer to Section 1.2.3) of short tandem repeats (PCR-STR) based DNA profiling has dramatically improved the speed of DNA profiling. DNA testing previously took 6 to 8 weeks, currently, can be performed in less than 24 hours (Butler, 2005; Carracedo, 2005). The sensitivity of the PCR-STR technique was a catalyst in the advancement of forensic DNA analysis, as it allowed DNA samples of limited quantity to be analyzed. 15

16 Other methods have also been introduced, such as mitochondrial DNA (mtdna) analysis. mtdna is found in the mitochondria organelle in the cytoplasm of a cell, and is inherited from the mother, with no contribution from the father (Siegel et al., 2000). Although it is not as time-effective and has a reduced power to discriminate, mitochondrial DNA analysis is very useful in forensic cases that involve severely degraded DNA samples or paternityrelated issues (Butler, 2005) (Figure 2). Figure 2: Comparison between the DNA marker methods used for DNA profiling. These markers are divided into four quadrants based on their power of discrimination between individuals and the speed of analysis (Butler, 2005). 1.2 METHODOLOGY OF DNA PROFILING WITH STR MARKERS The introduction of PCR-based assays for single STR and multiplex STR DNA markers has revolutionized the processes of DNA profiling. Currently, the PCRbased assays for single and multiplex STR techniques are the most commonly used methods to obtain the DNA profile by forensic laboratories. This is because these assays more time-effective than other methods and can obtain profiles from much smaller and degraded DNA samples, which is the case in most forensic samples. These assays now form the basis of standard methods 16

17 for DNA analysis of forensic sample that are particularly effective for mixtures and biological materials containing limited number of DNA or degraded DNA. PCR technology allows DNA profiling from different biological sample types such as blood, semen, hair roots, bones, saliva, and skeletal remains (Scharf, 1986; Jeffreys et al., 1988). The DNA profile of a sample is usually obtained by defined processes depending on biological sample types. These processes involve biological, technological and genetic steps (Figure 3). Figure 3: Overview of DNA profiling protocol (Source: Butler, 2005). As shown in Figure 3, the first step in any forensic DNA profiling assay is sample collection. This Step adheres to the principles that preserve the chain of custody. Followed by the biological step includes DNA extraction, DNA quantification and DNA amplification. The technological steps include the separation and detection of the PCR products using methods such as fluorescent detection using capillary electrophoreses (CE). Finally, the genetic steps consist of the comparison of a sample genotype to other sample s results in order to determine if there is a match (Butler, 2005). The following sections 17

18 contain a review of the steps involved in processing forensic DNA samples with STR markers Sample Collection and DNA Extraction Depending on the case scenario, samples collected either from a known or unknown contributor (victim and/or suspect) from the crime scene can be used as evidentiary samples. Once the sample has been collected DNA extraction is carried out using one of a number of validated methods. The cell contains a number of substances in addition to DNA, and these substances may inhibit the PCR amplification. Therefore DNA extraction methods aim to separate the DNA from the cellular material and remove any PCR inhibitors that may be present in the specimen (Akane et al., 1994). The different approaches to DNA extraction all begin with some form of cell lysis, followed by deproteinization and recovery of DNA. The most common extraction methods used in forensic cases are based on the organic solvents such as phenol/chloroform or inorganic methods such as Chelex and Qiagen methods depending on the nature of the chemicals used. The choice of the extraction method for a particular sample depends on many factors such as the sample quality and quantity, the type of surface that the sample is on and the kind of cells present (McClintock, 2008; Rudin & Inman, 2002). The organic phenol/chloroform method has been used for many years; it is more likely to obtain cleaner and larger pieces of DNA than most other methods. It may be a more appropriate extraction method used for degraded samples, or when samples contain a large amount of contaminants that affect the DNA profiling (Rudin & Inman, 1997; Walsh et al., 1992). This method uses Sodium Dodecyl Sulphate (SDS) to rupture cell membranes, Proteinase K to denature the proteins and expose the DNA and a phenol/chloroform mixture to separate proteins and lipids from the DNA (Lee et al., 1991; Eminovic et al., 2005). 18

19 The alternative inorganic DNA extraction method (Chelex ) was introduced in 1991 and since then it has become popular among forensic scientists to economically recover high yields of DNA in a fast, simple and safe manner (Walsh et al., 1992). Chelex is an ion-exchange resin that is added as a suspension to the samples. It composed of styrene-divinylbenzene resin containing Iminodiacetic acid groups. Iminodiacetate act as chelating groups in binding polyvalent metal ions, such as magnesium, which can protect the DNA from being destroyed by nucleases enzymes (Butler, 2001). In this method; a Chelex suspension, usually 5%, is added to the sample and boiled for several minutes. This step denatures the DNA, disrupts the cell membranes and releases the DNA. Centrifugation is used to separate the Chelex resin and cellular debris from the supernatant that contains the DNA (Rudin & Inman, 2002; Walsh et al., 1992). The Chelex method is well suited to PCR-based testing because it produces single-stranded DNA. Another common inorganic DNA extraction method performed by forensic laboratories is the Qiagen DNA extraction system. This method relies on the selective binding properties of a silica-based membrane. It allows the separation of different classes of nucleic acids, such as genomic DNA, RNA and plasmid DNA, by successive elution steps using simple salt buffers (Karp et al., 1998). As shown in Figure 4, the main steps in this method are: Lysis the sample is lysed under highly denaturing conditions at elevated temperatures in the presence of Proteinase K and Buffer ATL. Binding selective binding of the desired nucleic acid to the membrane of the column under low salt conditions. Washing washing of the membrane with buffers of moderate salt concentration to remove impurities, and the nucleic acids remain bound to the membrane of the column. Elution DNA is eluted from the membrane using a high salt buffer, and isopropanol precipitation for desalting. 19

20 Figure 4: General procedure for isolation of DNA using Qiagen anionexchange resin column. Other non-organic DNA extraction methods include magnetic beads and FTA paper (Montpetit et al., 2005; Tack et al., 2005). The magnetic beads extraction method uses a specific magnetic resin that captures DNA without the need for extensive washings to remove contaminants that would normally be added to lyse cells (Montpetit et al., 2005). The FTA paper is an absorbent cellulosebased paper that contains chemicals that prevent DNA degradation and preserve the paper from bacterial growth (Tack et al., 2005). These methods represent new approaches for automation procedure for DNA extraction with minimal cost (Côté, 2008; Tack et al., 2005) DNA Quantification DNA quantification is a very important step in obtaining accurate and reproducible STR profiles. The success of PCR amplification is dependent on the amount of starting material, as too much or insufficient DNA template can result in profiles that are difficult to interpret (Butler, 2005). One of the earliest methods of quantitation of a small amount of DNA was the use of yield gels. Other methods now common in most forensic laboratories include slot-blot, spectrophotometry, QuantiBlot and quantitative real-time-polymerase chain reaction (q-pcr) methods (Siegel et al., 2000; Walsh et al., 1992). The QuantiBlot techniques has been found to be reliable and sensitive, however, this technique is time consuming and labour intensive (Richard et al., 2004). Recently, the introduction of quantitative real-time-pcr assays in the form of kits has greatly improved the speed and accuracy of DNA quantification compared to earlier methods such as QuantiBlot (Christian et al., 2007). 20

21 Currently the Quantifier Human DNA Quantification Kit has become the standard DNA quantification method used in many forensic laboratories (Butler, 2005). The Quantifier Human DNA Quantification kit uses a real-time PCRbased process and was designed for the quantification of human genomic DNA in forensic samples. It can detect as little as 32 picograms of DNA using 2μL of sample per assay (Green et al., 2005). The Quantifier Human DNA Quantification kit combines two assays. First, the human DNA-specific assay detects the human telomerase reverse transcriptase htert locus located on chromosome 5 (5p15.33). The htert locus is detected as a single copy gene like the STR loci which enables better calculation of the availability of amplifiable copies of the STR alleles in samples. The second assay is the Internal PCR Control (IPC). This control is included as an internal quality control to verify that the polymerase, the assay, and the detection instrumentation are working correctly (Green et al., 2005). The starting point for quantitative PCR depends on the cycle number at which the fluorescent signal crosses the threshold, which is defined as the Threshold Cycle, or C t. Data from the exponential phase amplification is used to construct the curve as this phase directly correlates to the amount of starting DNA (Figure 5). For example to calculate the amount of starting template in a sample, Ct value, Figure 5 shows the increasing fluorescence from 2 samples, A and B. Sample A has a smaller Ct value compared to B and therefore contains a higher amount of starting template since it require less cycles to reach its exponential phase. Figure 5: The qpcr amplification plot. The Cycle at which the plot crosses the threshold known as the Ct value. 21

22 There is an exact inverse mathematical relationship between the starting copy number of target sequence molecules and the resulting C t. The standard curve of C t versus concentration of the standard dilutions of a known concentration is then used to calculate the DNA concentrations of samples (Green et al., 2005) Polymerase Chain Reaction (PCR) Amplification The development of the PCR method has improved the sensitivity of STR marker analysis. The concept of the PCR process was first explained by Kleppe in 1971 (Kleppe et al., 1971). However, the Polymerase Chain Reaction (PCR) as a technique was developed by Mullis in 1985 (Mullis, 1987; Saiki et al., 1985; Butler, 2005). Mullis managed to develop a method to replicate DNA in vitro which replicate the same way as DNA is replicated in every cell of an organism (Kolilinsky & Liotti, 2005). PCR enables the amplification of short segments of a longer DNA molecule by a cycling process using DNA polymerase and oligonucleotide primers as shown in Figure 6. This allows researchers to produce millions of copies of a specific DNA sequence (Saiki et al., 1985). It is a sensitive and rapid amplification technique that requires a heat-resistant polymerase enzyme and the ability to alternately denature double-stranded DNA molecules and renature the complementary single strands (Lodish et al., 2003). The first practical application of PCR for genetic testing was described by Saiki and colleagues in This involved a sample of DNA being analyzed for the presence of genetic disease mutations (Saiki et al., 1985). Since then, the development of PCR as a basic component of molecular biology has been used extensively for a wide range of fields. Some of these fields are: forensic DNA casework (Gill, 2002), mass disasters, missing persons and immigration issues. The PCR method is also used for a wide rang of health and research issues such as diagnosis of genetic disorders and disease susceptibility, detection of infectious diseases, DNA cloning procedures (Boehnke et al., 1989), hereditary studies and paternity testing (Chengtao & Richard, 1999). 22

23 The essential components of a PCR reaction consist of two primers, which flank and define the region to be copied, template DNA that will be copied, the different four dntps (deoxynucleotide triphosphates), a thermostable DNA polymerases and a reaction buffer (Saiki et al., 1985). The main component of the buffer is magnesium, which acts as a cofactor for enzyme activity, and a buffer to maintain a neutral ph (Figure 6) (Kolilinsky & Liotti, 2005). The primers used for the amplification are designed with a unique base sequence that depends on a particular location or set of locations on the strand of DNA required to be isolated. The DNA is copied over and over until a sufficient quantity exists for testing (Rudin & Inman, 2002). The amplified product, which is known as an amplicon, can then be analyzed for size, quantity, and sequence or used in further experimental procedures. PCR comprises cycling conditions that are repeated between times, resulting in an exponential increase of the target sequence. Each cycle consists of three steps: melting, annealing and extension (Figure 6). These steps are usually performed on an automated thermal cycler and are repeated for a number of cycles depending on the product desired and the reaction type (Butler, 2005). The initial step in the PCR heats the target DNA to 95 C or higher for 15 seconds to 2 minutes. In this step, the double strand melts to form singlestranded DNA. In the annealing step, the temperature is reduced to approximately 40 to 60 C. At these temperatures, the primer molecules anneal to the single-strand template. The final extension step involves the synthesis of new DNA with the aid of a heat-stable DNA polymerase such as Taq Polymerase and a mixture of the four bases (dntps) in the presence of magnesium, to direct the rebuilding of double-stranded DNA segments that are identical to the starting template (Atchison & Georgalis, 1990). dntps are usually a mixture of datp, dctp, dgtp, and dttp, at equimolar concentrations. The Taq DNA Polymerase is the standard PCR enzyme responsible for synthesizing or extending the new DNA strand (Figure 6). 23

24 Figure 6: The PCR amplification steps Polymerase Chain Reaction (PCR) Advantages and Limitations The introduction of PCR-STR amplification to forensic laboratories has added speed and flexibility in obtaining DNA profiles. A significant result can be obtained even from samples containing little or degraded amounts of DNA, which is the case in many forensic samples. In addition, using human-specific primers can prevent the amplification of fungal and bacterial DNA contamination. PCR-STR multiplexing amplification permits more than one region to be amplified simultaneously. The STR loci are usually selected to have a small size range, keeping all amplified alleles smaller than 350 base pairs, which simplifies interpretation of results (Frégeau & Fourney, 1993; Kimpton et al., 1993; Urquhart et al., 1995). 24

25 One major disadvantage of PCR amplification is the generation of false positives (Kwok, 1989). This may be caused by primers annealing to sequences present in contaminating DNA derived from a source other than the sample being diagnosed or, particularly from previously amplified material. Contamination can influence PCR results, particularly in the absence of proper handling techniques and contamination controls. Consequently, sample contamination is one of the most serious problems facing DNA profiling using PCR-based methods and proves to be a rate-limiting step in obtaining useful forensic DNA profiles (Rand et al., 1991; Montiel et al., 2001; Berryman, 2003; Dent, 2006; Frumkin, et al., 2009) Forensic DNA Amplification Kits Commercial kits are now available for easy PCR setup and amplification. The CTT triplex kit (Promega Corporation) consisting of three STR markers (CSF1PO, TPOX and TH01) was one of the first commercially available STR kits capable of multiplex amplification. This kit was introduced in 1994 and had a matching probability of 1-in-500. Since then, the technology for DNA profiling kits has rapidly evolved. Kits are now available to simultaneously amplify in a single multiplex reaction up to 16 STR markers. These STR markers use multiple colour fluorescent detection tags and can be analyzed on automated DNA sequencers (Butler, 2005). Multiplexing DNA amplification kits have been developed for the following reasons: to achieve the highest power of individual discrimination, to preserve limited evidentiary samples by utilizing the smallest portion of evidence possible for analysis, to minimise contamination and to improve the opportunities for sharing data between forensic laboratories worldwide (Chantal et al., 2003). The major suppliers of such kits used by the forensic laboratories are Applied Biosystems and Promega Corporation, both located in the USA. Other suppliers are Serac and Biotype, located in Germany. Some commonly used kits are Profiler Plus, Cofiler, Identifiler and PowerPlex 16 (Butler, 2005). 25

26 The primers and STR loci have been validated for forensic use in many studies. And these kits using these loci have been subjected to rigorous quality control testing to ensure reliable performance (Holt et al., 2002) AmpFlSTR Profiler Plus DNA Profiling Kit In Australia, the AmpFlSTR Profiler Plus PCR amplification kit made by Applied Biosystems in conjunction with automated florescence-based allele detection is used routinely to produce DNA profiles from forensic samples (Pachette et al., 2002). This kit offers high sensitivity and accuracy of the DNA profiling from samples containing less than 1 ng of DNA, gives results in 24 hours or less, and simplifies the interpretation of the DNA profile (Butler, 2005; Frégeau & Fourney, 2003). The AmpFlSTR Profiler Plus PCR amplification kit uses a 10-loci multiplex in which 10 loci of the nuclear genome are simultaneously examined. The 10 loci comprise 9 STR and segments of the homologous gene known as the Amelogenin locus, found on the X- and Y-chromosomes. As shown in Table 1, these sets of markers have been chosen after a great deal of research to verify that the primer pair are compatible and can work well in combination with each other during multiplex PCR conditions (Wallin et al., 2002; Krenke et al., 2002). Also these loci have been chosen on different chromosomes to avoid the complicating factors of co-segregation and linkage disequilibrium between alleles of the loci (Frank et al., 2001). The Amelogenin gene is located on both X- and Y-chromosome with a range of 104 to107 bp from the X chromosome and 110 to113 bp from the Y chromosome, and that can be used for sex determination. The ratio of Amelogenin X and Y PCR products can be helpful in interpreting mixtures containing male and female DNA, such as in sexual assault cases, as well as for missing persons and mass disaster investigations. 26

27 Table 1: AmpFlSTR Profiler Plus ID Loci and Allele Information Locus Designation Chromosome Location Allelic Ladder Alleles Dye Label Common Sequence Motif D3S1358 3p 12, 13, 14, 15, 16, 17,18, 19 5-FAM TCTA (TCTG) 1-3 (TCTA) n vwa, 12p12-pter 11, 12, 13, 14, 15, 16, 17, 18, 5-FAM TCTA (TCTG) 3-4 (TCTA) n 19, 20, 21 FGA 4q28 18, 19, 20, 21, 22, 23, 24, 25, 5-FAM (TTTC) 3 TTTT TTCT (CTTT) n 26, 26.2, 27, 28, 29, 30 CTCC (TTCC) 2 Amelogenin X: p X, Y JOE Not Applicable Y: p11.2 D8S , 9, 10, 11, 12, 13,14, 15, 16 JOE (TCTR)n c 17, 18, 19 D21S , 25, 26, 27, 28, 28.2, 29, JOE (TCTA) n (TCTG) n [(TCTA) , 30, 30.2, 31, 31.2, 32, TA(TCTA) 3 TCA(TCTA) , 33, 33.2, 34, 34.2, 35, TCCA TA] (TCTA) n 35.2, 36, 38 D18S51 18q21.3 9, 10, 10.2, 11, 12, 13, 13.2, JOE (AGAA) n 14, 14.2, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26 D5S818 5q , 8, 9, 10, 11, 12, 13, NED (AGAT) n 14, 15, 16 D13S317 13q , 9, 10, 11, 12, 13, 14, 15 NED (GATA) n D7S820 7q , 7, 8, 9, 10, 11, 12, 13, 14, 15 NED (GATA) n n represents the variable number of core sequence motif repeats on different chromosomes. 5-FAM (Blue), JOE (Green) and NED (Yellow) Separation and Detection of PCR Product (STR Alleles) The particular location of a STR marker on the chromosome is referred to as its locus or loci for more than one marker. Each chromosome will have some differences in the DNA at the same locus, referred to as alleles (Atchison & Georgalis, 1990). These alleles are inherited; one from the mother and one from the father, and individuals DNA profiles show either one or two alleles at each locus. If the individual DNA profile shows two alleles at a particular locus, he or she is usually assumed to be heterozygotic, meaning that the individual has inherited different alleles from each parent. On the other hand, if the 27

28 individual DNA profile shows one allele, this mean that the individual is homozygotic, meaning that they have received two copies of the same allele, one from each parent. The separation medium of the STR marker on the chromosome may be in the form of a slab gel or a capillary. STRs can be detected using various methods, such as fluorescent dye labelling, silver staining, or fluorescent dye staining (Edwards & Gibbs, 1994). Forensic laboratories commonly use the fluorescent dye labelling that can permit automation and high throughput analysis (Rudin & Inman, 2002). Because the multiplex PCR process is the most effective and reliable when amplification products are the same size, the fluorescent-labels dyes with 5-FAM, JOE, or NED NHS-ester are used to detect PCR-STR multiplexes. These fluorescent-labels dye are detected as fluorescent blue, green, or yellow respectively, and are attached to the PCR primers present in the AmpFlSTR Profiler Plus Kit, as shown in Figure 7 (Jobling & Gill, 2004). A real-time imaging system is used to detect these fluorescent-labels on automated sequencing equipment such as ABI 3770 Genetic Analyser (Applied Biosystems). These types of instruments use capillary electrophoresis to separate the DNA fragments and determine their sizes according to the internal size standard, for example GS 500 (Applied Biosystems). The internal size standard usually included with each run as a control reference standard to give accurate readings of DNA length when the marker and unknown fragment have the same sequence and the same size (Figure 7). The different colours differentiate the STR loci alleles within the same size range. Figure 7: Schematic diagram shows the fluorescent dye label color and relative PCR product size ranges for various STR loci found in The AmpFlSTR Profiler Plus. 28

29 In addition, allelic ladders, for instance AmpFlSTR allelic ladders, consist of the known alleles for a particular locus, and are used for accurate genotyping. These genotypes are assigned to sample alleles by comparing their sizes to those obtained for the known alleles in the AmpFlSTR allelic ladders and it is recommended that they be used in each set of capillary injections (Applied Biosystems, 2000; AmpFlSTR Profiler Plus User Manual). When analysing the amplified STRs using a DNA sequencer, alleles at particular loci are represented in the form of peaks on an electropherogram. The alleles at a locus are represented by different numbers of repeat sequences, and so will be of different size. The alleles present at a specific locus are known as a genotype, whilst the combination of genotypes across multiple loci is known as a DNA profile (Butler, 2005; Rudin & Inman, 2002). For example, Figure 8 shows nine STR primer pairs plus a gender-typing locus present in the AmpFlSTR Profiler Plus kit. These STR primer pairs are represented by different numbers of repeat sequences known as alleles in the AmpFlSTR allelic ladders. Figure 8: GeneMapper Software plot of the AmpFlSTR Profiler Plus allelic ladder. 1.3 THE STR MARKERS USED FOR DNA PROFILING STR markers are the most popular marker used for DNA profiling. This is due to their wide distribution in the human genome, high level of polymorphism, and smaller fragment size compared to VNTR marker as shown in Figure 9 (Lechair 29

30 & Fregeau, 2004). In addition, the increased number of the STR markers used to detect genetic differences between individuals, provide a rich source of markers for individual identification between unrelated and even closely related individuals (Butler, 2005). Figure 9: Schematic diagram illustrate the differences between the VNTR and the STR markers. STR sequences are usually termed according to the length of the repeat unit. STR loci consist of repeated segments of 2 to 6 bases; these are termed di-, tri-, tetra-, penta-, and hexanucleotide repeats, and have 2, 3, 4, 5 and 6 nucleotide repeated units respectively (Jin et al., 1994). Among the various types of STR locus, tetranucleotides (4-bp repeats) are the repeat units currently used for human identification as shown in Figure 9 (Mifflin, 2003; Jobling & Gill, 2004). These tetranucleotides repeat loci are stable and polymorphic, with heterozygosity values for some loci >0.9 (Frank el al., 2001). In addition, in comparison to dinucleotide and trinucleotide STR markers, tetranucleotide STR markers are able to reduce the stutter product formation, and are easier to resolve with size-based electrophoretic separation. In general, the main characteristics of STR loci used in human identification include high power of discrimination, low stutter formation, low mutation rate, robustness and reproducibility of results when multiplexed with other markers. They also have alleles that are reasonably well distributed in any given population, have a high level of variability within a locus with low match 30

31 probability and must include male-specific Y chromosome STRs for analysing male-female mixtures (Carracedo & Lareu, 1998; Jobling & Gill, 2004; Gill et al., 1997) STR Markers Commonly Use in DNA Profiling Currently, a set of standardized STR markers are used for DNA profiling to provide DNA profile validity across a wide number of jurisdictions. DNA profiling results cannot be effectively shared unless all forensic laboratories use the same markers and the same conditions. Accordingly, in 1994, a committee of Forensic experts, known as the DNA Commission of the International Society of Forensic Haemogenetics (ISFH) issued the first guidelines for designating STRs alleles. Today this committee is known as the International Society of Forensic Genetics (ISFG) (Bar et al., 1997). According to ISFG recommendations, thirteen core STR loci were chosen by the Federal Bureau of Investigation (FBI), to be the basis of the Combined DNA Index System (CODIS) of the national DNA database. These STR loci are CSFIPO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D185S51, D21S11 and Amelogenin STR locus (Budowle et al., 1998). CODIS was developed specifically to enable public forensic DNA laboratories to create searchable DNA databases of authorized DNA profiles. By testing these core STR loci, the average of random match probability is less than one in a trillion among unrelated individuals (Chakraborty et al., 1999) Issues Related to STR Analysis Even though the STRs combined with the multiple-colour fluorescent tags used for DNA profiling have greatly enhanced the ability for individuals identification, biological and technical issues associated with DNA profiling, that may have a profound effect on the genotype results obtained from STR analysis, are still present (Butler, 2005). 31

32 For example, one of the technical issues is that pull-up (sometimes referred to as bleed-through) is caused by a failure of the fluorescent technology and analysis software to discriminate between the different dye colors during the generation of sample genotypes. A signal from a locus labeled with blue dye might mistakenly be interpreted as a yellow or green signal, thereby creating false peaks at the yellow or green loci, see Figure 10 (Butler, 2005). The peaks produced by this artifacts may be large enough to be confused with an actual peak or to mask actual peaks (Butler, 2005). Figure 10: Misleading DNA profile caused by pull-up peaks Artifacts. Other technical issues can affect genotype analyzers, including the presence of noise, spikes and blobs peaks. These peaks are referred to as artifact peaks. For example, Figure 11 shows how Noise, is the term used to describe small background peaks that occur due to a variety of factors such as air bubbles, urea crystals or sample contamination. On the other hand, spikes are narrow peaks usually attributed to fluctuations in voltage or the presence of minute air bubbles in the capillary. And blobs are false peaks that arise when some colored dye becomes detached from the DNA and gets picked up by the detector. Blobs are usually wider than real peaks and are typically only seen in one color. Although many technical artifacts are clearly identifiable, standards for determining whether a peak is a true peak or a technical artifact are often rather subjective, leaving room for disagreement among experts (Figure 11) (Butler, 2005). 32

33 One biological issue is the stutter artifacts (Figure 11). Stutter artifacts occur as a by-product of the PCR amplification process, and they are usually less or more than the size of the main band by one or more repeat lengths. They are small peaks that occur immediately before the real peak (Walsh et al., 1996) (Figure 11). The amount of DNA template used for PCR amplification may affect the amount of stutter artifacts present in the genotype results. In addition, the probability that stutter peaks will form increases as the repeat size decreases. Figure 11: Misleading DNA profile caused by stutter, blob and noise artifacts peaks. Artifacts peaks caused by air bubbles, urea crystals or sample contamination can be confused with an actual peak or can mask the actual peaks of the tested sample (Applied Biosystems, 2000) Issues and Challenges Related to Mixtures of STR The presence of a mixture of STRs is one of the major challenges for DNA profiling. Mixtures of STRs arise when two or more individuals contribute DNA to the sample being tested, resulting in more than two alleles present at a particular heterozygous marker locus. This is a common occurrence in forensic cases and in some mixtures it may not be detected easily as compared to other mixtures especially if homozygous marker alleles are present see Figure 12 (Butler, 2005). 33

34 Figure 12: Interpretation of DNA profile containing possible mixtures of STR. A: include two alleles; this might be interpreted as a mixture of samples if the STR is homozygous, and also could be interpreted as single source if the STR was heterozygous. Such samples will not be easily detectable. B and C; these STR markers are definitely a mixed sample since there are more than two alleles present (Butler, 2005). The difference between STR profiles from single source samples and mixed source samples can be determined according to STR allele peak patterns (Butler, 2005). For example, STR allele peak patterns for heterozygous samples from a single-source will have stutter peaks that are less than 15% of the associated allele peak height/area, and will have a peak height ratio of greater than 70% between the lower and higher quantity allele peak as shown in Figure 13 (Gill et al., 1997). Mixed samples, on the other hand, have STR allele peaks that fall in the range of 15% to 70% of the highest peak at a particular STR locus (Butler, 2005). Figure 13: A Comparison between the two different STR allele peak patterns obtained from single source and mixed sources of heterozygous samples. A: a single-source sample that usually has stutter peaks less than 15% of the associated allele peak height and greater than 70% between the lower and higher quantity allele peak (Gill et al., 1997). B: a mixed sample including a peak greater than 15%, which means that it is not a stutter, and at the same time, the percentage between the alleles peaks is less than 70%. 34

35 1.4 DNA CONTAMINATION DNA evidence relies on a statistical approach based on population genetics and empirical testing (Saks & Koehler, 2005). DNA evidence has become the gold standard of forensic testing, but there are still some concerns surrounding the admissibility of the DNA profiling in court. The initial concerns were focused on the nature of the technique itself. Currently, the main problem facing the analysis of forensic DNA evidence is contamination. DNA contamination in a forensic context is usually defined as the unintentional addition of an individual s physiological material or DNA during or after collection of the sample as evidence (Rudin & Inman, 2002). As shown in Figure 14, modified revision from Gill 2002, DNA contamination can take place accidentally through inadvertent crime scene contamination by witnesses, police and others; or by secondary transfer of DNA through personto-person or person-to-object contact; and by laboratory contamination with PCR amplicons (carryover contamination) (Gans, 2003; Gill, 2002). DNA contamination can also take place by a person s DNA deliberately being planted at a crime scene or DNA evidence being masked using PCR amplicons to contaminate the biological samples at a crime scene (Berryman, 2003; Dent, 2006; Frumkin, et al., 2009,) (Figure 14). Figure 14: Timeline illustrating the potential of DNA transfer before, during or after a crime. 35

36 1.4.1 Accidental Contamination One of the most serious problems facing DNA profiling analysis is accidental sample contamination in the laboratory, and has been a continual problem for laboratories performing forensic procedures and detection of infectious agents (Pellett et al., 1999; Scherczinger et al., 1999). The sources of such contamination of evidence samples can be either with genomic DNA from the environment, contamination between samples during preparation or with PCR products from prior amplification (refer to Figure 14), known as carryover contamination (lygo et al., 1994). Many of these sources of potential contamination are easily removed if standard protocols are followed. Some of these protocols include investigators and other personnel wearing protective clothing when investigating the crime scene and using clean equipment when handling the evidence (Rutty et al., 2003). As well, laboratory staff should wear appropriate clothing such as laboratory coats, face masks and disposable single use gloves. PCR amplicon carryover contamination from samples processed in the laboratory is more difficult to control and more effort is required to prevent its occurrence. Separate rooms for pre- and post-pcr amplification areas and using a dedicated set of pipettes and pipette tips for each amplification area are the most efficient means of preventing PCR contamination. DNA specimen extraction and preparation areas should be separated from those where amplicons are created and analysed (Koupparis, 2002). Using UV light and bleach to decontaminate the equipment and the working surfaces is highly effective in minimizing the chances of contamination. Using amplification controls can also help detect carryover amplicon contamination. Controls usually include a negative control reaction reagent blank consisting of all reagents used during sample processing except the DNA template, to show that contamination has not influenced the results (Kwok & Higuchi, 1989; Gill, 2002). A positive control, which includes a genomic DNA of known concentration, helps in verifying the operation of the amplification process. Limited research has identified the potential problems of human DNA contamination either on the cadaver (Rutty, 2002), from the instruments (Rutty 36

37 et al., 2000), or from the work surfaces within the mortuary (Toledano et al., 1997). Other sources of accidental contamination can occur from the plasticware contaminated at the manufacturing source (Gill, 2002; Gans, 2002; Rutty et al., 2003) Deliberate Contamination Deliberate DNA contamination is one type of contamination that has yet to be fully researched by forensic scientists. In this study, deliberate DNA contamination refers to the introduction of DNA by individuals unassociated with the crime (Figure 14). It can be by means of a person s DNA being maliciously planted at a crime scene - such as a hair, blood or a cigarette-butt or by the DNA evidence found at the crime scene being masked using PCR amplicons. Of these, amplicon contamination is potentially the most concerning (Koupparis, 2002; Berryman, 2003; Dent, 2006; Frumkin, et al., 2009,). PCR amplicons can be introduced into a crime scene by an aerosol and they affect a DNA profile by masking the original DNA profile or giving a false impression of a mixture The previous studies (Berryman, 2003; Dent, 2006) examined the effects of contaminating simulated crime scenes with a concentrated solution of PCR amplicons. The results showed that PCR amplicons can produce false DNA profiles and are able to survive for at least two weeks in a typical crime scene environment, although further testing beyond this two week time period to determine the effect and long term persistence of PCR amplicons, and the influence of different surface types was not carried out (Berryman, 2003; Dent, 2006). Deliberate DNA contamination problem is further complicated by the increased sensitivity of the current generation DNA profiling kits that use the PCR-STR method to detect very low amounts of DNA. When contaminations are detected, a forensic laboratory has no way of knowing if it occurred by accident during collection or the samples were deliberately contaminated before arriving at the laboratory. There is no guarantee that a crime scene has been secured before the arrival of the police; therefore, the prospect of fraudulent planting of 37

38 DNA is a potential issue in every investigation and trial involving DNA identification evidence (Figure 14). Recent concerns with DNA fraudulence have been with the masking of DNA evidence. DNA evidence can be masked by adding or spaying amplicons made from another person s DNA to contaminate the DNA evidence at the crime scene. The resultant amplicons solution usually contains a high concentration of target allele molecules, and the DNA analysis results from that solution may yield the profile of the second person, rather than that of the original contributor (Dent, 2006; Koupparis, 2002). Moreover, DNA evidence can be faked using artificial DNA developed with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can be then applied into genuine human tissues and planted in crime scenes (Frumkin, et al., 2009). Deliberate DNA contamination using PCR amplicons would require scientific knowledge but could be carried out anywhere with the use of a commercial kit. These kits can be purchased by a member of the public or by someone with access to PCR facilities. Amplicon solutions can be kept stable at room temperature for several weeks and will stay fresh for months in a refrigerator and years when stored in a freezer (Koupparis, 2002). Such amplicons could then be distributed by diluting them into water and using an aerosol such as a perfume bottle. This form of amplicon contamination has been found to be both possible and easy to accomplish; therefore, it is important to remember that physical evidence is circumstantial and there is no guarantee that it may be fraud-proof until the matter of deliberate contamination is addressed (Gans, 2003). Currently, procedures have been implemented to prevent accidental contamination in the laboratory, as well as to prevent contact of extraneous DNA with evidentiary samples either at the crime scene by investigators or at laboratories by the analysts. However, there are serious concerns about deliberate contamination of DNA evidence (Berryman, 2003; Frumkin, et al., 2009). 38

39 1.5 PROJECT AIMS The occurrence of false and misleading DNA profiles resulting from accidental or deliberate contamination is well-known and well-documented (Mifflin, 1997; Berryman, 2003; Frumkin, et al., 2009). Deliberate DNA contamination is one aspect of contamination that has serious implications for the subsequent downstream DNA profiling analysis, and this has yet to be fully researched by forensic scientists. This study addresses some of the issues associated with the deliberate contamination of DNA evidence, or amplicon contamination of crime scene samples prior to sample collection. The aims of this study were as follow; 1 Investigate re-amplified amplicon characteristics. 2 Detect the persistence of PCR amplicons added directly to blood and hair samples for an extended period of up to six months. 3 Determine the persistence of PCR amplicons sprayed directly on to blood and hair samples under simulated crime scene conditions. 4 Test the effect of different surfaces (such as wood, carpet, metal, glass, plastic, tiles and clothing) on the resulting DNA profiles of contaminated blood samples. 39

40 CHAPTER 2: MATERIALS AND METHODS 2.1 Sample Collection and Preparation Samples were gathered from two known volunteers for this study. Blood samples were collected by venipuncture in 4 ml BD Vacutainer heparin tubes (Becton, Dickinson and Company) while hair was removed from the head hair strands with attached follicular tag to ensure the present of DNA for extraction procedures. The liquid blood samples were used directly or spotted on to cotton undergloves as shown in Figure 15. The bloodstain patches were prepared by spotting 20 μl approximately 1 cm 2 in diameter. Squares of 2mm 2 were used for the DNA extraction. The hair root samples were cut to approximately 2 cm in length and placed in a container. Both of the samples (the bloodstain and the hair root) were left on the lab bench allowed to air dry. Three replicates were prepared for each sample one of which was used as a control. A separate set of samples was prepared for the second time series experiments (Figure 15). Figure 15: Preparation of the biological samples used in this study. The Bloodstain samples were spotted on to cotton under-gloves, and the hair samples with attached follicular tag were cut to approximately 2 cm in length and placed in a container. 40

41 2.2 The Characteristic of Genomic Blood and Hair DNA Profiles DNA Extraction and Quantification DNA was extracted from bloodstain patch (2 mm), liquid blood (2 μl), and hair (a set of three hair roots), using Chelex as outlined in the Users Manuals (Applied Biosystems Foster City, CA USA) [see Appendix A and B for detailed method]. The samples were referred to as SB, B and H respectively. DNA extraction from each sample was repeated three times. DNA was quantified using the Quantifier Human DNA Quantification Kit as recommended by Applied Biosystems (Applied Biosystems Foster City, CA USA) [see Appendix C for detailed method]. DNA quantification is particularly important for AmpFlSTR Profiler amplifications, where optimal results are obtained using a range of ng of input DNA. Adding greater than 2.5 ng of DNA can result in too much PCR product (amplicons) that will exceeded the detection range of the instrument used to detect and analyze the PCR product (Walsh et al., 1992). A set of standards with known DNA concentration was used in every run to ensure correct readings prior to measuring test samples. All PCR preparation was carried out in a laminar flowhood in a dedicated PCR room. All tubes, pipettes and pipette tips were irradiated under ultraviolet light for 10 minutes to minimise contamination of reagents and other DNA samples, and were kept in a dedicated PCR room. The master mix was prepared for each sample as follows: Reaction Components Volume (μl) Quantifiler Human Primer Mix 10.5 Quantifiler PCR Reaction Mix 12.5 DNA Template 2.0 Total Volume (per reaction) 25.0 Real-time PCR Amplifications were carried out for 40 cycles using the Rotor- Gene 3000 (Corbett Life Science, Sydney, NSW) in 0.2 ml, thin-walled PCR tubes. 41

42 The cycle parameters were as the follows: Thermal Cycler Protocol Initial Denaturation C Denaturation C Annealing C Fluorescent Acquisition End of Annealing (FAM/Sybr, CY5, JOE and ROX) Amplification Conditions Samples were amplified using AmpFlSTR Profiler Plus Amplification kit, or AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems Foster City, CA USA) allowing amplification of multiple STR loci in a single reaction mixture. In addition, positive and negative controls were included in the amplification run. The positive control was one of the standard DNAs, K562 or 9947A, to confirm successful amplification; and the negative control included pyrogenfree injection water instead of a DNA template, to detect any unintentional contamination. AmpFlSTR Profiler Plus PCR Amplification kit The reaction master mix of the AmpFlSTR Profile Plus was prepared for each sample by adding 1 ng of DNA template to a total sample volume of 10 μl, using distilled water as outlined in the Profiler Plus User Manual (Applied Biosystems, 2000), except that a 25 µl reaction volume was used instead of the recommended 50 µl final reaction volume. The reaction master mix was prepared as follows: Reaction Components Volume (μl) AmpFlSTR Profile Plus PCR Reaction Mix 9.5 AmpFlSTR Profile Plus Primer Set 5.0 AmpliTaq Gold DNA Polymerase 0.5 DNA Template (0.1 ng/ μl) 10.0 These PCR amplification conditions were used consistently throughout this research. The reaction mixtures were subjected to a hot start at 95 C for 10 minutes in order to activate the AmpliTaq Gold DNA polymerase (Applied Biosystems Foster City, CA USA). Amplification was carried out for 28 cycles 42

43 using the following parameters, as recommended by the AmpFlSTR Profiler Plus User Manual (Applied Biosystems), with a slight change of 11 minute from 10 min in the initial incubation and a change in the annealing temperature to 55 C from 59 C, as used by Schoske (Schoske, 2004). The thermal cycler protocol was as follows: Thermal Cycler Protocol Initial Incubation C Denaturation 94 C Annealing 55 C Repeat for 28 cycles Extension 72 C Final Extension C Hold at 25 C forever AmpFlSTR Identifiler PCR Amplification Kit The reaction master mix of the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems Foster City, CA USA) was prepared for each sample by adding 1 ng of DNA template to a total sample volume of 10.0 μl as well, using distilled water as outlined in the AmpFlSTR Identifiler User Manual (Applied Biosystems, 2000). The reaction master mix was prepared as follows: Reaction Components Volume (μl) AmpFlSTR Identifiler PCR Reaction Mix 10.5 AmpFlSTR Identifiler Primer Set 5.5 AmpliTaq Gold DNA Polymerase 0.5 DNA Template (0.1 ng/ μl) 10.0 Amplification was carried out for 28 cycles using the following parameters, as recommended by the AmpFlSTR Identifiler User Manual (Applied Biosystems). The thermal cycler protocol was as follows: Thermal Cycler Protocol Initial Incubation C Denaturation 94 C Annealing 59 C Repeat for 28 cycles Extension 72 C Final Extension C Hold at 25 C forever 43

44 All amplifications were conducted using a Perkin Elmer GeneAmp PCR System 9700 thermal cycler (GMI, Inc. Ramsey, Minnesota, USA) and 0.2 ml thin-walled MicroAmp Reaction Tubes Detection of DNA after PCR Amplification Following the amplification, the ABI Prism 3730 DNA Sequencer was used for allele separation and detection. The master mix was prepared as recommended by the User Manual (Applied Biosystems 2000). The ABI GeneScan 500 Rox Internal Lane Size Standard was added to the reaction mixture to determine the DNA fragment sizes. The master mix was as follows: Reaction Components Volume (μl) PCR product 1.0 GeneScan 500 Rox 0.1 Hi-Di Formamide 11.8 Total Volume 12.9 GeneMapper version 3.7 (Applied Biosystems) was then used to display the different colours as separate panels of data and to determine the exact length of the DNA profile at a minimum threshold of 50 relative fluorescent units (RFU). 2.3 Preparation of Contaminant Amplicons The standard genomic DNAs, K562 (Promega Corporation, Madison, WI) and 9947A (Promega Corporation, Madison, WI) were used for PCR amplicon production. These amplicons were prepared from 1 ng of genomic DNA template using the AmpFlSTR Profiler Plus PCR Amplification Kit as stated in Section DNA profiles were obtained as stated in Section These amplicons were then added or sprayed directly onto bloodstain and hair samples as contaminant source. 44

45 2.3.1 Re-amplification of Diluted Contaminant Amplicons The aim of this experiment was to observe the characteristics of different diluted amplicons when they re-amplified and to determine the best dilution factor to use for contaminating the biological samples. Genomic K562 amplicons were diluted in a serial manner using autoclaved deionised water. The dilutions were 10-1, 10-2, , 10-5 and fold. An aliquot of 10 μl from each diluted amplicon was subject to a second round of PCR amplification using the AmpFlSTR Profiler Plus PCR Amplification Kit as stated in Section 2.2.2, and the DNA profiles were obtained as stated in Section Effect of time on the Presence of Amplicons in Blood and Hair DNA Profiles after Direct Contamination The following time line experiments sought to determine the effect of PCR amplicons on the resultant DNA profiles of the blood and hair samples after sample contamination. Diluted PCR amplicons were added directly to the blood and hair samples prior to DNA extraction and analysis. DNA was extracted from contaminated and control samples after different time periods and these were labeled as shown in Table 2. Table 2: Labels of the biological samples extracted after each time period. No Sample Label 1 Hair K562 Amplicons H Hair K562 Amplicons H Bloodstain K562 Amplicons BS Bloodstain K562 Amplicons BS Negative Bloodstain Control NBS 6 Negative Hair Control NH Two test trials were set up for this experiment. The first trial was set up with ten time periods (Table 3); immediate DNA extraction (T 0 ), 24 hours (T 1 ), 4 days 45

46 (T 2 ) and extraction after 1(T 3 ), 2(T 4 ), 4(T 5 ), 5(T 6 ), 7 (T 7 ), 10(T 8 ) and 12 (T 9 ) weeks. The second trial was prepared at a later date under the same conditions as the first time trial. Six time trial periods for this longevity test were used (Table 4); immediate extraction (T 0 ), 2(T 1 ), 4 (T 2 ), 5 (T 3 ) 7 (T 4 ) and 10 weeks (T 5 ) The Addition of 10-3 and 10-4 K562 Amplicons to Blood and Hair Samples Based on the results obtained from Section 2.3.1, K562 amplicons 10-3 and 10-4 dilutions were used in this experiment. From 10-3 and / or 10-4 K562 diluted amplicons, 1.0 μl was added directly to dried bloodstain samples, while hairroot samples were immersed in to the K562 amplicons 10-3 or 10-4 dilution tubes for 10 seconds and placed in a container. Both of the contaminated samples (the bloodstain and the hair root) were separately kept on the lab bench. Control samples were separately prepared. The samples were exposed to an open unprotected environment as this is usually the case in real crime scenes. DNA extraction of the contaminated biological samples was carried out as the time trial proposed; the first sample set was extracted immediately (T 0 ) following the addition of PCR amplicons; and the remaining samples were kept on the lab bench and extracted at the appropriate times (Table 3). Negative controls (uncontaminated bloodstain and hair) were also included in each time trial periods. Two sample set were used in each time trial time as shown in Table 3. 46

47 Table 3: Set of samples extracted from the first trail time period after direct contamination. First Trial Time Periods T0 T1 T2 T3 T4 T5 T6 T7 T8 T9 First Sample Set H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 Second Sample Set H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 Controls NBS NBS NBS NBS NBS NBS NBS NBS NBS NBS NH NH NH NH NH NH NH NH NH NH DNA extraction and quantification were carried out as stated in Section DNA profiles were obtained as stated in Sections and The second time trial was carried out under the same conditions as the first time trial series. Two replicates and one control sample were included in each of the time periods for both time test trials. Table 4: Set of samples extracted from the second trail time periods after direct contamination. Second Trial Time Periods T0 T1 T2 T3 T4 T5 First Sample Set H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 Second Sample Set H10-4 H10-4 H10-4 H10-4 H10-4 H10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 BS10-4 H10-3 H10-3 H10-3 H10-3 H10-3 H10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 BS10-3 Controls NBS NBS NBS NBS NBS NBS NH NH NH NH NH NH 47

48 2.5 Effect of time on Presence of Sprayed Amplicons in Blood and Hair DNA Profiles under Simulated Crime Scene Conditions The aims of these experiments were to determine the presence of the PCR amplicons in crime scene samples. As well as to examine the effect of spraying amplicons on the resulting blood and hair DNA profiles under simulated crime scene conditions. In addition, trial experiments were performed to examine the amplicons survival using the spray method for amplicon contamination. This method of application was chosen as it is considered to be the most likely method criminals would use to distribute PCR amplicons at a crime scene Preparing the Crime Scene Model Two crime scene models were assembled on two different bench coat sheets, one sheet for each type (Figure 16) K562 and A diluted amplicons (Promega Corporation, Madison, WI) were used to contaminate the forensic samples (hair and bloodstain) under a simulated crime scene condition. An amount of 30 μl of undiluted amplicons was aliquoted into the 50 ml spray bottle and autoclaved distilled water was added to make up 30 ml of 10-3 amplicon dilution. The volume of a single pump of the spray bottle was approximately 100 µl. A trial test was carried out using a spray bottle filled with coloured water to spray solution on a white background so that the droplets would be highly visible (Appendix D). Based on this, the best height and the distance from which the amplicons should be sprayed onto the forensic samples were determined, to ensure the even spread of droplets with uniform size on the surface. These were found to be at 30 cm height from the surface with an angle of 120 o in a retort stand and 27 cm away from the stand as shown in Figure 16. The surface area of the spray was calculated to be approximately cm 2. It was calculated that the concentration of amplicons was 0.28 μl /cm 2. The estimated amount of amplicons on the bloodstain was 0.06 μl for every 2 mm diameter bloodstain patch. 48

49 Figure 16: Crime Scene Model Preparation. Refer to Appendix D for the preparation steps Spraying Amplicons onto Crime Scene Samples The 10-3 K562 and/or A diluted amplicons were chosen as the contaminant in this test based on the results obtained from Section Each test was carried out by spraying three full single pumps of the spray bottles (300 μl in total) over the samples. DNA extraction of the contaminated crime scene samples was carried out as the time trial periods. The first sample set was collected and extracted immediately (T 0 ) after spraying the PCR amplicons; the second set was collected one week later (T 1 ); the third set was collected after two weeks (T 2 ); and the fourth set was collected after 5 weeks (T 3 ), and the last set was collected after seven weeks (T 4 ). Negative and positive controls were included in each time period. The positive control was the crime scene samples placed on a separate bench, with 10-3 K562 and / or A diluted amplicons (2 μl) being added directly onto the samples to ensure that they were contaminated. The negative control was crime scene samples with no contamination as shown in Figure

50 Figure 17: Set-up of the Simulated Crime Scene Model. The crime scene setup included the positive and negative controls. Each set of samples included: a positive and negative control bloodstain and hair sample; two contaminated bloodstain samples, one with 10-3 K562 and the other one with A; and four contaminated hair root samples two with K562 and two with 9947A (Table 5). One of the of contaminated hair root samples from both contaminated amplicons was subjected to a wash step before DNA extraction. All samples were extracted and quantified as stated in Section DNA profiling was carried out as stated in Sections and

51 Table 5: Set of Samples Extracted After Each Time Period under the Simulated Crime Scene Conditions No samples Label 1 (-ve) Negative Bloodstain Control NBS 2 (+ve) Positive Bloodstain K562, 3X spray Control PBSK562 3 (+ve) Positive Bloodstain A, 3X spray Control PBS9947A 4 Bloodstain K562, 3X spray BSK562 5 Bloodstain A, 3X spray BS9947A 6 (-ve ) Negative Hair Control NH 7 (+ve) Positive Hair Control K562, 3X spray PHK562 8 (+ve) Positive Hair Control A, 3X spray PH9947A 9 Hair K562 3X spray (washed) HK562W 10 Hair K562 3X spray (unwashed) HK562UW 11 Hair A 3X spray (washed) H9947AW 12 Hair A 3X spray (unwashed) H9947AUW 2.6 Contamination of Bloodstain Samples Deposited on Different Surfaces The aim of this experiment was to test the effect of different surface types on the resulting DNA profiles of the contaminated blood samples. The surfaces were included; wood, metal, tiles, plastic, glass, carpet and fabric (Figure 18). The bloodstains were prepared by spotting 20 μl from the fresh blood on each surface and allowing to dry for two hours. 51

52 Figure 18: Seven different materials were used to examine the recovering ability of the blood DNA profile and amplicon contamination Spraying 10-3 K562 Amplicons on Blood Samples Diluted K562 amplicons 10-3 were used to contaminate the bloodstains on each surface. Amplicons were prepared as stated in Section 2.3. An amount of 30 μl from 10-3 K562 amplicons was aliquoted into the 50 ml spray bottle and autoclaved distilled water was added to make up 30 ml of 10-3 K562 amplicon dilution. Each surface was subject to three direct sprays as stated in Section The crime scene model was assembled as stated in Section Positive and negative controls were included; the positive control was a cotton swab that had been sprayed with amplicons directly, while the negative control was a cotton swab without anything. In addition, uncontaminated bloodstain was included for comparison (Table 6) Contaminated Blood DNA Profiles Using Identifiler From each surface, one bloodstain was swabbed using a wet cotton swab five minutes after spraying the amplicons. DNA was extracted and quantified as stated in Section DNA amplification was carried out using the 52

53 AmpFlSTR Identifiler PCR Amplification Kit as stated in Section DNA profiles were obtained as stated in Sections The samples were labeled as shown in Table 6. Table 6: Labels of the Bloodstain Samples No samples Label 1 Bloodstain K562 Swab from the wood BSK562 W 2 Bloodstain K562 Swab from the metal BSK562M 3 Bloodstain K562 Swab from the tiles BSK562T 4 Bloodstain K562 Swab from the plastic BSK562P 5 Bloodstain K562 Swab from the glass BSK562G 6 Bloodstain K562 Swab from the carpet BSK562C 7 Bloodstain K562 Swab from the fabric BSK562F 8 (+ve) cotton swab + K562 3x spray (+ve) 9 (- ve ) cotton swab (-ve) 10 Bloodstain (control) BS 53

54 CHAPTER 3: RESULTS 3.1 The Characteristics of Biological Sample Profiles Genomic K562 and 9947A Standard DNA Profiles The standard genomic K562 and 9947A DNA profiles were obtained using 1ng DNA templates as stated in Sections and Both profiles were as expected (Table 7) compared to the provided information in the manual. As shown in Figure 19; A, the K562 DNA profile at the D21S1 locus shows three peaks, al29, al30 and al31, and an imbalance of the peaks height FGA heterozygous locus. The abnormal characteristics of the K562 DNA profile are due to the mutation of the K562 cells, since it is derived from myelogeneous leukemia cells. The 9947A DNA profile (Figure 19; B) shows similarity of heterozygous locus allele s peaks height across five loci, and double the heterozygous loci peak height within the homozygote loci at the other five loci. Peaks on each electrophoregram are labeled with allele s repeat numbers in a particular individual s profile at each specific locus. Both DNA profiles showed only an X peak representative of a female profile as expected. A summary of genomic K562 and 9947A DNA profiles are listed in Table 7. Table 7: Genomic K562 and 9947A DNA Profiles Locus Fragment size K A range (bp) DNA Profile DNA Profile Amelogenin X:104, Y: 109 X X X X D3S D8S D2S vwa D21S D13S FGA D7S D18S Table 7 illustrates the fragment range size for each locus. The STR markers are listed from the smallest to largest fragments. The abnormal characteristics of the K562 DNA profile at the D21S1 locus shows three peaks, al29, al30 and al31, are due to the mutation of the K562 cells, since it is derived from myelogeneous leukemia cells. 54

55 A : Genomic K562 DNA profile B: Genomic 9947A DNA profile Figure 19: Genomic profiles of the standard K562 and 9947A DNA. In the results shown above, DNA profiles were obtained from K562 (A) and 9947A (B) standard DNA using the multiplex STR typing assay 'AmpFlSTR Profile Plus. This assay examines nine autosomal STRs and a region of the SRY gene found on the X and Y chromosomes that can be used for sex determination. The lengths of the amplified DNAs are shown by the scale from 100 bp to 310 bp at the top of the figure. And the relative fluorescence intensities (RFU) values are scaled from 0 RFU up to 6000 RFU. 55

56 3.1.2 The Characteristics of the Genomic Blood and Hair DNA Profiles Blood and hair DNA profiles were obtained from uncontaminated samples (Sections 2.2.1, and 2.2.3) are shown in Figure 20. The alleles were determined using RFU values of the blood and hair DNA profiles as shown in Table 8. The electrophoregrams of blood and hair STR profiles represent traces associated with the three sets of fluorescent dyes (blue, green, yellow). Peaks on each electrophoregram are labeled with repeat numbers in a particular individual s profile at each specific locus. The blood DNA profile (Figure 20; A) shows both X and Y peaks represent a male profile as expected, since the donor of the sample was male. The hair DNA profile (Figure 20; B) shows only an X peak representative of a female profile as expected also since the donor for hair samples was female. The RFU values of the homozygote loci in both profiles (blood and hair) show approximately double the height of the heterozygote loci, and similar peak heights across the heterozygote loci. This is the normal characteristic expected to be obtained from uncontaminated profiles. Table 8: Genomic blood and hair DNA profiles Locus Fragment Size Range (bp) Hair DNA Profile Blood DNA Profile Amelogenin X:104, Y: 109 X X X Y D3S D8S D2S vwa D21S D13S FGA D7S D18S Table 8 illustrates the fragment range size for each locus. The STR markers are listed from the smallest to largest fragment 56

57 A: Genomic Blood DNA Profile B: Genomic Hair DNA Profile. Figure 20: Genomic Blood and Hair DNA Profiles. In the results shown above, DNA profiles were obtained from biological samples using the multiplex STR typing assay 'AmpFlSTR Profile Plus kit. (A) represent blood DNA profile obtained from a male sample, while (B) represent the hair DNA profile obtained from female sample. The lengths of the amplified DNAs are shown by the scale from 100 bp to 310 bp at the top of the figure. And the relative fluorescence intensities (RFU) values are scaled from 0 RFU up to 6000 RFU.

58 3.1.3 The Characteristics of the Re-amplified DNA Amplicons The characteristic of the re-amplified PCR amplicons from the genomic K562 DNA for sample contamination was studied. A series of K562 DNA profiles were obtained from different dilutions of K562 amplicon as stated in Section (10-1, 10-2, , 10-5 and 10-6 ). This experiment was performed to observe the characteristics of the K562 re-amplified amplicon profiles and to determine the appropriate dilution factors to be used as a contaminant source (see Figure 21). Figure 21: Electropherograms Showing the K562 Amplicon DNA Profiles Panels A to F represent the amplicon K562 DNA profile obtained from 10-1, 10-2, , 10-5 and 10-6 diluted K562 amplicon respectively. The fluorescence intensity of the PCR products obtained from using 10-3 and 10-4 K562 amplicons were above the threshold and no missing amplification at the larger markers were present (panel C and D). However, the fluorescence intensity of the PCR products exceeded the detection threshold of the DNA Sequencer when 10-1 and 10-2 K562 amplicons were used (panel A and B). While there was missing amplification at the larger markers, and so only a partial DNA profiles were obtained when 10-5 and 10-6 K562 amplicons were used to generated the DA profiles. 58

59 The DNA profiles obtained from 10-3 K562 amplicons showed a clear and full amplification for all STR markers as shown in Figure 21; Panel C. DNA profiles obtained from 10-4 K562 amplicons (Figure 21; Panel D) showed similar results but lower relative fluorescence intensities (RFU) values. The DNA profiles generated from 10-1 and 10-2 K562 amplicons could not be determined due to the strong signals of the fluorescence intensity caused by the large number of amplicons (Figure 21; Panels A & B). This resulted in signals which exceeded the detection threshold of the 3730 DNA Sequencer and prevented correct determination of DNA profiles. The DNA profiles generated from 10-5 and 10-6 K562 amplicons showed a lack of amplification of the larger markers such as D13S317, FGA, D7S820 and D18S51, resulting in partial or incomplete DNA profiles (Figure 21; Panels E & F). Based on the above results, 10-3 and 10-4 dilutions of K562 amplicons were used in subsequent experiments to contaminate the blood and hair samples. The amount of PCR amplicons was estimated on the amount of DNA in the gel as shown in Figure 22.. Figure 22: Gel Electrophoresis show image of PCR product Lane 1; 100 bp DNA ladder (Progma ) ; lane 2; hair, lane 3; blood were added for comparison; lane 4K562 amplicons; and Lane 5; water negative control. The 200bp band in the 100 bp marker ladder contains 25ng (25ng/ μl). Based on this value the total amount of PCR amplicon DNA was estimated to be

60 ng. Since 5 μl of PCR amplicons was added to the gel, the DNA concentration of amplicons in the PCR reaction was estimated to be 100ng/ μl. One μl of the PCR amplicons was used to make the dilution of 10-3 and 10-4 as stated in Sections The concentration of K562 amplicons in the 10-3 and 10-4 dilutions was approximately 100pg/1μL and 10pg/1μL respectively 3.2 Examine the Effect and Presence of PCR Amplicons on the Blood and Hair DNA Profiles after Direct Contamination The Effect of K562 DNA Amplicons on Blood DNA Profile The effect of K562 amplicon contamination on blood DNA profiles was determined by comparing the blood DNA profile obtained prior to and following the addition of K562 amplicons to blood samples. The uncontaminated control samples at each time period produced DNA profile with the correct allele designation for the blood sample. Each contaminated bloodstain sample that was analyzed exhibited an alteration of the genomic blood profile compared to the uncontaminated bloodstain sample up to a certain time period as shown in Figure 23. The impact of amplicon contamination on blood DNA profile was either adding allele unique to 10-4 K562 DNA (black stars), dropout (become undetectable) of the original blood alleles (green stars) and the addition of extra alleles into blood DNA profile that are originally not present in both profiles (blue stars). Taking into consideration that some alleles are present in both DNA profiles (red stars), a complete 10-4 K562 DNA profile in addition to the genomic blood DNA profile was present (Figure 23). The blood DNA profile from the contaminated blood sample also shows that X allele peak height (31517 RFU) was much higher than the Y allele peak height (1394 RFU) of the contaminated blood sample (Table 9). The reason for this is that the X allele amplification was carried out using DNA templates from both samples (blood and 10-4 K562) while the Y allele was amplified using only the blood DNA templates. The imbalance of the peaks height between the X and Y 60

61 of the contaminated blood DNA profile can change the genotype from male (the original blood genotype) to a mixture of female contributor and a minor contributor of uncertain gender. Furthermore, some of the peaks height from the 10-4 K562 amplicons and blood genomic DNA approached the same height, such as D21S11 locus and D13S317 (Table 9). This similarity of the peaks height could be easily interpreted as a mixed profile and not as amplicon contamination. The result shows that such contamination can mask the correct interpretation of the blood DNA profiles. Similar result to 10-4 K562 amplicons contamination was obtained when 10-3 K562 amplicons was used as a contaminant source. The same amplicon contamination effect was observed within the blood DNA profile, confirming that, within the range of dilutions examined, the different dilution had little or no effect on the contamination results. 61

62 Control blood DNA profile The resultant of blood DNA profile shows the impact of 10-4 K562 amplicons contamination. This figure illustrates the appearance of a mixed profile. The peaks marked with black stars represent the allele peaks unique to 10-4 K562 amplicons. The peaks marked with green stars represent dropout of the original blood alleles. The peaks marked with blue stars represent the new allele peaks to both profiles, and the peaks marked with red stars represent the allele found on both profiles. The control blood DNA above was added for comparison. Figure 23: Electropherogram Shows the Genomic Blood DNA Alteration after 2 Weeks of 10-4 K562 Amplicons Contamination. 62

63 Table 9: The RFU Values of the Contaminated Genomic Blood DNA with 10-4 K562 Amplicons after Two and Ten Weeks. Locus Genomic Blood Genomic K 562 Bloodstain +K562 (2 weeks) Bloodstain +K562 (10 weeks) Allele Height Allele Allele Height Allele Height Amelogenin X Y X X X Y X Y D3S D8S D5S vwa D21S D13S FGA D7S D18S The numbers highlighted in red represent the peak heights of the corresponding 10-4 K562 diluted amplicons, while the numbers highlighted in blue represent the peak heights corresponding to blood and K562 amplicons and the numbers highlighted in yellow represent the peak heights corresponding to extra alleles. The STR markers are listed from the smallest to largest fragments. 63

64 3.2.2 The Survival of K562 DNA Amplicons in Contaminated Blood DNA Profile The blood DNA profiles of the contaminated bloodstain samples after immediate addition of amplicons (T 0 ), 24 hours (T 1 ), 4 days (T 2 ), 1 week (T 3 ) and 2 weeks (T 4 ) periods showed alteration of blood DNA profile (Figure 23). On the other hand, the blood DNA profiles extracted from the contaminated bloodstain samples after a period of 5 weeks (T 4 ) shows less impact of 10-4 K562 contamination. The study also shows that the presences of 10-4 K562 amplicons alleles were present up to a period of 10 weeks (T 8 ) after contamination (Figure 24). The RFU values of the peaks contributed by the 10-4 K562 amplicons were smaller than RFU values of the peaks corresponding to the blood DNA profile (Table 9). As a result it was possible to differentiate between the genomic blood which has the higher RFU values than the 10-4 K562 amplicon alleles with the lower RFU values. In addition, the RFU values of the X and Y were approximately similar, which indicates no change in the apparent gender genotype. A simple illustration of the contaminated results of the blood sample is shown Figure 25. The results of the DNA profiles obtained from three different blood samples (control, contaminated sample after 2 and 10 weeks) demonstrated the impact of 10-4 K562 amplicons contamination. Figure 25 shows that 10-4 K562 amplicons contamination after the 10 weeks was not capable of masking or altering the original blood DNA profile. The RFU values of the X allele in the Figure 25 were truncated to (RFU) in order to better demonstrate the other STR markers. 64

65 Control blood DNA profile Figure 24 shows the comparison between the genomic blood DNA profile resulting from the addition of 10-4 K562 diluted amplicons (right) and control blood sample (Top) after a period of 10 weeks show that the allele peaks unique to 10-4 K562 amplicons (black stars) are still presence within the blood DNA profile but with less impact of 10-4 K562 amplicons contamination. The peaks marked with black stars represent the allele peaks unique to 10-4 K562 amplicons. Figure 24: Electropherogram Shows the Contaminated Genomic Blood DNA Profile after Ten Weeks of Contamination with 10-4 K562 Amplicons. 65

66 Figure 25: The Impact of 10-4 K562 Amplicon Contamination on Blood DNA Profile. The above diagrams illustrated the impact of 10-4 K562 amplicon contamination onto blood DNA profile after two weeks, and after ten weeks of contamination. The control blood DNA profile was added for comparison. The black and gray columns represent the STR markers unique to genomic blood DNA, the red columns represent the STR markers unique to 10-4 K562 amplicons, and the yellow column represents the extra allele s presence in the blood DNA profile. 66

67 3.2.3 The Effect of K562 DNA Amplicons on the Hair DNA Profile The effect of K562 amplicons contamination on the hair DNA profile was determined by comparing the contaminated hair DNA profile with the uncontaminated hair DNA profile. The results obtained from the time trail experiments of amplicon contamination of hair samples (Section 2.4.1) showed that the 10-4 K562 amplicons were carried through the Chelex extraction method and subsequently re-amplified along with the genomic hair DNA. However, the impact of 10-4 K562 amplicon contamination on hair DNA profiles was not as noticeable compared to the impact of 10-4 K562 amplicon contamination on blood DNA profile (Figure 26). The peaks heights of the 10-4 K562 amplicon were smaller than the peaks height corresponding to the hair DNA profiles. In addition, the peaks height of the homozygote loci in hair DNA profile shows double the height of the heterozygote loci and similar peaks height across the heterozygote loci (Figure 26). The similarity between the peaks height at a particular locus represents the normal characteristic usually expected to be obtained from the uncontaminated profile, which was not the case here. The hair DNA profile obtained from the contaminated samples shows additional alleles corresponding to K562 amplicons; however, the presence of amplicon alleles has no effect on hair DNA profile interpretation. Similar results were obtained when 10-3 K562 amplicons was used as a contaminant source. The same amplicon contamination effect was observed within the hair DNA profile, confirming that different dilutions had little or no effect on the contamination results. 67

68 Control Hair DNA Profile Figure 26 shows the 10-4 K562 amplicon contamination after 1 week & 5 weeks. The peaks marked with black stars represent the alleles unique to 10-4 K562 amplicon, and the peaks marked with red stars represent the allele found on both profiles. The control hair DNA above was added for comparison. Figure 26: Electropherogram Shows the Contaminated Genomic Hair DNA Profile after One Week of 10-4 K562 Contaminations. 68

69 3.2.4 The Survival of K562 DNA Amplicons in Contaminated Hair DNA Profile The control samples at each of these time periods produced DNA profiles with the correct allele designations for hair sample. The result from the time line experiments after immediate addition of amplicons (T 0 ), 24 hours (T 1 ), 4 days (T 2 ), 1 week (T 3 ) and 2 weeks (T 4 ) periods showed partial K562 DNA profile in addition to hair DNA profile (Figure 26). However, after 5 weeks (T 6 ) of amplicon contamination, the hair DNA profile showed only one or two peaks corresponding to the 10-4 K562 amplicons profile (Figure 27). This indicates the survival of amplicons decreases with time. The result also shows there was a loss of amplification of the larger STR markers. This is possibly due to DNA degradation during the course of time experiments. In general, the presence of amplicon contamination within the hair DNA profile had little or no effect; consequently the hair DNA profiles obtained from the contaminated hair samples were possible to be interpreted. The impact of 10-4 K562 contamination on hair is summarized in Appendix E. Also a summary of the RFU values correspond to the contaminated and uncontaminated genomic hair DNA profile is provided in Appendix F. Figure 27: Electropherogram Shows the Contaminated Genomic Hair DNA Profile after Five Weeks of 10-4 K562 Contaminations. 69

70 3.3 Effect of Sprayed PCR Amplicons on Blood and Hair Samples under Simulated Crime Scene Conditions The Effect of Spraying 9947A and K562 Amplicons on Blood DNA Profile The standard genomic DNA samples, K562 and 9947A, were used to examine the effect of spraying the PCR amplicon directly to a bloodstain before the sample was extracted. In this experiment the results of the positive and negative PCR controls confirmed that the samples were not accidentally contaminated. The positive control was the 9947A genomic DNA and negative control was distilled water. The control blood sample from each time period shows a correct blood DNA profiles. The results of the blood DNA profiles obtained from the time line experiments showed that STR marker (e.g. D7S820 locus) consisted of more than two alleles (Figure 28). The extra alleles present in the DNA profile were peaks corresponding to 10-3 K562 amplicons profile, and were approximately the same height as blood alleles this caused the DNA profiles to appear as a mixture of two contributors. Under these conditions the resultant DNA profile was difficult to interpret (Figure 28). The result of the blood DNA profile also shows a significant difference between the peaks height corresponding to X and Y, the Y allele peak height was very small as compared to the X allele peak height. Such a result can mask the genotype of the blood DNA profile, and it will appear as female DNA profile (amplicon) instead of male DNA profile (blood) (Refer to Table 10 & Figure 29; green dye layer). In addition, some of the original blood alleles were not detected (Figure 29; blue stars). This is possibly due to the significant difference between the allele peaks height at a particular locus (for example vwa). This experiment showed that amplicon contamination of blood samples has a strong impact on the blood DNA profiles. This impact is seen across all STR markers. Amplicon contamination can produce false DNA profile interpretation, or mask the original profile. 70

71 3.3.2 The Survival of 9947A and K562 Amplicons in Contaminated Blood DNA Profile Full amplicon profiles were obtained from blood DNA profiles extracted from contaminated blood samples after immediate (T 0 ), 1 week (T 1 ) and 2 weeks (T 2 ) periods after spraying the PCR amplicons (Section 2.5.2). The study also shows that the impact of amplicon contamination on blood DNA profiles begins to decrease after 5 weeks of amplicon contamination that is contamination effect decreased with time. The result of spraying 10-3 K562 diluted amplicons into bloodstain samples shows that PCR amplicons are able to survive for up to 7 weeks in a typical crime scene conditions (Table 10 and Figure 30). Although amplicon alleles were still present even after 7 weeks, the amplicon allele peak heights were generally smaller than the allele peaks height of the blood DNA profile. These peaks height corresponded to the amplicon alleles after 5 weeks and up to 7 weeks, within the contaminated blood DNA profiles, usually dismissed by the software as stutter or artifact peaks. A summary of amplicon DNA and contaminated blood DNA profiles and their alleles RFU is shown in Table 10. A similar result was obtained when A was used. Amplicon contamination was still present after a period of 7 weeks as shown in Figure 31. However, the peak heights corresponding to 9947A amplicon were smaller than the blood DNA peaks, thus the contaminated blood DNA profile was still able to be interpreted. 71

72 Figure 28: Electropherograms Show the Results of Spraying the PCR Amplicons onto Bloodstain Samples. The blood DNA profiles were obtained from the bloodstain samples before and after it was sprayed with A and/or 10-3 K562 amplicons. Panel 1: Represents the genomic blood DNA profile. Panel 2: Represents the genomic K562 DNA profile. Panel 3: Represents the resultant contaminated genomic blood DNA profile with 10-3 K562 amplicons. Panel 4: Represents the genomic 9947A DNA profile. Panel 5: Represents the resultant contaminated genomic blood DNA profile with A amplicons. The peaks marked with black star represent the allele peaks unique to each amplicons present in the resulting genomic blood DNA profile. And the peaks marked with red star represent the peak alleles belonging to both profiles. The X- and Y- axes indicate fragment size (bp) and the signal intensity (RFU) respectively. 72

73 Figure 29: The Contaminated Genomic Blood DNA Profiles with 10-3 K562 Amplicons under Different Time Periods. The figure of the three dyes above show the STR locus plus a gender-typing locus present in the profile plus kit. These STR loci are fluorescent labeled with 5-FAM, JOE and NED NHS-ester dyes which are detected on automated sequencing ABI PRISM instrument as blue, green and yellow fluorescence respectively. The black stars represent the contributed amplicon alleles present in the genomic blood DNA profile, the peaks marked with blue stars represent the missing alleles from the original blood DNA profiles, the peaks marked with green stars represent the extra alleles and the red stars represent the alleles belonging to both profiles. The X- and Y- axes indicate fragment size (bp) and the signal intensity (RFU) respectively. 73

74 Control blood DNA profile Figure 30: The Contaminated Blood DNA Profiles with 10-3 K562 Amplicons after Five and Seven Weeks. The figure shows the results of the contaminated blood DNA profiles extracted from the contaminated bloodstain with 10-3 K562 amplicons after a period of five and seven weeks. The black stars represent the contributed amplicon alleles and the red stars represent the alleles belong to both profiles. The X- and Y- axes indicate fragment size (bp) and the signal intensity (RFU), respectively. The control blood DNA above was added for comparison. 74

75 Figure 31: The Comparison between the Pre and Postcontaminated Blood DNA Profiles with A Amplicon after Seven Weeks. The Electropherograms of the three dyes show the nine STR primer pairs plus a gender-typing markers present in the AmpFlSTR Profile Plus. Panels 2, 4 and 6: Represent the contaminated blood DNA profile. Panels 1, 3 and 5: Represent the control blood DNA profile. The peaks marked with black stars represent the allele peaks unique to A amplicon, and the red stars represent the peak alleles belong to both profiles. The X- and Y- axes indicate fragment size (bp) and the signal intensity (RFU), respectively. 75

76 Table 10: A Summary of Amplicon and Contaminated Blood DNA Profiles and their Respective RFU Values. Locus Genomic Blood Genomic K 562 Genomic blood + K562 Amplicons 0 hrs Genomic blood + K562 Amplicons 1 week Allele Height Allele Height Allele Height Allele Height Amelogenin X Y X X X Y X Y D3S D8S D5S vwa D21S D13S FGA D7S D18S Locus Genomic blood + K562 Amplicons 2 weeks Genomic blood + K562 AmpliconsGenomic blood + K562 Amplicons 5 weeks Genomic blood + K562 Amplicons 7 week Allele Height Allele Height Allele Height Allele Height Amelogenin X Y X Y X Y X Y D3S D8S D5S vwa D21S D13S FGA D7S D18S The numbers highlighted in red represent the peaks height of the corresponding 10-4 K562 diluted amplicon, while the numbers highlighted in blue represent the peaks height corresponding to blood and K562 amplicons and the numbers highlighted in yellow represent the peaks height corresponding to additional alleles. The STR markers are listed from the smallest to largest fragments. 76

77 3.3.3 The Effect of Spraying 9947A and K562 Amplicons on Hair DNA Profile The standards K562 and 9947A were used to examine the effect of spraying the PCR amplicon directly to hair samples before the DNA extraction. The ability to detect 10-3 K562 or A after amplicon contamination of unwashed hair samples was determined in this study (Figure 32; Panel B). The results of the unwashed contaminated hair DNA profiles obtained from the time line experiments showed that the peaks height contributed by the 10-3 K562 or A amplicons were approximately the same as the peaks height corresponding to the hair profile causing the profile to appear to be from two contributors (Figure 32; Panel B). Complete 9947A amplicon and hair profiles were seen in the contaminated hair DNA profile extracted from the unwashed hair samples (Figure 33). There was no alteration of the hair gender type due to the fact that both sources of the DNA templates in this study were generated from genomic female DNA. A similar result was obtained after using K562 amplicons (Refer to Appendix G). In contrast, this study shows no amplicon peaks detected in genomic hair DNA profile extracted from washed hair samples (Figure 32; Panel C). There were no significant difference between the hair DNA profiles extracted from the washed contaminated hair sample and the controlled uncontaminated hair sample (Figure 34). However, there was a drop-off in RFU values corresponding to hair DNA profile (Table 11) The survival of 9947A and K562 Amplicons in contaminated Hair DNA Profile According to the results of this study, the alteration of the hair DNA profile through both times trails up to 7 weeks (T 4 ), as stated in Section , occurs only when the washing step was not performed before the DNA extraction. This result demonstrates that the washing for hair samples before DNA extraction, as recommended by the Chelex User s Manual, can eliminate amplicon carryover contamination under the conditions used in this study. A comparison 77

78 between the hair DNA profile extracted from washed and unwashed samples can be seen in Appendices H & I. Figure 32: Electropherograms Showed the Comparison between the contaminated Hair DNA Profiles with and without the washing step. The figure above show the pre and post-contaminated hair DNA profiles. Complete amplicon and hair alleles were seen in the contaminated hair DNA profiles extracted from unwashed samples (Panels B). While Panels (C) show the resulting genomic hair DNA profiles extracted from washed samples. Panels (A) represent the control hair DNA profile. The peaks marked with black stars represent the allele peaks unique to each amplicons present in the hair DNA profile. The peaks marked with red stars represent the allele found on both profiles. The X- and Y- axes indicate fragment size (bp) and the signal intensity (RFU), respectively. The red peaks are the DNA size standards. 78

79 Figure 33: The Contaminated Genomic Hair DNA Profile Extracted from Unwashed Hair Sample. The Electropherograms of the three dyes show the nine STR primer pairs plus a gender-typing markers present in the AmpFlSTR Profile Plus. These STR markers illustrate the impact of amplicon contamination on hair DNA profile extracted from unwashed contaminated hair. The peaks marked with black stars represent the allele peaks unique to A amplicon DNA alleles. The peaks marked with red stars represent the allele peaks unique to hair DNA alleles. 79

80 Figure 34: The Contaminated Genomic Hair DNA Profile with A Extracted from Washed hair sample. The Electropherograms of the three dyes show the resultant hair DNA profile obtained from the contaminated hair sample after the washing step shows similar DNA profile obtained from the control hair sample. 80

81 Table 11: The Comparison between Genomic Hair DNA Profile Before and After A and/ or 10-3 K562 Amplicon Contamination under the Simulated Crime Scene Conditions, and their RFU Values. Control Hair Control K652 Hair +10^-3 K562 Unwashed Hair+10^-3K562 Washe Locus Allele Height Allele Height Allele Height Allele Height Amelogeni X X 8737 X X X X X X D3S D8S D5S vwa D21S D13S FGA D7S D18S Control Hair Control 9947A Hair +10^ A Unwashed Hair + 10^ A Washed Locus Allele Height Allele Height Allele Height Allele Height Amelogeni X X 8737 X X 5646 X X X X D3S D8S D5S vwa D21S D13S FGA D7S D18S The numbers highlighted in red represent the peaks height of the corresponding diluted amplicon, while the numbers highlighted in blue represent the peaks height found in both blood and amplicon and the numbers highlighted in yellow represent the peaks height corresponding to additional alleles. The STR markers are listed from the smallest to largest fragments. 81

82 3.4 The Influence of Different Surface Types on the Contamination of Blood DNA Profile This experiment was conducted to examine the effect of different surface type on the recovering ability of the blood DNA profile and amplicon contamination. Bloodstain samples were deposited on seven different surfaces (wood, metal, tiles, plastic, glass, carpet and fabric); these were then sprayed with 10-3 K562 amplicons and extracted by Chelex. The AmpFlSTR Identifiler PCR Amplification Kit was used for DNA profiles determination as shown in Section The results show that the swab samples recovered from the rough porous surface such as carpet or fabric contain smaller quantity than the non-porous smooth surface type such as metal or glass (Figure 35). The results of the study also showed that the electropherograms of the DNA profile recovered from the smooth surfaces have better results (higher peaks) than rough surfaces, as shown in Figure 35 panels 7 and 8. However, the result shows that the different surfaces types have no influence on the presence of amplicons contamination within the blood DNA profiles under simulated crime scene conditions (Figure 35). 82

83 Figure 35: Electropherograms Show the Comparison between Contaminated Blood DNA Profiles with 10-3 K562 amplicons Swabbed from Different Surfaces. In the above figure, Panel 1: Represents the resultant genomic blood DNA profile. Panel 2, 3, 4, 5, 6, 7 and 8: Represent the contaminated blood DNA profile swabbed from wood, metal, tiles, plastic, glass, carpet and fabric surfaces respectively. The peaks marked with black stars represent the allele peaks unique to 10-3 K562 amplicons present in the resulting genomic blood DNA profile. The X- and Y- axes indicate fragment size (bp) and the signal intensity (RFU) respectively. 83

84 CHAPTER 4: DISCUSSION 4.1 Introduction The PCR-STR analysis of forensic samples enables the exponential amplification of very small amounts of DNA. Typically, 1 ng or less DNA templates can be analyzed and high-quality DNA profiles produced. This level of sensitivity allows forensic scientists to extract and amplify DNA from minute or degraded samples. At the same time, the highly sensitive PCR technique creates an opportunity for criminals to use amplicons to deliberately contaminate biological evidence. This study investigated the deliberate contamination of the biological samples with amplicons and the resulting effect of this contamination on the DNA profiles of the forensic samples. The following chart summaries the steps undertaken in this study (Figure 36). Figure 36: Summary of research outcomes assessed in this project. 84

85 4.2 Characterisation of Biological and Standard K562 and 9947A DNA Amplicons Profiles In this study two common forensic samples, blood and hair DNA were extracted with the Chelex method using low amounts of biological samples (3 mm blood stain or 2 μl whole blood, or three hair-root), to mimic typical crime scene samples. The genomic blood, hair, K562 and 9947A DNA profiles were obtained with the AmpFlSTR Profiler Plus amplification kit from 1 ng of templates DNA. These profiles were used as reference profiles for comparison between contaminated and uncontaminated samples in this study. Studying the characteristics of blood, hair, K562 and 9947A DNA profiles showed a standard characteristic of uncontaminated DNA profiles as expected. The homozygous locus shows a single peak that is approximately twice the height of alleles seen at a heterozygous locus within the same dye color. This is due to the doubling of the signal from two alleles of the same size (Figures 19 and 20). These profiles confirm the absence of contamination in the PCR reaction mix or any accidental contamination through the DNA extraction, amplification and STR analysis. Before using K562 and 9947A amplicons as a contaminant source, DNA profiles from the amplicons under different dilutions, in the range of 10-1 to 10-6, were obtained. The DNA profiles obtained with different dilutions of the K562 amplicons showed different results due to the input amount of the DNA templates used for amplification. Complete DNA profiles were obtained with both 10-3 and 10-4 dilution of K562 amplicons when they were re-amplified. There was no lack of amplification of the larger alleles and the RFUs values were within the normal range for analysis by the 3730 DNA Sequencer. However, the amplicon DNA profiles obtained from 10-1 and 10-2 dilutions were impossible to determine because the smaller sized markers, such as Amelogenin, D3S1358, D8S1179 and D5S818, were amplified in such an amount that the RFUs peaks exceeded the detection threshold (50 RFU) of the 3730 DNA Sequencer. Similarly, when 10-5 and 10-6 dilution of the K562 amplicons were used, incomplete K562 DNA profile was obtained due to the lack of amplification of the larger markers, such as D13S317, FGA, D7S820 and 85

86 D18S5 as seen in Figure 21. This is probably due to the selective amplification of smaller DNA fragments over the larger fragments present in an amplification reaction (Butler, 2005). Based on the above results, the 10-3 and 10-4 diluted K562 amplicons were used to measure the survival of PCR amplicons and to determine the effect of adding amplicons to biological samples. 4.3 The Effect of PCR Amplicons Applied Directly on the Biological Samples The results of the deliberate DNA contamination with 10-3 K562 amplicons indicated that the alteration of DNA profile which was obtained from contaminated biological samples with PCR amplicons can be easily achieved. Furthermore, the K562 amplicons were effectively carried through the Chelex extraction methods and subsequently re-amplified along with the genomic DNA in detectable amounts. The study demonstrates that the resultant DNA profile of the genomic blood DNA combined with amplicons show both K562 amplicon and genomic blood DNA profiles (Figure 23) and appeared to be a mixed profile. Usually the mixtures of STRs arise when two or more individuals contribute DNA to the sample being tested, resulting in more than two alleles present at a particular heterozygous marker locus (Butler, 2005). The genomic blood DNA profile shows more than two alleles present at the heterozygous STR markers such as D3S1358, vwa, and D7S820, and more than one alleles present at the homozygote markers such as FGA and D5S818 (Figure 23). In this case, the DNA profile of the original blood sample could not be determined because it was impossible to distinguish between the genomic blood and amplicon DNA profiles. The impact of amplicon contamination not only affected the original sample DNA profile by introducing alleles from amplicons into the sample DNA profile. It also introduced extra alleles that are not found in the amplicons or sample DNA profiles (Figure 23). It is likely that some of these extra alleles are stutter peaks since they are one repeat smaller than very strong allele peaks (eg: locus D3S1358 al 15, vwa al 15, FGA al 19 and D8S1179 al 11). Other additional 86

87 peaks do not have the typical characteristics of stutter peaks. These peaks were one or more repeats larger (eg: locus D3S1358 al 19, D18S51 al 17, D7S820 al 15/15 and D13S317 al 13) or smaller (D5S818 al 10) than the expected alleles. Although the reason for the appearance of these unique alleles is unknown, it is possible that they are the result of changes in the PCR reaction caused by the introduction of previous PCR reaction containing amplicons into the DNA profiling reactions. For instance, the amplicons (or failed extension products) may have acted as PCR primers and caused the appearance of the additional alleles. Similar results are commonly noted when performing nested PCR with single primers, where additional bands are commonly observed. It should be noted that additional alleles such as these occurred only in reactions where previous PCR amplifications were added to the reaction.. These results led to difficult interpretation of DNA profile because of the presence of too many alleles. The impact of amplicon contamination also caused a dropout of some original alleles of the sample DNA profile. Allele dropout is defined as the condition where an allele cannot be visualized, the small peaks height are simply not measured but are still present. It is not yet clear whether it is an extreme form of variable amplification, but this can lead to the false positive result that the genotype is a homozygote instead of heterozygote. The most obvious effect of amplicon contamination is the sexgender type alteration. In this study the amplification of the X allele was carried out using both K562 and blood DNA templates, producing much larger amount of X (RFU) than Y (RFU). Therefore when it come to comparing the sample gender type of interest with a reference sample it maybe interpreted as a mixture of a major female contributor and a minor contributor of uncertain gender. Results like this could be used to mask DNA profiles or produce false DNA profile that can incriminate another individual or to mislead an investigation. The impact of amplicon contamination on hair DNA profiles is much lower than the effect on blood samples (Figure 26). This maybe because of the application methods, the hair samples were immersed in a solution containing amplicons for 10 second, while, amplicons (1.0 μl) had been added directly to dried bloodstain samples (Section 2.4.1). The amount of amplicons that contaminate the hair samples is unknown, which may not be in sufficient quantities to alter 87

88 the hair DNA profile. The DNA profile extracted from the contaminated hair samples showed that the majority of RFU values of the peaks contributed by the K562 amplicons were too small compared to the RFU values of the peaks corresponding to the hair profile and was dismissed as stutter peaks (Figure 26). Therefore the correct hair DNA profile was still able to be obtained from the contaminated samples. When amplicons were added to hair and blood samples the RFU from both reamplified diluted K562 amplicons and genomic K562 were compared, significant variation of RFU values between the markers of the re-amplified diluted K562 amplicons. There was a general decrease in fluorescent intensity as the marker fragment size increased, as a result this could be used as a method to differentiate between the amplicon and genomic DNA profiles. 4.4 The Effect of Amplicons Sprayed Directly on the Biological Samples under Simulated Crime Conditions Diluted amplicons were applied to typical forensic samples (blood and hair) by spraying directly to dried bloodstain and hair samples (Section ). This method of application seemed to be the most likely way in which criminals would distribute amplicons at a crime scene. The resultant profiles obtained from contaminated bloodstain samples contain both amplicon (10-3 K562 and A) and genomic blood DNA components. The majority of peak heights of the genomic blood and amplicon DNA profiles were similar within each locus; this can be seen in Figure 27 Panel 3 and 5. As a result, the DNA profiles indicate a mixture of two samples. Furthermore, the RFU value of the X allele was higher than the Y alleles; again this maybe interpreted as a mixture of a major female contributor and a minor contributor of uncertain gender. In contrast, no amplicon peaks were observed in the genomic hair DNA profiles obtained from washed contaminated hair samples comparing to hair DNA profile obtained from unwashed contaminated hair samples. An example of this 88

89 is shown in Figure 32 Panel C. The absence of amplicon contamination after the wash step demonstrates the efficiency of the step in reducing the amount of contamination. It is one possible way to control amplicon contamination and other sources such as dirt, grime etc. This step could be suitable to be applied to other types of the biological sample evidence (excluding the liquid substances) before the DNA extraction. 4.5 The Presence of Amplicons in the Biological DNA Profiles The contaminated biological samples were tested by direct application at the laboratory and spraying amplicons under the simulated crime scene conditions over different time periods. The estimated amount of amplicons on the bloodstain under the simulated crime scene conditions (0.06 μl for every 2mm) indicates that only minute amounts of contaminant amplicon are required to change the original DNA profile. This can be used to mask the original DNA profile thus producing false DNA profile. This is a very serious concern and represents a potential issue in every investigation and trial, concerning DNA identification evidence. The study showed that PCR amplicons using only 0.06 μl / 2mm can survive for at least seven weeks in a typical crime scene environment. However, after the five weeks period, there was a drop-off of fluorescence intensity of the amplicons alleles especially at the larger markers which represents a decrease in amplification of these markers (Figures 30 and 31). This maybe due to amplicon DNA degradation since they consist of short DNA fragments In addition, the study demonstrates that PCR amplicons are able to survive for a period of ten weeks after being directly added onto genomic blood DNA samples prior to DNA extraction, and five weeks within the hair DNA profile. However, it shows that the impact of amplicons contamination after four weeks has less interference in the resultant DNA profile. This indicates the survival of amplicon decrease with time. As shown in Figure 24, the blood DNA alleles can be recognized from the amplicons alleles as they higher. And the contaminated 89

90 hair DNA profile showed only one or two peaks corresponding to the 10-4 K562 amplicons profile (Figure 27). For a period of up to four weeks, it was difficult to differentiate between the blood and amplicon alleles as the majority of peak height of the genomic DNA and amplicons were similar within each locus. This can be seen in Figure 29. As a result, the DNA profiles indicate a mixture of two samples. Furthermore, the RFU value of the X allele was higher than the Y allele, and again this may be interpreted as a mixture of a major female contributor and a minor contributor of uncertain gender. 4.6 The Influence of Different Surface Texture on amplicon Contamination of Bloodstain samples under Simulated Crime Conditions To gain a better understanding of varies surfaces effect on DNA profile and amplicon contamination. DNA profiles were obtained from metal, fiber, wood, glass, tiles and plastic surfaces. The results show that the blood DNA profiles obtained from smoother and non porous surfaces such as glass provide a clear profile than the rough and porous surfaces such as carpet (Refer to Figure 35), and this could be a result of sample concentrations. There is a possibility that DNA recovery from the carpet was not adequate for PCR amplification, after all, the amount that had been added to the surface was 20 μl. These results demonstrate that, if possible, it is always preferable to collect evidential samples from smooth non-porous surfaces than the rough surfaces at a crime scene. However, all contaminated blood samples showed that the DNA profiles recovered from the different surface types have the same contaminant level. This indicates that there is no significant effect of surface on the presence of amplicons contamination. 90

91 CHAPTER 5: CONCLUSION Recent studies show that DNA evidence can be faked using artificial DNA with any desired genetic profile (Berryman, 2003; Dent, 2006; Frumkin, et al., 2009). This artificial DNA can be then applied into genuine human tissues and planted at crime scenes. The current study shows that the deliberate contamination of the biological samples with amplicons under simulated crime scene conditions resulted in an alteration to the DNA profile of the original genomic DNA. After examining the bloodstains and hair samples, at different time periods, it was concluded that contaminating amplicons are carried through the standard Chelex DNA extraction method and readily amplified in subsequent PCR reactions. The resultant DNA profile contained both amplicon and genomic DNA components. This shows that there were more than two alleles at each locus which could be interpreted as a mixture at the forensic laboratory when the results are interpreted by the guideline published by Clayton 1998 (Clayton et al., 1998). Not only do immediate additions of PCR amplicons to samples alter the genomic DNA profiles, amplicons remain present in the genomic DNA profiles even after a period of up to seven weeks under the conditions used in this study. However, the alteration of the genomic DNA with amplicon contamination is not obvious after a period of five weeks. Amplicon alleles were still present but there was a drop-off of fluorescence intensity of the alleles and these peaks could be dismissed as stutter peaks. The DNA profile obtained from the contaminated hair samples showed no evidence of amplicon contamination when samples were subjected to a preliminary wash step as stated in the Chelex DNA extraction method. These results demonstrated that preliminary washing of hair samples before DNA extraction removed amplicon contamination, and could possibly be used to remove amplicon contaminated from other forensic evidence such as skin or nails. However, the effect of using a higher amplicon concentration (for example undiluted or 10-1 or 10-2 amplicon dilutions) may give different results. 91

92 The results of the current study showed that the surface type can influence the recovery of biological samples; for example, less amplicon contamination was recovered from rough porous surfaces than the smoother non-porous surfaces. However, in all cases the recovery of contaminant amplicons was observed from the swabbed surfaces. Previous studies show that the use of substrate controls in DNA analysis has limited value (Gill, 1996). The results of the current study, in contrast, suggest that swabs from different surfaces at the crime scene, such as from an area adjacent to a sample or stain, may be useful in detecting the presence of amplicon contamination at crime scenes. Finally, this study demonstrates that only small amounts of PCR amplicons (0.06 μl / 2mm) are required to alter profile of genomic DNA and this can be easily used extensively by criminals to alter the DNA profiles of the forensic evidence samples at the crime scene. 92

93 CHAPTER 6: FUTURE STUDIES This study has shown that the alteration of DNA profile obtained from contaminated biological samples with PCR amplicons under a simulated crime scene conditions can easily be obtained. However, this study shows also no alteration of contaminated hair DNA using only 10-3 amplicon dilution after a washing step. It will be challenging for a future study to examine if more amount of amplicons can have impact on hair DNA profile even after conduct the washing step. Although it is very difficult to anticipate all future developments for forensic DNA profiling, single nucleotide polymorphisms (SNPs) markers could be examined for that purpose, and may play a valuable role in the future of identification individuals (Butler, 2005). SNPs are variations in the DNA sequence in individuals that occur when a single nucleotide is altered at a certain point in the genome sequence (Butler, 2005). The short PCR products (<100bp) containing SNPs could be more effective to analyse than STRs that have amplicons as large as 300 to 400bp (Butler, 2005). More studies are required using SNPs in forensic DNA profiling research. Furthermore, amplicon contamination may not be limited to crime scene samples; it might also be possible to compromise suspect s samples collected by the police after the commission of a crime (Koupparis, 2002). Koupparis states that amplicons can be added either by a mouthwash containing amplicons before a swab is taken by the police officer, or by applying an oilbased product such as Brylcreem combined with amplicons. In the latter case a hair sample, even after a wash step, will still remain contaminated and both sample analyses will provide false DNA profiles. It may be necessary in the future for a study to be conducted to examine how effectively these methods can alter DNA profiles, and to test amplicon longevity under these conditions. Finally the possibility of intentional contamination of forensic samples is a concern in every investigation and trial concerning DNA identification evidence. Currently, there are no protocols in place to detect and/or prevent amplicon 93

94 contamination at a crime scene, this serious problem has to be considered as well as developing new methods to detect and control such contamination. 94

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102 i. APPENDICES APPENDIX: A The Chelex Extraction Method for Hair (Applied Biosystems) Reagents to be prepared for extraction: Autoclaved deionised water 5% Chelex 10mg/mL Proteinase K 1. Transfer the hair with clean forceps to a dissecting microscope and examine the hair for the presence of sheath material. 2. Wash the hair to reduce surface dirt and contaminants as follows: a) Fill a clean 50mL beaker with autoclaved deionised water. b) Pick up a single hair with a pair of clean forceps. c) Wash each hair to be analysed separately by immersing in fresh deionised water. 3. Return the hair to the dissecting microscope and using a clean scalpel, cut a 1cm portion from the root end of the hair. 4. Add the hair sample, root end at the bottom, to 200µL of 5% Chelex in a 1.5mL microcentrifuge tube. 5. Add 2µL of 10mg/mL Proteinase K. Mix gently. 6. Incubate the sample at 56 C for at least 6-8 hours or overnight. 7. Vortex the sample at high speed for 5-10 seconds. 8. Spin the sample in a microcentrifuge for seconds at 10,000-15,000 x g (maximum speed) at room temperature. 9. Incubate the sample in a boiling water bath for 8 minutes. NOTE: Check that the hair is completely immersed in the Chelex solution before boiling. 10. Vortex the sample at high speed for 5-10 seconds. 11. Spin the sample in a microcentrifuge tube for 2-3 minutes at maximum speed at room temperature. The sample is now ready for DNA quantitation and the PCR amplification process. 12. Store the remainder of the sample at 2-6 C or 15 to 25 C. To re-use, thaw at room temperature and repeat steps

103 APPENDIX: B The Chelex Extraction Method for Blood (Applied Biosystems) The following protocol can be used with whole blood and bloodstains. Reagents to be prepared for extraction: Autoclaved deionised water 5% Chelex TE buffer 1. Pipette 1mL of autoclaved deionised water into an autoclaved 1.5mL microcentrifuge tube. 2. Add portion of bloodstain approximately 3 μl from the whole blood or 3mm from bloodstains (use of a larger portion is likely to result in PCR inhibition). 3. Incubate at room temperature for minutes. Mix occasionally by inversion or gentle vortexing. 4. Spin sample in a microcentrifuge for 2-3 minutes at 10,000-15,000 x g (maximum speed) at room temperature. 5. Carefully remove and discard as much supernatant as possible. Leave no more than 20 L of residual supernatant in the tube. Leave the fabric substrate in the tube with the pellet. 6. Add 5% Chelex to a final volume of 200 L. Vortex the sample briefly to mix. 7. Incubate the sample at 56 C for minutes. 8. Vortex the sample at high speed for 5-10 seconds. 9. Incubate the sample in a boiling water bath for 8 minutes. 10. Vortex the sample at high speed for 5-10 seconds. 11. Spin the sample in a microcentrifuge tube for 2-3 minutes at maximum speed at room temperature. The sample is now ready for DNA quantitation and the PCR amplification process. 12. Store the remainder of the sample at 2-6 C or -15 to -25 C. To re-use, thaw at room temperature and repeat steps

104 APPENDIX: C Quantifier Human DNA Quantification Kit (Applied Biosystems) Quantifiler Human DNA Identification Kit, Applied Biosystems To prepare the DNA quantification standards dilution series: 1. Label eight microcentrifuge tubes: Std. 1, Std. 2, Std. 3, and so on. 2. Dispense the required amount of diluent (PCR water) to each tube. 3. Prepare Std. 1 (using the minimum amounts in the table below): a. Vortex the Quantifiler Human DNA Standard 3 to 5 seconds. b. Using a new pipette tip, add the calculated amount of Quantifiler Human DNA Standard to the tube for Std. 1. c. Mix the dilution thoroughly. 4. Prepare Std. 2 through 8: a. Using a new pipette tip, add the calculated amount of the prepared standard to the tube for the next standard. b. Mix the standard thoroughly. c. Repeat steps 4a and 4b until you complete the dilution series. 104

105 To prepare the reactions: 1. Calculate the volume of each component needed to prepare the reactions, using the table below. Component Volume Per Reaction (μl) Quantifiler Human 10.5 Primer Mix Quantifiler PCR 12.5 Reaction Mix Total 23.0 Volume for X Reaction (µl) 2. Prepare the reagents: Thaw the primer mix completely, then vortex 3 to 5 seconds and centrifuge briefly before opening the tube. Swirl the Quantifiler PCR Reaction Mix gently before using. Do not vortex it. 3. Pipette the required volumes of components into an appropriately sized polypropylene tube. 4. Vortex the PCR mix 3 to 5 seconds, then centrifuge briefly. 5. Dispense 23 μl of the PCR mix into each 0.2μL PCR tube. 6. Add 2 μl of sample, standard, or control to the appropriate tubes. 7. Run samples on a Real-Time PCR machine (i.e. Rotor- Gene 3000) using the following cycling parameters Initial Denaturation 95 C Denaturation 95 C Annealing 60 C Fluorescent Acquisition End of Annealing Repeat for 40 cycles 105

106 APPENDIX: D The following steps were performed to determine the bottle position where the forensic samples will be contaminated (A) Assemble the bottle in the stand to determine the surface area using colored water. (B) Measured the surface area and calculated the distance and the height from which the amplicons will be sprayed onto the forensic samples. (C) Placing the forensic sample on the white sheet to be contaminated with the amplicons. 106

107 APPENDIX: E Control Hair DNA Profile Relative Flourescence Intensities (RFU) Amelogenin D3S1358 D8S1179 D5S818 vwa D21S11 D13S317 FGA D7S820 D18S51 STR Loci Hair + K562 amplicons (One week) Relative Flourescence Intensities (RFU) Amelogenin D3S1358 D8S1179 D5S818 vwa D21S11 D13S317 FGA D7S820 D18S51 STR Loci Hair + K562 amplicons (Five weeks) Relative Flourecent Intensities (RFU) Amelogenin D3S1358 D8S1179 D5S818 vwa D21S11 D13S317 FGA D7S820 D18S51 STR Loci The above diagrams illustrated the impact of the addition of 10-4 K562 amplicons on hair DNA profile after one week and five weeks. The control hair DNA profile was added for comparison. The black and gray columns represent the STR markers unique to genomic hair DNA and the red column represents the STR markers unique to 10-4 K562 amplicon. The RFU value of the X allele in the following diagrams were truncated to (RFU) allowing for the other STR markers to be showing. 107

108 APPENDIX : F The Relative Fluorescence Intensities (RFU) of the Genomic Hair, Genomic K562 and the Contaminated Hair DNA Profiles. Locus Genomic Hair Genomic K 562 Hair +K562 amplicons (one weeks) Hair +K562 amplicons (five weeks) Allele Height Allele Allele Height Allele Height Amelogenin X X X X X X X X D3S D8S D5S vwa D21S D13S FGA D7S D18S The numbers highlighted in red represent the peaks height of the corresponding 10-4 K562 diluted amplicon, while the numbers highlighted in blue represent the peaks height corresponding to blood and 10-4 K562 amplicons. The STR markers are listed from the smallest to largest fragments. 108

109 APPENDIX: G The Electropherograms of the three dyes show the nine STR primer pairs plus a gender-typing markers present in the AmpFlSTR Profile Plus Kit. Panels A: Represent the control hair DNA profile. Panels B: Represent the impact of 10-3 K562 amplicon contamination on hair DNA profile without the washing step. Panels C: Represent the impact of the washing step to eliminate the amplicon contamination. The peaks marked with black stars represent the allele peaks unique to 10-3 K562 amplicon alleles. 109