An Evaluation of Forensic DNA Profiling Techniques

An evaluation of forensic DNA profiling techniques currently used in the United Kingdom
Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester by
Eleanor Alison May Graham BSc (Hons), MSc
Forensic Pathology Unit
University of Leicester
October 2007 Abstract
Eleanor Alison May Graham
PhD thesis title: An investigation of Forensic DNA Profiling techniques currently used in the United Kingdom.
The work presented in this thesis uses contemporary methodology to address three gaps in the current understanding of forensic DNA profiling.
Preliminary work was undertaken to identify the most appropriate techniques for use in this thesis, given the equipment available. It was discovered that the QIAamp
DNA mini kit was suitable for DNA extraction from most sample types. PicoGreen dsDNA quantitation reagent was proven adequate for quantitation of DNA concentrations greater than 5ng/μl and the AmpFlSTR® SGM Plus™ PCR
Amplification system was used for all DNA profile generation.
These methods were then used to investigate the effect of peri-mortem blood transfusion on DNA profiling. It was hypothesised that donor leucocytes present in the administered blood products could result in mixed DNA profile generation in post-transfusion blood samples. This hypothesis is rejected after direct analysis of blood products and case examples.
The second question addresses the preservation of field-collected biological samples for disaster victim identification using DNA analysis. Two buffer solutions are tested for their ability to preserve soft tissue samples over a period of six months at room temperature. The results of DNA quantification and degradation analysis suggest that both solutions are capable of DNA preservation of 5 – 1000mg muscle tissue over a six month time period, allowing full standard DNA profile production.
Finally, this thesis examines the normal background level of non-self DNA present on the adult neck surface by sampling 24 volunteers. These observations are used to investigate whether background levels of non-self DNA interfere with DNA profile analysis and interpretation of physical assault situations. The results do not provide a conclusive answer, but augment our understanding of DNA transfer theory by highlighting the high level of non-self DNA normally present on the adult neck due to adventitious transfer, and the discrepancies from previously published DNA transfer theory. i

Dedication
For my Mum, Dad and Sister; Helen, Tom and Alice Graham
Mum
Dad
Sister
Me ii Acknowledgements
The vast majority of my thanks are directed towards Professor Guy N Rutty, for giving me the opportunity to undertake a project under his guidance and also for providing continuous support, both academically and personally throughout the duration of my PhD studentship.
The freedoms and responsibilities he has given me have allowed me to develop a professional confidence and self-belief that I could never have achieved without him.
I also express my appreciation to Dr J. Howard Pringle; our sometimes infrequent, but usually lengthy discussions have provided the additional academic support necessary to produce this thesis. My thanks are also given to the above and to Dr Jacqui Shaw for taking the time to read and comment on my thesis, despite their busy schedules they always found the time to provide the essential reviews and comments required to complete this work.
I would like to thank Mr Malcolm Rae for putting up with my, sometimes constant, harassment for suppliers of unusual items, last-minute finance plans for new bizarre projects and for everything related to Applied Biosystems! Also to Mrs Wendy Pitts, Mrs
Beverley Richardson and Mr Jim Strupish for being pictures of patience and fonts of all knowledge no matter what (occasionally stupid) questions were thrown their way.
Without a little help from my friends, this thesis would not have been started, yet alone completed. Thanks to my Scouse mates, Helen, Ann-Lou and Lynsey; through all of the highs and lows that life has presented, they are always there to keep me sane, usually with a pint or two in hand, Cheers guys! Thanks also to Vicky Bowyer for being there from the start and aiding the development of the lab, and to Vicki Martin (a.k.a. Number 2, in order of appearance, not preference!) for being my football and pub buddy and for making
Leicester a happier place to be.
Finally, I would like to express my utmost gratitude to the families of the deceased who have given permission for samples to be collected from their recently departed loved ones under times of great personal anxiety and to the patients of the Leicester Royal Infirmary undergoing the distress of amputation surgeries for allowing their limbs to be donated for use in this research. Also to the numerous members of the Department of Cancer Studies and Molecular Medicine who returned time and time again to volunteer their time and DNA. iii Contents
1. INTRODUCTION ...................................................................................1
1.1. FORENSIC SCIENCE................................................................................................. 2
1.2. FORENSIC DNA TYPING........................................................................................... 4
1.3. STR ANALYSIS........................................................................................................ 6
1.4. THE AMPFLSTR® SGM PLUS™ SYSTEM ................................................................ 8
1.5. ALTERNATIVE DNA MARKERS ................................................................................ 11
1.6. DNA EXTRACTION ................................................................................................. 12
1.7. DNA QUANTIFICATION ........................................................................................... 16
1.8. DNA PROFILE INTERPRETATION ............................................................................. 19
1.9. LOW COPY NUMBER DNA PROFILING...................................................................... 22
1.10. AIMS AND OBJECTIVES........................................................................................... 27
1.10.1. Aim ............................................................................................................. 27
1.10.2. Objectives................................................................................................... 27
2. MATERIALS AND METHODS ............................................................30
2.1. REAGENTS AND SUPPLIERS.................................................................................... 31
2.2. BUFFERS .............................................................................................................. 32
2.2.1. 10 x Alec Jeffreys (AJ) buffer..................................................................... 32
2.2.2. Tris Acetate EDTA (TAE) buffer................................................................. 32
2.2.3. Tris borate EDTA (TBE) buffer................................................................... 32
2.2.4. Tris EDTA (TE) buffer ................................................................................ 33
2.2.5. Lysis storage and transportation (LST) buffer............................................ 33
2.2.6. Lysis buffer A.............................................................................................. 33
2.2.7. Lysis buffer B.............................................................................................. 33
2.3. DNA CONTAMINATION PREVENTION........................................................................ 34
2.4. DNA EXTRACTION ................................................................................................. 35
2.4.1. QIAamp® DNA mini kit - Swab protocol .................................................... 35
2.4.2. QIAamp® DNA mini kit - Blood protocol .................................................... 36
2.4.3. QIAamp® DNA mini kit - Tissue protocol................................................... 37
2.4.4. QIAamp® DNA micro kit - Swab protocol .................................................. 38
2.4.5. Plucked head hair extraction...................................................................... 39
2.4.6. Chelex® extraction..................................................................................... 40
2.4.7. Microcon® centrifugal filter device, YM-100 of Chelex® 100 extracted DNA
................................................................................................................... 41
2.4.8. Ethanol precipitation of Chelex® 100 extracted DNA................................ 41
2.4.9. In house cell lysis DNA extraction.............................................................. 42
2.5. DNA QUANTIFICATION ........................................................................................... 43
2.5.1. PicoGreen® dsDNA and OliGreen ssDNA Quantitation Reagent ............. 43
2.5.2. Nanodrop – 1000 spectrophotometer ........................................................ 45
2.6. POLYMERASE CHAIN REACTION (PCR) ................................................................... 46
2.6.1. AmpFlSTR® SGM Plus™ DNA profiling PCR ........................................... 46
2.7. DNA VISUALISATION.............................................................................................. 47
2.7.1. 1% Agarose gel electrophoresis ................................................................ 47
2.7.2. 3% Agarose gel electrophoresis ................................................................ 47
2.7.3. DNA visualisation on the ABIPRISM™ 377XL DNA Sequencer ............... 48
2.8. DNA PROFILE INTERPRETATION ............................................................................. 50
2.9. STATISTICAL ANALYSIS .......................................................................................... 51
iv 3. PRELIMINARY WORK........................................................................52
3.1. INTRODUCTION...................................................................................................... 53
3.2. AIMS AND OBJECTIVES........................................................................................... 54
3.3. MATERIALS AND METHODS..................................................................................... 55
3.3.1. Swabbing technique................................................................................... 55
3.3.2. DNA extraction ........................................................................................... 56
3.3.3. DNA quantification ..................................................................................... 56
3.3.4. DNA profiling .............................................................................................. 57
3.4. RESULTS .............................................................................................................. 58
3.4.1. Results of swabbing method optimisation.................................................. 58
3.4.2. Results of DNA extraction method optimisation......................................... 62
3.4.3. Results of DNA quantification against a high range (0 – 100ng) standard
curve........................................................................................................... 66
3.4.4. Results of DNA quantification against a low range (0 – 10ng) standard
curve........................................................................................................... 69
3.5. RESULTS OF DNA PROFILING VALIDATION ASSESSMENT.......................................... 72
3.5.1. Fragment size consistency......................................................................... 72
3.5.2. Stutter analysis........................................................................................... 76
3.5.3. Balanced amplification of heterozygote loci............................................... 80
3.6. DISCUSSION.......................................................................................................... 83
4. BLOOD TRANSFUSION .....................................................................86
4.1. INTRODUCTION...................................................................................................... 87
4.2. AIM AND OBJECTIVES............................................................................................. 89
4.3. MATERIALS AND METHODS..................................................................................... 91
4.3.1. Sample collection ....................................................................................... 91
4.3.2. DNA extraction, Blood................................................................................ 92
4.3.3. DNA extraction, Muscle Liver ................................................................. 92
4.3.4. DNA extraction, Red blood concentrates................................................... 92
4.3.5. Theoretical levels of DNA required for detection in red blood cell
concentrates............................................................................................... 92
4.3.6. DNA quantification ..................................................................................... 93
4.3.7. DNA Profiling.............................................................................................. 93
4.4. RESULTS .............................................................................................................. 94
4.4.1. DNA extraction from packed red blood cells.............................................. 94
4.4.2. Test for detectable levels of cells/free DNA in red blood cell concentrates95
4.4.3. DNA profiling of transfused patients .......................................................... 97
4.5. DISCUSSION........................................................................................................ 100
5. PRESERVATION OF BIOLOGICAL SAMPLES IN THE FIELD.......104
5.1. INTRODUCTION.................................................................................................... 105
5.2. AIM AND OBJECTIVES........................................................................................... 107
5.3. MATERIALS AND METHODS................................................................................... 108
5.3.1. Tissue collection – amputated limbs ........................................................ 108
5.3.2. Tissue collection – soft tissue .................................................................. 108
5.4. STORAGE METHODS ............................................................................................ 109
5.4.1. Storage at -20°C ...................................................................................... 109
5.4.2. Oragene™ DNA collection pots ............................................................... 109
5.4.3. LST buffer................................................................................................. 110
5.5. DNA EXTRACTION ............................................................................................... 111
5.5.1. Oragene™ DNA purification protocol....................................................... 111
5.6. VISUALISATION OF EXTRACTED DNA TO ASSESS DNA PRESERVATION................... 111
5.7. AMPFLSTR® SGM PLUS™ DNA PROFILING PCR................................................ 111
5.8. ANALYSIS OF DNA PROFILING RESULTS................................................................ 112
5.9. RESULTS ............................................................................................................ 113
v5.9.1. Limb model............................................................................................... 113
5.9.2. Soft tissue samples .................................................................................. 120
5.10. ADDITIONAL WORK .............................................................................................. 123
5.11. DISCUSSION........................................................................................................ 125
6. DNA TRANSFER DURING MANUAL STRANGULATION ...............129
6.1. INTRODUCTION.................................................................................................... 130
6.2. AIMS AND OBJECTIVES ........................................................................................ 132
6.2.1. Phase I ..................................................................................................... 132
6.2.2. Phase II .................................................................................................... 132
6.2.3. Phase III ................................................................................................... 133
6.3. METHODS ........................................................................................................... 134
6.3.1. Volunteer recruitment............................................................................... 134
6.3.2. DNA extraction ......................................................................................... 134
6.3.3. DNA collection.......................................................................................... 135
6.3.4. DNA quantification ................................................................................... 135
6.3.5. DNA profiling ............................................................................................ 135
6.4. RESULTS ............................................................................................................ 137
6.4.1. Brush swabbing and point swabbing ....................................................... 137
6.4.2. Results of Phase I: Determination of inter- and intrapersonal patterns of DNA recovery from the neck surface. ...................................................... 139
6.4.3. Results of Phase II: mapping of normal background level of non-self DNA ..
................................................................................................................. 143
6.4.4. Results of Phase III: Analysis of DNA transfer by physical contact......... 148
6.5. DISCUSSION........................................................................................................ 157
7. CONCLUSION...................................................................................165
8. APPENDIX.........................................................................................171
8.1. RAW DATA FOR AMPFLSTR® SGM PLUS™ PCR AMPLIFICATION KIT VALIDATION
STUDIES171
8.2. SGM PLUS DNA PROFILING RESULTS FOR DECOMPOSED TISSUE FROM CASE 1...... 181
8.3. SGM PLUS DNA PROFILING RESULTS FOR DECOMPOSED TISSUE FROM CASE 2 ..... 183
8.4. QUESTIONNAIRE DESIGNED FOR PARTICIPANTS OF PHASE I, II AND III OF STRANGULATION PROJECT ................................................................................................ 185
8.4.1. Phase I questionnaire .............................................................................. 185
8.4.2. Phase II questionnaires............................................................................ 186
8.4.3. Phase III questionnaires........................................................................... 188
8.5. FULL INTERPRETATION FOR EACH SWAB ANALYSED DURING PHASE III OF STRANGULATION
PROJECT................................................................................................................. 191
8.6. LIST OF PUBLICATIONS......................................................................................... 199
8.6.1. First authored publications....................................................................... 199
8.6.2. Co-authored publications ......................................................................... 200
8.6.3. Co-authored book chapter ....................................................................... 200
8.6.4. Published abstracts.................................................................................. 201
8.6.5. Oral presentations.................................................................................... 202
8.7. ORIGINAL MANUSCRIPTS...................................................................................... 204
8.7.1. DNA Reviews: miniSTR’s......................................................................... 204
8.7.2. DNA Reviews: Non-human DNA.............................................................. 210
8.7.3. DNA Reviews: Lab-on-a-chip technology ................................................ 214
8.7.4. DNA Reviews: Automated DNA profile analysis...................................... 221
8.7.5. DNA Reviews: Forensic DNA on the Internet .......................................... 231
8.7.6. DNA Reviews: Disaster Victim Identification............................................ 236
8.7.7. DNA Reviews: Sex typing ........................................................................ 242
8.7.8. DNA Reviews: Hair .................................................................................. 249
8.7.9. DNA: Risk of Contamination .................................................................... 257
8.7.10. Investigation into the usefulness of DNA profiling earprints .................... 264
9. REFERENCES ..................................................................................277 vi Index of figures
Figure 1-1: Timeline of some major scientific advances that have helped to shape modern
forensic science Sourced from (Lane 1992) and .......... 3
Figure 1-2: Example of typical stutter pattern of alleles 24 and 26 at STR locus FGA....... 16
Figure 3-1: Illustration of the lay out of the non-absorbent board used for testing the efficiency of single and double swab techniques for the recovery of trace levels of control
DNA...................................................................................................................................... 55
Figure 3-2: Electropherogram image to show the full SGM Plus DNA profile generated from
material recovered by single swabbing of 5ng control DNA. ............................................... 59
Figure 3-3: Electropherogram image to show the full SGM Plus DNA profile generated from
material recovered by double swabbing of 5ng control DNA............................................... 60
Figure 3-4: Electropherogram image to show the full SGM Plus DNA profile generated after
a repeat PCR reaction performed on a second 7.5μl aliquot of DNA extracted after double
swab recovery of 5ng control DNA....................................................................................... 61
Figure 3-5: Bar chart to illustrate the results of standard and low copy number DNA
profiling after DNA extraction was performed on identical sterile cotton swabs loaded with
10ng control DNA................................................................................................................. 63
Figure 3-6: Bar chart to illustrate the results of standard and low copy number DNA
profiling after DNA extraction was performed on identical sterile cotton swabs loaded with
25ng control DNA................................................................................................................. 64
Figure 3-7: Standard curve generated for quantification of DNA by PicoGreen method.... 67
Figure 3-8: Standard curve generated for DNA quantification of low quantities using the PicoGreen method. .............................................................................................................. 70
Figure 3-9: Stutter percentages observed for 1ng control DNA 007 ampified for 28 cycles in
a total reaction volume of 25μl ............................................................................................. 77
Figure 3-10: Stutter percentages observed for 1ng control DNA 007 ampified for 34 cycles
in a total reaction volume of 25μl ......................................................................................... 78
Figure 3-11: Stutter percentages observed for 500pg control DNA 007 ampified for 28
cycles in a total reaction volume of 12.5μl ........................................................................... 79
Figure 4-1: Bar chart to show the results of SGM Plus PCR performed on DNA extracted
from 200μl RBC’s with the addition of differing numbers of fibroblasts. .............................. 95
Figure 4-2: Bar chart to show the results SGM Plus PCR performed on DNA extracted from
200μl RBC’s with the addition of differing amounts of free DNA. ........................................ 96
Figure 4-3: Electropherogram images of the DNA profiles produced from liver and blood
samples from case 5 ............................................................................................................ 99 vii Figure 5-1: DNA quality assessment by 1% agarose gel electrophoresis after 1, 12 and 36
weeks (a-c)......................................................................................................................... 115
Figure 5-2: Electropherogram image of degraded DNA amplified using SGM Plus PCR
amplification kit................................................................................................................... 116
Figure 5-3: Electropherogram images to show results of DNA profiling of 100mg muscle
stored in Oragene solution for 36 weeks showing no evidence of DNA degradation........ 118
Figure 5-4: Electropherogram images to show results of DNA profiling of 100mg muscle
stored in LST buffer for 36 weeks showing no evidence of DNA degradation................... 119
Figure 5-5: Graph to show the average percentage of each DNA profile observed after
amplification of 1ng ............................................................................................................ 121
Figure 5-6: DNA visualised by 1% agarose gel electrophoresis and stained with Ethidium
Bromide.............................................................................................................................. 122
Figure 5-7: Bar chart to show the amount of DNA recovered from LST buffer, incubated for
1 hour at room temperature, 56°C or 70°C after addition of 50mg muscle tissue. ............ 124
Figure 6-1: Deaths recorded as homicide during 2004/2005 with apparent method of death.
............................................................................................................................................ 131
Figure 6-2: Illustration describing brush swabbing (top) and point swabbing (bottom)
techniques investigated for colellection of DNA from the skin surface of adult volunteers 136
Figure 6-3: Bar chart illustrating the results of the percentage of volunteer’s DNA profile
that was recovered following QIAamp DNA mini kit extraction of brush swabbed adult neck
surfaces.............................................................................................................................. 137
Figure 6-4: Bar chart illustrating the results of the percentage of volunteer’s DNA profile
that was recovered following QIAamp DNA mini kit extraction of point swabbed adult neck
surfaces.............................................................................................................................. 138
Figure 6-5: Bar chart to illustrate the results of DNA profiling carried out after brush
swabbing of 5 volunteers ................................................................................................... 140
Figure 6-6: Bar chart to show comparison of DNA recovery between male (n = 10) and female (n = 11) volunteers during phase I of this investigation.......................................... 142
Figure 6-7: Scatter diagram to show percentage of DNA profile recovered after sampling
each volunteer compared to the time since the neck surface was last washed. ............... 142
Figure 6-8: Chart to illustrate the areas of the neck in which non-self DNA alleles were
detected during phase II..................................................................................................... 145
Figure 6-9: An example of a female/male two person DNA mixture observed after brush
swabbing of a female volunteer neck surface in area C. ................................................... 146
Figure 6-10: Bar chart to illustrate the difference between the background levels of non-self
DNA observed between single volunteers and volunteers with partners........................... 147 viii Figure 6-11: Bar chart to illustrate the percentage of DNA profile observed after brush
swabbing male and female necks during phase II............................................................. 147
Figure 6-12: Neck areas sampled during phase III of this investigation. .......................... 148
Figure 6-13: Summary of the results of DNA transfer observed during Phase III when
single volunteers were acting as the victim of manual strangulation ................................. 150
Figure 6-14: Summary of the results of DNA transfer observed during Phase III when
volunteers with partners were acting as the victim of manual strangulation...................... 151 ix Index of tables
Table 1-1: Characteristics of SGM Plus STR loci. Table adapted from AmpFlSTR SGM
Plus PCR Amplification kit manual and (Ruitberg et al. 2001)............................................. 10
Table 1-2: List of materials commonly collected for DNA analysis during forensic
investigations with reference to extraction methods ............................................................ 14
Table 2-1: Protocol for preperation of high range and low range standard curves ............. 43
Table 2-3: Cycling conditions for AmpFlSTR® SGM™ Plus PCR........................................ 46
Table 3-1: Relative fluorescent measurements observed for production of standard curve in
the 0 -100ng range............................................................................................................... 66
Table 3-2: Results of DNA quantification of known amounts of K562 control DNA ............ 67
Table 3-3: Relative fluorescent measurements observed for production of standard curve in
the 0 -10ng range................................................................................................................. 69
Table 3-4: Results of DNA quantification of known amounts of K562 control DNA ............ 70
Table 3-5: Results of fragment size variation of 1ng 007 control DNA amplified for 28 PCR
cycles in a total reaction volume of 25μl .............................................................................. 73
Table 3-6: Results of fragment size variation of 500pg 007 control DNA amplified for 34
PCR cycles in a total reaction volume of 25μl...................................................................... 74
Table 3-7: Results of fragment size variation of 500pg 007 control DNA amplified for 28
PCR cycles in a total reaction volume of 12.5μl................................................................... 75
Table 3-8: Results of Hb calculations performed on 10 replicate SGM Plus amplifications of 1ng template DNA in total reaction volume of 25μl.............................................................. 81
Table 3-9: Results of Hb calculations performed on 10 replicate SGM Plus amplifications of 1ng template DNA in total reaction volume of 12.5μl........................................................... 81
Table 3-10: Results of Hb calculations performed on 10 replicate SGM Plus amplifications
of 100pg template DNA in total reaction volume of 25μl...................................................... 82
Table 4-1: Summary of literature concerning DNA analysis of transfused patients............ 90
Table 4-2: Peri-mortem history of patients sampled for use in this investigation................ 91
Table 4-3: DNA Quantification for blood packs 1 – 5, performed in duplicate .................... 94
Table 4-4: Results of DNA quantification and SGM Plus DNA profiling performed on all
materials collected from 5 cadavers who had received transfused blood before death ...... 98
Table 5-1: Quantity of DNA from muscle removed from leg A and leg B in each
preservative medium over a 36 week period. .................................................................... 114
Table 5-2: Quantity of DNA recovered from soft tissue samples stored frozen at -20°C or at
room temperature in Oragene™ or LST buffer for 48 hours prior to DNA extraction ........ 120 xTable 6-1: Table to summarise questionnaires filled in by each volunteer, shedder status,
and DNA profiling results of phase II.................................................................................. 144
Table 6-2: Results of SGM Plus DNA profiling carry out on swabs taken from pair 1 and pair 2. ................................................................................................................................. 152
Table 6-3: Results of SGM Plus DNA profiling carry out on swabs taken from pair 3 and pair 4. ................................................................................................................................. 153
Table 6-4: Results of SGM Plus DNA profiling carry out on swabs taken from pair 5 and pair 6. ................................................................................................................................. 154
Table 6-5: Results of SGM Plus DNA profiling carry out on swabs taken from pair 7 and pair 8................................................................................................................................... 155
Table 6-6: Results of SGM Plus DNA profiling carry out on swabs taken from pair 9 and pair 10. ............................................................................................................................... 156 xi Abbreviations
AAdenine
ABI Applied Biosystems, Foster City, CA, USA
aDNA Ancient DNA
AFIS Automated fingerprint identification system
AJ buffer Alec Jeffreys buffer
ANOVA Analysis of variance
B.C. Before Christ
βME Beta mercaptoethanol
BSA Bovine serum albumin
CCytosine
CCD Charged coupled device
CPS Crown prosecution service
DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate dsDNA Double stranded DNA
DTT Diothiothreitol
DVI Disaster victim identification
EDTA Ethylenediamine tetra – acetic acid
EtBr Ethidium bromide
ExB Extraction blank
FBI Federal Bureau of Investigation
FSS Forensic Science Service ggram
GGuanine
GUSCN Guanidine thiocyanate
HCl Hydrogen chloride
HPLC High performance liquid chromatography
IAA Isoamyl alcohol
KCl Potassium chloride lLitre
LCN Low copy number
LOH Loss of heterozygosity xii LREC Local research ethics committee
LST Lysis storage and transportation
MMolar mM Millimolar
MFI Mass fatality incident
mg Milligram
MgCl2 Magnesium chloride
MHC Major histocompatibility complex
MLP Multi locus probe mtDNA Mitochondrial DNA
NaCl Sodium chloride
NaOH Sodium hydroxide
NDNAD National DNA database ng Nanogram
(NH4)2SO4 Ammonium sulphate
PCR Polymerase chain reaction
PGM Phosphoglucomutase pH Potential of Hydrogen
RFLP Restriction fragment length polymorphism
PEP Primer extension pre-amplification
RFU Relative fluorescent unit
SDS Sodium dodecyl sulphate
SGM Second generation multiplex
SLP Single locus probe
SNP Single nucleotide polymorphism
ssDNA Single stranded DNA
STR Short tandem repeat
TThiamine
TAE Tris acetate EDTA
TBE Tris borate EDTA
TE Tris – EDTA
Tris 2-amino-2 (hydroxymethyl)-1,3-propandiol
UKAS United Kingdom accreditation service
UK United Kingdom xiii μl Microlitre
μM Micromolar
UP Ultra pure
USA United States of America
UV Ultra violet
VNTR Variable number tandem repeat xiv 1. Introduction
“If we had called this „idiosyncratic Southern blot profiling‟, nobody would have taken a blind bit of notice. Call it „DNA fingerprinting,‟ and the penny dropped”
- Professor Sir Alec Jeffreys, February 2005, Café Scientifique, Leicester, UK
1
1.1.Forensic Science
The word „forensic‟ has its origins in the Latin language and is translated as „before the forum‟. Forensic science is therefore defined as the application of scientific knowledge and experimentation to legal contentions, be they civil or criminal matters. Forensic investigations may be carried out for cases up to 75 years old, after this time a case will be referred to as archaeological, as it is likely that, for example, the immediate family of a murder victim and indeed the murderer themselves may be deceased. The exact origins of forensic science are not clearly defined and the use of fingerprints as a mark of individualisation can be traced back centuries B.C. to ancient Chinese and Babylonian civilisations where they were used as an official seal and as currency. The first recorded autopsy was carried out in 44 B.C. on the corpse of Julius Caesar by the physician Antistius, who was able to determine which of the 23 stab wounds present to his body was responsible for his death.
The official origin of the discipline is often attributed to Song Ci, a Chinese medical
practitioner who in 1248 published the book, Hsi Duan Yu. This is translated as
„The washing away of wrong‟, and describes how scientific techniques such as entomology and medicine can be used to identify murder weapons and distinguish between drowning and strangulation as a cause of death. In the centuries that have elapsed since this publication, numerous key advances have been made, bringing the expertise of every imaginable scientific discipline to the constant battle to resolve civil and criminal disputes, identify the deceased and the perpetrators of criminal activity. This thesis focuses on the analysis of deoxyribonucleic acid