Overview and History of Forensic DNA
What is Forensic Science?
Forensic science is the application of scientific principles and techniques to matters of law, whether in criminal or civil cases. It encompasses a wide range of disciplines, including biology, chemistry, physics, and medicine, to analyze evidence and provide impartial scientific testimony in legal proceedings.
Edmund Locard, a pioneering French criminalist, is widely regarded as the founder of modern forensic science. He established one of the first forensic laboratories and developed many of the fundamental principles and methods used in forensic investigations today.
He formulated the Locard Exchange Principle: "Every contact leaves a trace."
This principle posits that when two objects or individuals come into contact, there is always a transfer of material between them. This exchange can occur in various forms, such as the transfer of fibers, hairs, fingerprints, or other microscopic evidence.
The Locard Exchange Principle forms the basis for trace evidence analysis in forensic science, emphasizing the importance of even the smallest pieces of evidence in reconstructing events and linking individuals to crime scenes.
He used fingerprints for human identification since 1901.
Recognizing the uniqueness and permanence of fingerprints, Locard advocated for their use in identifying individuals involved in criminal activities. His work helped establish fingerprint analysis as a standard forensic technique.
Forensic DNA analysis has been used since 1986.
ACE Principle
Analysis: This involves a thorough examination of the evidence to determine its nature, characteristics, and quality. The goal is to assess whether the evidence is suitable for further comparison and analysis.
Comparison: In this phase, the evidence from the crime scene is compared to a known reference sample from a suspect or other relevant source. This comparison aims to identify similarities or differences between the two samples.
Evaluation: Based on the analysis and comparison, a conclusion is drawn regarding the potential association between the crime scene evidence and the reference sample. This conclusion may include statements of inclusion (match), exclusion, or inconclusive results.
Forensic DNA Process
Determine if the DNA profile is of sufficient quality: Assessing the quality of a DNA profile involves evaluating factors such as DNA quantity, degradation, and the presence of inhibitors. A high-quality profile is essential for accurate and reliable comparisons.
Compare the DNA profile to reference samples: This step involves comparing the DNA profile obtained from the crime scene evidence to DNA profiles from known individuals, such as suspects or victims. The comparison aims to identify potential matches or exclusions.
Determine if someone is included in a DNA profile/mixture or excluded: Based on the comparison, a determination is made as to whether an individual can be included as a potential contributor to the DNA profile or excluded as a possible source.
Initial Uses of Forensic DNA
Early cases highlight how forensic DNA started to solve crimes.
1983: Linda Mann was murdered in the UK.
Semen was recovered with type A blood and an enzyme combo found in 10% of the population.
1986: Dawn Ashworth was murdered in a similar way.
The semen type was the same as in Linda Mann's case.
Richard Buckland, a 17-year-old with a learning disability, was a suspect.
He admitted to Dawn's murder but denied Linda's murder.
Alex Jeffries' Discovery
In 1984, Doctor Alex Jeffries made his first genetic fingerprint.- He determined whether individuals were related through RFLP technique.
Initially used for immigration cases to verify familial relationships.
Police sent DNA samples from the two murders to Doctor Jeffries.
Richard Buckland was excluded from both murders.
The tests were repeated twice with the same results to ensure accuracy and reliability.
Investigation and Colin Pitchfork
Richard Buckland was released after three months due to the DNA evidence that cleared him.
Police collected samples from men in the area in an attempt to identify the real perpetrator.
Two testing centers were set up to handle the large volume of samples.
Blood typing was done first, followed by RFLP testing for matches to narrow down potential suspects.
After eight months, 5,511 men were tested with no matches, indicating the perpetrator was not among those initially tested.
Colin Pitchfork asked a coworker to take the test for him to avoid suspicion.
The coworker confessed this information while drinking with friends, leading to Pitchfork's eventual identification.
Police investigated him and found his DNA matched the crime scenes, confirming his involvement in the murders.
Colin Pitchfork was sentenced to life in prison with no parole for thirty years for the murders of Linda Mann and Dawn Ashworth.
After thirty-three years, in 2021, he was released, but in 2022, he was recalled to prison for approaching young girls, raising concerns about his potential risk to the community.
Impact of Forensic DNA
Over 50,000,000 people have had their DNA tested in criminal investigations, contributing to both convictions and exonerations.
Convictions and exclusions have resulted, helping to bring justice to victims and prevent wrongful convictions.
The DNA Innocence Project has had over 375 exonerations as of January 2020, highlighting the power of DNA evidence to overturn wrongful convictions.
Additional Resources
A book called The Blooding explains the Colin Pitchfork case in detail, providing insights into the investigation and the impact of DNA evidence.
Optional readings of Doctor Jeffrey's research papers on RFLP offer a deeper understanding of the scientific principles and methods behind this technique. These documents will be provided for further study.
First Case of DNA Evidence
In 1989, the first case of DNA evidence using RFLPs was accepted by the Canadian court system in R versus McNally, marking a significant milestone in the use of forensic DNA in legal proceedings.
The RCMP lab did the work, and it was a sexual assault case.
A DNA profile from a semen stain was produced, providing key evidence linking the suspect to the crime scene.
The suspect voluntarily provided a sample that matched with a statistic of one in seventy billion, demonstrating the overwhelming strength of the DNA evidence.
During the trial, the accused changed his plea from not guilty to guilty in light of the DNA evidence against him.
A qualified expert in Canada may offer their opinion within their expertise, provided that the probative value of the testimony is not substantially outweighed by the prejudicial effect, ensuring that expert testimony is both relevant and reliable.
The court used a test of relevancy and helpfulness to the trier of fact to determine the admissibility of the DNA evidence.
First Conviction Using DNA Evidence
In 1991, The trial of the monster of Miramachi occurred, resulting in the first conviction based on DNA evidence in Canada.
Alan Leguerre was accused of torture, rape, and murder, facing severe charges for his alleged crimes.
Seminal fluid recovered from the deceased females matched Alain Leger's sample, providing crucial evidence linking him to the victims.
The random match probability (RMP) was one in three ten million and one in five million, indicating the rarity of the DNA profile and the high likelihood of Leger's involvement.
Forensic DNA Timeline in Canada
1994: Laboratories in North America transitioned from RFLP to STR technology, marking a shift to a more efficient and accurate method of DNA analysis.
1995: Bill C-one hundred four amended the Criminal Code of Canada and the Young Offenders Act.
Police can apply for warrants to take DNA samples from criminal suspects, expanding their ability to collect and analyze DNA evidence in criminal investigations.
Comparisons can be made to crime scene DNA profiles, allowing for the identification of potential suspects and the resolution of criminal cases.
1997: Bill C-one hundred four was reintroduced as Bill C-three.
DNA Identification Act came in and helped establish the National DNA Database in Ottawa, providing a centralized repository for DNA profiles and facilitating the identification of offenders and the resolution of crimes.
The database includes convicted offender samples and crime scene samples, enhancing its effectiveness in linking offenders to past and future crimes.
Updates to Canadian Forensic DNA
02/2008: Bill C-thirteen and C-eighteen came into force amending the Criminal Code and DNA Identification Act.
Certain offenses were added to the list of designated offenses where an individual needs to provide a DNA profile to the police to be checked against other samples in the DNA database, expanding the scope of DNA collection and analysis in criminal investigations.
July 2023: Approximately close to 700,000 DNA profiles and over 78,000 hits to date in the DNA database in Canada, demonstrating the growing use and effectiveness of DNA technology in law enforcement.
US vs. Canada
The US national DNA database is called CODIS (Combined DNA Index System).
The population of Canada was around 38,000,000, with 445,000 convicted offender profiles, representing approximately 1.2% of the Canadian population.
The U.S. has approximately three thirty million individuals with 15,600,000 convicted offender profiles and 4,800,000 arrestee DNA profiles, representing approximately 6.2% of the population.
Arrestee DNA profiles are only allowed in certain states in the United States due to legal and ethical considerations.
Humanitarian DNA Indices
December 2014: The DNA Identification Act was updated to include three new humanitarian DNA indices to the National DNA Data Bank in Ottawa. These databases came into effect in 2018.
Missing persons index: DNA profiles of missing persons to aid in their identification and recovery.
Human remains index: DNA profiles from found human remains to help identify unidentified bodies.
Relatives of the missing person index: DNA profiles from relatives of missing persons to facilitate comparisons and potential matches.
New victims index and voluntary donors index were developed to further enhance the capabilities of the DNA database.
As of July 2023 there have been 74 identifications, and 24 investigative leads from the indexes as mentioned above, demonstrating the positive impact of humanitarian DNA indices in resolving missing persons cases.
Restriction Fragment Length Polymorphisms (RFLP)
RFLP is an older method replaced by short tandem repeats (STRs) due to its time-consuming nature and lower efficiency.
This method will be explained further for reading next week.
The process would take weeks, whereas STRs can take hours or one or two days, making STRs a more practical choice for forensic DNA analysis.
First, DNA is extracted and quantified to ensure sufficient material for analysis.
VNTR Markers and Restriction Enzymes
Individuals have different DNA sequences at VNTR markers, which are regions of DNA with variable numbers of repeating units.
Restriction enzymes cut the DNA at specific sequences, creating fragments of different lengths based on the individual's VNTR profile.
VNTR repeat sections: Top has three repeats, bottom has four, illustrating the variability in repeat numbers among individuals.
Electrophoresis
The chopped-up DNA is run on an agarose gel, which separates fragments based on size. This process is called electrophoresis.
Large fragments remain at the top, while smaller fragments travel to the bottom, creating a pattern of bands that can be visualized and analyzed.
Southern Blot
The DNA separated in the gel is transferred to a nylon membrane to create a permanent record of the DNA fragment pattern.
A highly alkaline solution is used to make the double-stranded DNA single-stranded, allowing it to bind to the nylon membrane.
UV light cross-links the single-stranded DNA onto the membrane, ensuring that the DNA fragments are permanently attached.
Excess probes are removed to reduce background noise and improve the clarity of the results.
Autoradiography
The membrane is placed on X-ray film, recording band locations through the emission of radioactive signals from the DNA probes.
This is scanned, and the DNA locations are compared to identify matches or differences between samples.
Multi-locus fingerprinting involves analyzing multiple VNTR loci simultaneously to create a unique DNA profile for each individual.
In 1998, a way to reduce bands was found, that way a comparison between samples is easier.
RFLP Process Steps
(a) Gel electrophoresis with DNA in sample wells to separate DNA fragments based on size.
(b) Nylon membrane placed on the gel to transfer the DNA fragments from the gel to the membrane.
(c) DNA transfers to the nylon membrane, creating a permanent record of the DNA fragment pattern.
(d) Hybridization of the radioactive DNA probe to bind to specific DNA sequences on the membrane.
(e) Placing the X-ray film on the gel to capture the radioactive signals emitted by the DNA probes.
(f) Exposure of the X-ray film with DNA band locations, revealing the DNA fragment pattern and allowing for comparison between samples.
Visualizing RFLP Results
The example shows one locus at a time and is usually repeated four times using stripping and re-probing to analyze the DNA at four locations, analyzing four different markers to increase the accuracy and reliability of the analysis.
Autoradiographs can take one week to develop due to the low levels of radioactivity emitted by the DNA probes.
The overall testing time can be several weeks, making RFLP a time-consuming method compared to more modern techniques.
Interpreting RFLP Gels
Rows A, F, and H: size standards used to determine the size of the DNA fragments.
Row B: positive control to ensure that the RFLP process is working correctly.
Row G: crime scene sample containing DNA from an unknown source.
Rows C, D, and E: potential suspects; bands line up with suspect E, suggesting that suspect E may be the source of the DNA in the crime scene sample.
Four Locations in the Human Genome
D1, D2, D5, and D10 at the bottom represent different loci on different chromosomes.
D1S7 is on the first chromosome, D2S44 is on the second chromosome, D5S110 is on the fifth chromosome, and D10S28 is on the tenth chromosome, indicating the chromosomal location of each VNTR marker.
Interpretation of RFLP Results
Bands do not line up: no statistics needed.
The unknown sample did not come from the suspect/person of interest/victim, indicating that the suspect can be excluded as a possible source of the DNA.
Inconclusive: results of the question sample are not good enough to analyze due to poor quality or other factors.
Match/inclusion: bands align, statistics are required.
The unknown sample came from the suspect, suggesting that the suspect may be the source of the DNA.
The unknown DNA sample originated from another person with a matching profile by coincidence, which is a possibility that needs to be statistically evaluated.
Calculate the random match probability to assess explanation and determine the likelihood of a coincidental match.
Forensic DNA experts do not decide guilt or innocence; that is the court's role, emphasizing the importance of the legal process in determining the outcome of a case.
Imaginary Murder Trial
Items of interest to test with forensic DNA to gather evidence and reconstruct the events of the crime:
Knife blade and handle (separately) to determine if there is any DNA evidence linking the suspect to the weapon.
Inside and outside of wine glasses to identify potential DNA sources and determine who may have been present at the crime scene.
Basics of Forensic DNA
Forensic DNA was developed in 1986 by Doctor Alex Jeffries, revolutionizing the field of forensic science and providing a powerful tool for identifying individuals.
It's often recognized as the gold standard in human identification due to its accuracy and reliability.
Based on accepted principles of molecular biology and population genetics (Mendelian inheritance, Hardy Weinberg equilibrium), ensuring the scientific validity of DNA analysis.
A single-source, good-quality crime scene DNA profile allows unambiguous identification to a reference sample, providing strong evidence in criminal investigations.
Challenges arise with mixed or degraded DNA profiles, requiring careful interpretation and statistical analysis.
DNA Composition
DNA stands for deoxyribonucleic acid, the molecule that carries genetic information in all living organisms.
The "deoxyribo" part is related to a pentose sugar ribose, missing an oxygen at position two, which is a key structural feature of DNA.
The nucleic acid part includes the phosphate backbone with nitrogen bases, forming the building blocks of DNA.
Four bases: guanine (G), cytosine (C), thymine (T), and adenine (A), which pair up in specific combinations to encode genetic information.
The location of DNA is referred to as locus (singular) or loci (plural), which are specific positions on a chromosome.
Short Tandem Repeats (STR) Markers
An SCR loci one of the markers used in forensic DNA profiles is called D8S1179.
Indicates that the DNA marker is on the eighth chromosome.
The S indicates it's a single copy.
1,179th marker on the D8 chromosome.
TPOXX or SO1 are named after introns, non-coding regions of a gene, which can also be used as DNA markers in forensic analysis.
Motif Repeats
Short tandem repeats have a repeating unit known as a motif (e.g., AGTC, ATTC), which is a short sequence of DNA that is repeated multiple times in a row.
Usually four base pairs long (tetranucleotide repeat), making them easy to amplify and analyze using PCR.
If there are nine repeats at D8, it's a nine allele; if 12, it's a 12 allele, indicating the variability in repeat numbers among individuals.
Different people have different alleles, allowing individualization and the ability to distinguish between individuals based on their DNA profiles.
Each person has two copies of each chromosome, one from each parent, which means that each person has two alleles at each STR locus.
Alleles & Genotypes
Each person has two alleles at each marker, one inherited from each parent.
If the individual has a nine and 12 at D8, that's heterozygous, meaning they have two different alleles at that locus.
If the person has a 99, they're homozygous, meaning they have two identical alleles at that locus.
Without parental testing, it's impossible to know which allelic version was inherited from each parent, making it challenging to determine the exact source of a particular allele.
Forensic DNA Testing Process
Blood stain at the crime scene, which is a common source of DNA in forensic investigations.
Extraction recovers DNA from human cells, separating it from other cellular components.
Quantify the amount of DNA present to ensure that there is enough DNA for successful analysis.
Amplify the DNA using polymerase chain reaction (PCR) to create millions of copies of the DNA region of interest.
PCR Process
Two primers complementary to the DNA on each side of the region of interest (short tandem repeat locus) to initiate the amplification process.
Double-stranded DNA is separated by heating, allowing the primers to bind to the single-stranded DNA.
Primers bind, and DNA polymerase adds complementary nucleotides, extending the primers and creating new copies of the DNA region.
Millions of copies of DNA (amplicons) are produced through repeated cycles, allowing for the detection and analysis of even small amounts of DNA.
Capillary Electrophoresis
Amplicons are separated and labeled with dyes for detection.
Separation occurs in a thin capillary through a polymer at different rates based on size, allowing for the resolution of DNA fragments that differ by only a few base pairs.
A laser excites the dye as the amplicons pass through a glass window, causing the dye to emit light.
The light emitted is captured and recorded, allowing for the detection and quantification of the DNA fragments.
The output is an electropherogram (DNA profile), a visual representation of the DNA fragments and their relative amounts.
DNA Profile Visualization
A DNA profile is an electropherogram, a visual representation of the data generated during capillary electrophoresis.
Analysis software separates out the different dye colors, allowing for the identification of the different STR loci.
SCR loci are separated based on their size, allowing for the determination of the alleles present at each locus.
Example shows two markers, d 16 and d two, in the blue dye channel, illustrating how different STR loci are visualized in a DNA profile.
Details about Alleles and Fragment Lengths
The two markers or two loci circled contain a nine and an 11, indicating the presence of two different alleles at those loci.
Below the circles that say nine and eleven, the 600 represents the peak height of the alleles, which is proportional to the amount of DNA present.
The fragment lengths are 120 and 128 base pairs, indicating the size of the DNA fragments containing the alleles.
The nine allele is 120 base pairs long, while the 11 allele is 128 base pairs long, illustrating the difference in size between the two alleles.
There is a difference of eight base pairs which shows us there is a four base pair repeat in the allele, confirming that the difference in size is due to the number of repeats in the STR.
The individual is heterozygous for this locus, meaning they have two different alleles at this location in their genome.
On the other hand, a homozygous locus can is shown on the right. And as it's twice as high as the nine and eleven alleles to the left
Case Study
DNA swabs from knives and wine glasses will be looked at. I will now present the results.
Sample 01A (knife blade): Yielded sufficient DNA for a full profile for comparison.
The donor of this profile matches the victim, Mary Smith 05a, indicating that the victim's DNA was found on the knife blade.
The random match is at least one in 1,000,000,000,000, providing strong evidence that the DNA on the knife blade came from the victim.
Sample 01B (knife handle): Yielded sufficient DNA for a partial profile.
The accused, Jane/John Doe, cannot be excluded as a possible source, suggesting that the accused may have handled the knife.
Statistics and Additional Evidence
The statistic provides one piece of the puzzle for the judge and the jury in the case, and other factors, other pieces of information, other disciplines of forensic evidence need to be considered as well, emphasizing the importance of considering