Forensic Biology/DNA Domenic Daniel Panetta

Forensic Biology/DNA
Domenic Daniel Panetta, BSc, MSc
Forensic Biologist: Center of Forensic Science
Case #: 2371823September 24th 2018
Table of Contents
TOC o “1-3” h z u Case #: 2371823 PAGEREF _Toc524897326 h 1CURRICULUM VITAE PAGEREF _Toc524897327 h 3The History of DNA PAGEREF _Toc524897328 h 5Forensics and DNA PAGEREF _Toc524897329 h 6STR Analysis PAGEREF _Toc524897330 h 8In Depth PAGEREF _Toc524897331 h 8Forensics in Canada PAGEREF _Toc524897332 h 10The Case PAGEREF _Toc524897333 h 11Evidence PAGEREF _Toc524897334 h 12Results PAGEREF _Toc524897335 h 14Conclusion PAGEREF _Toc524897336 h 16References PAGEREF _Toc524897337 h 17
CURRICULUM VITAEDomenic Daniel Panetta (BSc, MSc)
Center of Forensic Science
Biology Services
Education:
1995-1999 Holy Names Catholic High School – Windsor, ON
1999-2003University of Windsor – Windsor, ON – Bachelor of Forensic Science with specialization in Biology
2003-2005University of Toronto – Master of Science in Forensic Analysis
Professional Experience:
2003-2005 Deep Genomics – Research Technician – Toronto ON
2005-Present Forensic DNA Analyst – Center of Forensic Science
Professional Memberships:
Association of Forensic DNA Analysts and Administrators – General member since 2006Canadian Society of Forensic Sciences – General member since 2007
Conferences Attended:
Canadian Society of Forensic Science 2018 Conference
Investigative Excellence Through Professional Collaboration
Forensic Research and Technology
Association of Forensic DNA Analysts and Administrators
The History of DNADeoxyribonucleic acid, more commonly known as DNA, is the carrier of genetic information and is present in all living organisms. It was discovered in the late 1800s by Friedrich Miescher. Before the twentieth century it was believed that proteins were the genetic material carriers. It wasn’t until the structural discovery of DNA by James Watson and Francis Crick in 1953, that led scientists to believe DNA is the actual genetic material. (Griffiths, Wessler, Carrol, ; Doebley, 2015) DNA consists of three main parts. These parts being a phosphate group, a sugar group, and one of four nitrogen bases. A strand of DNA needs a nucleotide linked into the chain, as well as the phosphate groups and sugars alternating. (Green 2015) As previously mentioned, there are four different types of nitrogen bases. These bases being Adenine (A), Thymine (T), Guanine (G), and Cysteine (C). (Green 2015) DNA is the blueprint of life, and the order of the nitrogenous bases are what determine our characteristics. These bases are read and are then used to make proteins which are the molecules that do most of the work in our bodies. (Green 2015) For instance, if the sequence of nucleotides reads ATGCCT this might code for brown hair, whereas the sequence ATGCTT might code for black hair. (Green 2015)
DNA is located within any cell that contains a nucleus including, but not limited to saliva, blood, etc. Throughout the entire human body, DNA can be found tightly packaged in a form known as a chromosome in the nucleus of the cell. When the cell replicates the information that is contained within the DNA, it is passed on to the daughter cells it creates. This means that our DNA stays with us for our entirety. Every person has their own specific sequence of codons making us all unique with distinctive traits. This uniqueness implies that our DNA, when extracted and compared, can be used to prove for or against us in the court of justice.

Forensics and DNAThe main objective of a forensic scientist is to collect evidence, analyze it, and present it in a court to obtain the rules of the law. This includes incriminating a person on a crime they have committed, or exonerating a person from being wrongfully accused. DNA plays a crucial role when it comes to evidence. As previously mentioned, DNA is unique to each individual. This individualization is important to forensic scientists because when an unknown sample is found at the scene of a crime, a known sample may be compared to confirm the identity. Before the discovery of DNA in the field of forensics there were several other methods used to identify criminals. One of the first scientific systems of documenting personal identification was discovered in the late 1800s. (Manarin 2017) The method was referred to as Bertillonage, and was discovered by Alphonse Bertillon. The method worked by measuring an individual’s anthropometric measurements and recorded on file. It wasn’t until 1891 when the method was replaced by the use of fingerprints by Dr. Hans Gross. (Manarin 2017) Fingerprinting made the identification process of criminals easier and opened doors for scientists to further develop techniques and methods.
Dr. Alec Jefferys was a genetics professor at Leicester University, with interest in the DNA variations, and the evolution of gene families. He had a goal to create markers to track positions of genes. (Knaap 2018) In the early 1980s, Dr. Jefferys’ lab was one of the first to produce an explanation for the term restriction fragment length polymorphisms, also known as RFLPs. (Knaap 2018) RFLPs are sections of the DNA that are on a particular chromosome that have been cut into fragments from a specific enzyme. Within the same species the lengths of these section differ based on the individual. These lengths are due to the combination of base pair sequences. It wasn’t until the year 1984 when Jeffreys successfully tested the use of DNA analysis for identification purposes. When the DNA of several persons was processed, different band patterns were obtained. (Knaap 2018) This helps in the field of forensics to separate individuals based on their unique characteristics.
The study of serology is analyzing target bio-chemicals and cells that define the distinctive functional characteristics of different bodily fluids. For example, the hemoglobin in blood, and spermatozoa in sperm. When these bodily fluids get deposited in crime scenes they can be collected and analyzed to identify the perpetrator or exclude an individual from a trial.
The first case that incorporated DNA in its trial was in the year 1986. It involved Dr. Alec Jeffreys, his research and innovation on DNA analysis. It was a murder of two 15-year-old girls. A tip led the police to a man who confessed for only one of the murders. After the investigation began Jeffreys was sent blood from the suspect and semen samples from both victims. The semen samples matched to each other, but didn’t match to the blood sample from the suspect. This was the first exoneration in a case. After a genetic manhunt was held, an anonymous tip led the police to a man who paid a man to give blood under his name. They found the individual who was responsible for the death of the two girls. This method was then to be named DNA fingerprinting.
DNA profiling is the second generation typing system. This is a more recent development and is used today in the field of forensics analysis. The DNA profiling method works by looking at one area a single locus on a specific chromosome. (Knaap 2018) It is viewed as a set of numbers representing the DNA variants, at target locations. These are known as loci and work as an address on DNA.
STR AnalysisTandem repeats are categorized based on the size of the repeat region. Variable Number Tandem Repeats, VNTR’s, are regions that have approximately nine to 80 base pairs. Short Tandem Repeats, STR’s, are regions that contain two to five base pair repeats. STR analysis examines the smaller regions of repeated sequences and therefore does not require large quantities of DNA, and can also use degraded DNA. STR analysis is done with the technique of polymerase chain reaction, PCR. With STR analysis and PCR together, DNA typing has become more efficient, and is more reliable which creates a higher degree of incrimination. (Knaap 2018)
DNA is extracted chemically from the sample by breaking down the components of the rest of the cell to isolate the DNA from the nucleus. Copies of the DNA are made through amplification, a PCR technique. It uses an enzyme to make unlimited number of copies of DNA to have sufficient quantity from minute samples. The extracted DNA fragments of interest are taken from the same regions on a particular chromosome in different individuals and then analyzed.
In Depth
When performing an analysis of DNA, it is essential that sterile utensils are used as any contact can alter the results and tamper evidence. This is crucial because when given evidence from a crime scene there isn’t an unlimited supply, there is only what is collected so it is important to follow directions precisely. As mentioned previously, DNA is located tightly packaged within the nucleus. In order to extract the DNA, a lysis solution and Proteinase K would be added to the sample. The lysis solution will aid in getting the DNA out from the nucleus into solution. Proteinase K is an enzyme that breaks bonds between the amino acids in the proteins. The tube will then be incubated to increase the activity of Proteinase K. After protein precipitation will be added and vortexed. A separate tube will be prepared with isopropanol, which is used to precipitate DNA. The tube must then be centrifuged and the supernatant transferred to the prepared isopropanol tube. The isopropanol and supernatant tube will then be centrifuged. After the centrifugation, the supernatant will be discarded and 70% ethanol would be added and centrifuged once again, followed by removing the supernatant again. The pellet would then be dried using a SpeedVac. Distilled H2O will then be added and then the preparation for amplification will begin. A mastermix will be added to the tube. A mastermix is a solution that aids in the process of amplification. It contains Taq DNA polymerase, dNTPs, -62865331660500MgCL2, primers, and other components to aid in PCR amplification. The tubes will then remain in the thermocycler until the cycle is complete. Once completed, ddH2O would be added, along with Green Buffer which contains loading dye, DNA sample, as well as a restriction enzyme. The samples would be loaded into the agarose gel and will run for 40 min at 180 V. The gel contains Ethidium Bromide which intercalates with the DNA and glows under UV light. When the Gel is finished the image produced will be of the cuts from the enzymes in solution. The ladder on the image will show where the cuts on the band are. When the unknown DNA matches a known sample it can be inclusive evidence in a trial but is not enough information to convict a suspect.
Forensics in CanadaIn the year of 1988, DNA was introduced to Northern America. The introduction of DNA analysis created a lot of controversy in the court. This is because a lot of start-up companies did not have proper equipment or training to successfully conduct experiments. It took time to finally be accepted into the court as valid evidence. 30 years ago, the first DNA evidence was used in a Canadian court. (Blais 2013) It was a murder case in Edmonton, which compared DNA samples of an alleged sex offender with evidence collected. The results that came from the lab back fired in court, and instead of proving the suspect guilty, it cleared his name. This is because the blood samples taken from the suspect and the semen found at the crime scene did not match. (Blais 2013) Avril Inglis, an Edmonton Crown prosecutor, described DNA analysis as an evidence that is understandable and honorable in the justice system.

“A DNA analysis happens in a lab. It’s removed. Its peer reviewed. It’s solid quantifiable evidence that can be relied on and it is relied on, on a regular basis. And when you hear the numbers often used by a DNA analyst, you know what you are hearing. When you hear that a random match probability between the sample seized at a crime scene and the sample taken from the suspect is one in a billion, you know how reliable that evidence can be. It’s valuable to the courts. It’s valuable for juries. And because of the modern information age and everyday TV shows that people are watching, it’s also understandable to everybody who hears the evidence.” – The Toronto Sun
In Ottawa 1989, a full year later, a rape case was the first case that used DNA evidence to convict a criminal in Canada. The suspect was accused of allegations, and denied any involvement in the crime, but agreed to provide a DNA sample of his hair, blood, and saliva. Once tested the samples matched the semen found on the victim’s nightgown and bedspread and was placed under arrest. (Blais 2013) In court, the judge praised the DNA evidence and made it admissible, the rapist pleaded guilty and was sentenced. In 2000, the national DNA databank became operational in Ottawa. (Blais 2013) Inglis touches on the importance of storing DNA to help solve crimes in the future as well as being prepared to eliminate suspects by already having their DNA on file.
“When an offender is sentenced for particular kinds of crimes, it can be part of their sentencing order that their DNA sample be provided to the police so that it can be uploaded onto that DNA databank. And clearly, that’s proved to identify many culprits over the years, including offenders in cases where we wouldn’t otherwise have known who to suspect.” – The
This is important in the field of forensics to store all DNA collected from the criminals to quickly access DNA files. If a crime were to be committed and DNA samples from the scene were collected, they could be processed through the databank and if a match was found, the criminal could be convicted faster.
The CaseOn the night of August 24th 2018, a worried mother called the police after not hearing from her daughter when expecting a call after a long day. A search was conducted within the home and the body of the missing person was found. The investigators photographed, collected evidence, and documented the scene of the crime. The body was identified, and once permission was granted from the family, a forensic autopsy was conducted.
Throughout the scene of the crime many pieces of evidence were collected. Including the suspects clothing and samples. Each was delivered to my office, by technician Halpert, in proper containers where I then began my investigation. Evidence included items of clothing and other items around the room that seemed to be of question and valuable to the scene. There were two tips given to the police regarding suspects to the crime. The two suspects were taken into custody for questioning, and agreed to supply DNA evidence for testing. I was also provided with a DNA sample from the victim.
Evidence-177165198945500Technician Halpert delivered to me a bloody T-shirt that belonged to the victim. The shirt was covered in blood and was packaged in a brown paper evidence bag with the use of plastic gloves. The bag was folded twice, and taped, with an evidence tag filled out on the bag. The tag was signed by technician Halpert, and contained the collection date and item number. This ensured that the chain of custody has been recorded and there was no tempering of evidence. The paper bag was used because if the evidence is not completely dry and put in a container where it cannot breathe then microorganisms can destroy the evidence. As mentioned previously, blood is a crucial piece of evidence because it contains DNA which can be compared and used to either exonerate or place a suspect at the scene of the crime.

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In my lab I removed the shirt from the bag, on the shirt I noticed two different locations of blood. I swabbed the sample with a cotton swab, in both locations with different swabs, and used an indicator called phenolphthalein which proved the stains to be blood. I then began my investigation.
I removed a small sample of the blood and placed it in a micro tube. I then began the procedures of extracting the DNA. This includes the lysis of the cell to remove DNA from the nucleus. Proteinase K will be added to break apart the bonds between amino acids and proteins. Further steps will be taken as previously mentioned, until ready for the PCR to be completed. The amplified DNA was mixed with Ethidium Bromide and loading dye, and then loaded into the agarose gel. Also loaded into the gel is a ladder to show where the cuts are located. The gel ran for 40 min under 180V and then captured with UV light.
This procedure was done with both spots of blood on the shirt to determine if both are from the same source.
In a separate bag delivered to my lab was the undergarments of the victim. A swabbing or cutting was taken from Item 2 for DNA analysis. The sample was cut into three cuttings from the crotch area. Each sample was then placed into separated wells on a spot plate, all labeled accordingly. Each well is filled with sodium acetate buffer, and added is alpha naphthyl acid phosphate calcium salt. (Elwell 2009) Fast Blue B solution is then added to each well and will produce a purple colour when showing a positive result. A slide would be made for any well showing a colour resemblance for a 3+ or higher. A 4+ being Dark purple, and a 3+ being purple. (Elwell 2009) If there are visible sperm under the microscope the DNA must be extracted from the sperm using a mild detergent that bursts non-sperm cells. Once the non-sperm cells are busted, water is rinsed and only the sperm cells that acquire DNA remain and are then busted open with a stronger detergent to recover the DNA. A PCR is then done on the DNA and ran through a gel to get the restriction fragments to be tested to the other DNA collected from the crime scene.

ResultsBloodstain PCR

Provided are two images of the PCR Gel Electrophoresis that were conducted in the experiment. The left shows the Gel produced from the samples of the scene. The unnumbered lane resembles the ladder which shows all the alleles. The restriction enzymes cut DNA differently based on the person. These cuts appear as bands on the gel. The smaller the number the farther it travels down the band. The sizes can be measured using the ladder. The image on the right was created from the same markers in a format to resemble a clearer picture. The sample from the crime scene was ran and produced specific cuts. We ran the victim’s blood as well as two suspects. It is clear to see that all the strands of the DNA match to the sample found at the crime scene. It is my opinion, that the DNA found at the scene of the crime belongs to suspect number one.
Sperm Test
As previously mentioned when testing for sperm a sample must be placed in wells containing sodium acetate buffer, alpha naphthyl acid phosphate calcium salt, and Fast Blue B solution. When there is a positive indication for sperm a colour change will occur.
0-381000This picture shows the colour range of the evidence. A slide was made for all wells showing 3+/4+, a purple and dark purple colour.
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28257546990Crime Scene
Crime Scene
2794005080000131127549530Suspect 2 Suspect 1
00Suspect 2 Suspect 1
13087354953000After extracting the DNA from the samples a PCR was ran. The PCR shows the sample collected from the crime scene as well as the DNA from both the suspects. It is in my opinion that the DNA found on the victim’s clothing matches perfectly with suspect number one.
ConclusionIn my expert opinion, Suspect One’s DNA was found at the scene of the crime. This was exemplified through two different DNA types, from his blood, and from semen deposited on the victim’s undergarments. The techniques performed worked effectively in getting inclusive and exclusive results for the two suspects. Since the DNA from suspect one appeared on the victims clothing it is apparent that the suspect has come in contact with the victim, whereas suspect two can be exonerated for not showing any DNA evidence at the scene of the crime.
ReferencesGreen, Eric D. “Deoxyribonucleic Acid (DNA) Fact Sheet.” National Human Genome Research Institute (NHGRI), National Human Genome Research Institute, 2015, www.genome.gov/25520880/deoxyribonucleic-acid-dna-fact-sheet/.

Anthony J.F. Griffiths, University of British Columbia; Susan R. Wessler, University of California, Riverside; Sean B. Carroll, Howard Hughes Medical Institute, University of Wisconsin–Madison; John Doebley, University of Wisconsin–Madison. (2015). Introduction to genetic analysis. New York, NY :W.H. Freeman & Company,
Brian Manarin, Forensic Evidence in Context: Cases, Materials and Commentaries (Toronto: Thomson Reuters Canada Limited, 2017)
Wade Knaap, (2018) Forensic Identification: DNA Evidence (Forensic Biology) PowerPoint Retrieved from https://blackboard.uwindsor.ca/webapps/blackboard/content/listContent.jsp?course_id=_81018_1&content_id=_784389_1&mode=resetBlais, Tony (2013, December 06). DNA: 25 years in Canadian courtrooms. Retrieved September 14, 2018, from https://torontosun.com/2013/12/06/dna-25-years-in-canadian-courtooms/wcm/50162083-5f92-4c5a-ad6a-6bb0dbe43af9
Elwell, Jenny (2009, December 01). Technical Procedures Manual: Body Fluid Identification. NCSBI Forensic Biology Section