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laquanna_lewis__final_project.pptxForensic Evidence and Genetic Profiles
The Evolution of Genetic Fingerprinting/Profiling
The Use of Genetic Profiling in Forensic Evidence
Forensic Evidence and Genetic Profiles
DNA fingerprinting was discovered late in the late 20th century by Alec Jefferies. Initially DNA fingerprinting was developed as a way to establish paternity. Over the last 30 years since its discovery DNA fingerprinting has revolutionized forensic investigations. Today forensic DNA analysis helps to convict criminals, exonerate the wrongly accused, and identify victims of crime, disasters, and war.1 The current method is standardized on Short Tandem Repeats (STRs) and lineage markers (Y chromosome, mitochondrial DNA) but it all began with Restriction Fragment Length Polymorphism (RFLP) methods.
Origin of DNA Fingerprinting
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Forensic Evidence and Genetic Profiles
Restriction Fragment Length Polymorphism (RFLP) based methods became the first scientifically accepted forensic DNA profiling in the United States. RFLP was a good place to start but these methods were hindered by difficulties to reliably compare genetic profiles from different sources, they required large amounts of DNA compared to todays methods, and the DNA had to be recovered in a pristine form from a well preserved crime scene. RFLP methods can not be obtained from a single nucleated cell or from highly degraded DNA they can’t be quickly generated or automatically compared so starting in the early 1990s DNA fingerprinting methods based on RFLP analysis were gradually replaced by methods like STR which are based on PCR and have improved sensitivity, speed, and genotyping precision.1
RFLP to STR Evolution Overview
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Forensic Evidence and Genetic Profiles
In the “original” DNA fingerprinting method radio-labeled DNA probes containing either minisatellite or oligonucleotide sequences are hybridized to DNA using a restriction enzyme.1 The minisatellite is separated by agarose electrophoresis and immobilized on a membrane by southern blotting. The oligonucleotide probes are immobilized directly in a dried gel. After the separation process is performed the radio-labeled probes create a hybrid set of minisatellites or oligonucleotide stretches (depending on the material used) and genomic DNA.1 After washing away excess probe and exposing to X-ray film the variable fragments of genomic DNA can be visualized. Once visualization is established their profiles can be compared between individuals. This method was widely used in the UK and USA.
Minisatellite
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Image of Minisatellite Fingerprinting
The image (left) shows the use of minisatellite DNA for the purpose of DNA fingerprinting.
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Forensic Evidence and Genetic Profiles
In Multi-Locus Probe Profiling the DNA is digested with a selected enzyme then subjected to gel electrophoresis. The DNA is further broken down by alkali treatment and transferred onto a nylon membrane by Southern or Vacuum blotting.2 Once blotted exposure to UV light is used to affix the DNA to the membrane. MLP detects sets of 15 to 20 variable fragments per individual. Multi-locus fingerprinting had several limitations that led to its evolution into Single-locus profiling (SLP). First, the DNA quality issues made the exact matching between bands too difficult. Second, fragment association inside one DNA fingerprint profile was not known. Third, obtaining optimal profiles required large amounts of DNA despite these limitations MLP made successful contributions to solving crimes, and establishing kinship in court cases until the mid 1990s.1
Multi-Locus Probe (MLP)
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Forensic Evidence and Genetic Profiles
Image of MLP Fingerprinting
Using the MLP Southern Blot of DNA samples collected from a crime scene blood stain and 7 suspects which one do you think committed the crime? How sure can you be using this method?
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Forensic Evidence and Genetic Profiles
In Single-Locus Probe Profiling a single hypervariable locus is detected by a specific single-locus probe using high stringency hybridization1. The simple pattern created by a single locus probe is no longer specific to an individual. So most often 4 SLPs are used to yield 8 alleles of 4 independent loci per person. These create the specific DNA profile for comparison. This method requires a small amount of DNA, (~10 ng) roughly 1/10 of that needed for MLP. SLP has been validated through experiments and forensic casework, however, SLP like its 2 predecessors is still limited by the quality and amount of DNA available for comparison.
Single-Locus Probe (SLP)
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Forensic Evidence and Genetic Profiles
Image of SLP Fingerprinting
The image (left) shows the MLP method vs. the SLP method.
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Forensic Evidence and Genetic Profiles
Short Tandem Repeats (STRs) are microsatellites and the basis for the current US standard in DNA profiling. STR profiling is more sensitive than the RFLP methods and is used to fill the CODIS DNA Database. Forensic DNA profiling is currently performed using STR markers, 13 STR loci are matched in the US analysis. The microsatellites used in STR are comparable to the minisatellites used in RFLP. STRs have much shorter repeat tracts than the earlier methods and are easier to amplify with polymerase chain reactions (PCRs).1 A unique genetic code can be obtained from less than 3 nucleated cells and STRs can be produced using severely degraded DNA specimens. The probability that two people will have identical markers at 13 different STR loci exceeds one in a billion.1
Short Tandem Repeats (STR)
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Image of STR Fingerprinting
STR focuses on the number of repeats of specific nucleotide chains. The chains are unique to individuals
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Forensic Evidence and Genetic Profiles
Lineage Markers are characteristic molecules for cell lineages. The Y chromosome and mitochondrial DNA lineage markers are most commonly used to supplement STR Profiling.
Only men carry a Y chromosome in the form of XY. Females have only X chromosomes in the form of XX. Most human chromosomes have the ability to swap or share DNA between themselves. This is not true for Y chromosomes. All the information in a man's Y chromosome is passed to his son(s).3
Most DNA is contained in chromosomes inside the nucleus, but mitochondria have their own DNA in very small quantities. Mitochondrial DNA contains 37 genes in total but 13 of these genes are used to create adenosine triphosphate (ATP) which is the main function of the mitochondria. The other genes make transfer RNA (tRNA) and ribosomal RNA (rRNA), which are chemical cousins of DNA.4
Lineage Makers
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Image of Lineage Markers
The Y STR markers shown left will be identical in a males of the same lineage as demonstrated above
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Forensic Evidence and Genetic Profiles
The USA standardization of 13 STR Loci was used to implement CODIS. CODIS is a DNA database that is commonly used to identify criminals and link crimes together. On the right is an image of the 13 CODIS STR Loci identifying the chromosomal positioning of each. The possibility of 2 individuals having identical makers in these 13 Loci is more than 1 in a billion excluding identical twins.1
Advantages of the latest in DNA fingerprinting
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Forensic Evidence and Genetic Profiles
Case Study: Overview
During a 25-year span (1981–2006) the Bike Path Rapist murdered and/or raped at least four women, and raped between 9-15 girls and women in and around Buffalo, New York.5
In 1985 Anthony Capozzi was convicted of 2 murders also occurring in the Buffalo, NY area. The rapes shared the same modus operandi of the above mentioned rapes. Capozzi maintained his innocence while in prison, but was repeatedly denied parole for over two decades.5
A surviving rape victim contacted police in 1981 2 days after she was raped stating she saw her attacker at the mall. She obtained the license plate of the vehicle he was driving.
Wilfredo Sanchez Caraballo, the car's owner, was questioned but he provided a solid alibi for the rape. 25 years later Caraballo was interviewed again and admitted that on the day the car was identified by the victim he had lent it to his nephew, Altemio Sanchez.5
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Forensic Evidence and Genetic Profiles
Despite the solid alibi presented by Wilfredo Sanchez Caraballo a DNA sample was taken from him and compared to that of the Bike Path Rapist. Though the sample excluded Wilfredo as the rapist it also showed that another man from his family was in fact the rapist. Presented with this information Wilfredo admitted that the Car registered to him was not in his possession on the date it was identified by the surviving rape victim. When he confirmed the car was being driven by his nephew that day the investigation went full force towards Altemio Sanchez. Ironically the victim who's report led to the original and subsequent questioning of Wilfredo was obtained from the case files of Anthony Capozzi.5
22 years later the DNA of Anthony Capozzi was tested against samples collected at the rapes he was imprisoned for which exonerated him for the crimes. It also proved that the rapes he was sentenced for were committed by the Bike Path Rapist.5
Case Study: Catching the Right Man
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Forensic Evidence and Genetic Profiles
DNA from Altemio Sanchez was obtained by the Bike Path Task Force. This task force was comprised of the Amherst Police Department, Erie County Sheriff's Office and the Federal Bureau of Investigation (FBI)5 and created solely to find and convict the Bike Path Rapist. They acquired the silverware, a glass, and a napkin used by Sanchez while out to dinner with his wife. They submitted the items to the Erie County Forensic Lab for testing. The DNA samples matched those previously taken from semen samples collected from a murdered victim of the Bike Path Rapist. Finally they had succeeded in catching the Bike Path Rapist.
Case Study: Acquiring the Sample
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Applying what we have learned it is safe to say that the method of profiling used to convict Altemio Sanchez was comprised of STR profiling and supplemented with Y-Chromosome linage marker detection.
These conclusions are based on the fact that lip stains from silverware and a drinking glass were used to collect the DNA sample from Mr. Sanchez. Such a small sample could only be used to obtain a DNA profile using STR.
The surviving victim who correctly identified Mr. Sanchez at the mall and provided the license plate number led to the arrest and DNA testing of Altemio’s Uncle. Using the Y-Chromosome lineage marker the analysis proved that the uncle was innocent but the rapist/killer was closely related to him. This information led to the uncle’s confession of allowing his nephew to borrow his car on the day in question.
Case Study: Conclusions
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What DNA Profile method is being used?
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Based on the Southern Blot shown is the defendant innocent or guilty?
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Applying What We Learned
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RFLP Innocent
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Applying What We Learned
What DNA Profiling method is pictured? ___________________________________
Based on the nucleotide repeat being counted can we assume the participants are related? ___________________________________
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STR Yes relation is a good assumption with that many repeats of the same nucleotide sequence
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Applying What We Learned
What DNA Profile method is being used?
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Based on the Southern Blot shown is the accused innocent or guilty?
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MLP Guilty
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Sources:
Presentation:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831584/
http://www.forensicmag.com/articles/2004/09/dna-forensics-rflp-pcr-str-and-beyond
http://web.stanford.edu/~philr/Bachman/DNABachman3.html
https://ghr.nlm.nih.gov/mitochondrial-dna
https://en.wikipedia.org/wiki/Altemio_Sanchez
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Sources:
Images:
https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjamMrQy57MAhUCDywKHc7_CP8QjB0IBg&url=http%3A%2F%2Fwww.yourarticlelibrary.com%2Fbiotechnology%2Fdna-fingerprinting-5-steps-involved-in-dna-fingerprinting-in-forensic-medicines%2F33495%2F&bvm=bv.119745492,d.bGg&psig=AFQjCNEKi3FmdiW7MKtLvHABAlhG1AYR9A&ust=1461288924639176
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Sources:
https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwi7nNWxzJ7MAhVKb5oKHSPLDksQjB0IBg&url=http%3A%2F%2Fslideplayer.com%2Fslide%2F5679851%2F&bvm=bv.119745492,d.bGg&psig=AFQjCNEKi3FmdiW7MKtLvHABAlhG1AYR9A&ust=1461288924639176
https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjglfub0J7MAhVDiiwKHTlyCPoQjB0IBg&url=https%3A%2F%2Fwww.igenea.com%2Fen%2Fy-chromosome&bvm=bv.119745492,d.bGg&psig=AFQjCNF6QxH4oBSHsTvSUJ_QGJJCPDOazQ&ust=1461290159266448
https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjZ7ryl0J7MAhUnOJoKHTwfBVQQjB0IBg&url=http%3A%2F%2Ffreepages.genealogy.rootsweb.ancestry.com%2F~bricker%2Fy-chromosome.htm&bvm=bv.119745492,d.bGg&psig=AFQjCNF6QxH4oBSHsTvSUJ_QGJJCPDOazQ&ust=1461290159266448
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