DNA Fingerprinting and Forensic Analysis PDF

DNA Fingerprinting and Forensic Analysis Dr. Mona Rady DNA fingerprinting and forensic analysis - ILOs Define DNA fingerprinting. Outline the process of collecting and preparing a DNA sample to be used as evidence. List some factors that can degrade DNA evidence and some of the precaution...

DNA Fingerprinting and Forensic Analysis Dr. Mona Rady DNA fingerprinting and forensic analysis - ILOs Define DNA fingerprinting. Outline the process of collecting and preparing a DNA sample to be used as evidence. List some factors that can degrade DNA evidence and some of the precautions required to maintain the reliability of DNA evidence. Describe the steps in RFLP analysis. Describe the PCR method. Explain how DNA fingerprints are compared and evaluated. Compare two or more DNA fingerprints and determine if they are matches. Describe the use of DNA fingerprinting techniques in paternity testing. GUC WS2022 2 What is meant by forensic science? Forensic science is the intersection of law and science. It can be used to condemn the guilty or exonerate the innocent. Many court cases hinge on scientific evidence provided by forensics. Throughout the years, scientists have developed technologies to uncover facts in criminal investigations, and the law has been quick to embrace these new technologies as they became available. GUC WS2022 3 History of the use of scientific technologies in forensic analysis Photography Fingerprints DNA Fingerprinting GUC WS2022 4 DNA fingerprinting A collection of methods for detecting an organism’s unique DNA pattern. A DNA “fingerprint” is the unique signature found in each person’s genetic makeup. A primary tool used in forensic biotechnology. Forensic biotechnology is a powerful tool for law enforcement that can lead to the inclusion or exclusion of a person from suspicion, based on DNA evidence. DNA fingerprinting can be accomplished using trace amounts of tissue, hair, blood, or body fluids left behind at a crime scene. 5 GUC WS2022 DNA fingerprinting It was first used in 1987 to convict a rapist in England but is now routinely introduced as evidence in court cases throughout the world to convict criminals as well as to free those wrongly accused of a crime. DNA Fingerprinting for a Murder Case! This photo shows the results of DNA fingerprinting techniques comparing the DNA from blood stains on a defendant’s clothes to the DNA fingerprints of a victim’s blood. DNA fingerprinting cannot always be used to determine definitely that an accused person has committed a crime. In this case, DNA fingerprinting provided evidence that the defendant could be linked to the crime scene, although it does not mean that the defendant was guilty of the murder. 6 GUC WS2022 What is a DNA “fingerprint”? qThese two aspects of DNA that make DNA fingerprinting possible: Its uniform nature in a single individual the genetic variability between individuals GUC WS2022 7 What is a DNA “fingerprint”? qEvery human being carries a unique set of genes. qThe chemical structure of DNA is always the same, but the order of the base pairs in chromosomes differs in individuals. qThe novel assemblage of the 3.3 billion nucleotides formed into 23 pairs of chromosomes gives each of us a unique genetic identity. qEvery cell contains a copy of the DNA that defines the organism as a whole, even though individual cells have different functions (cardiac muscle cells keep our hearts beating, neurons transmit the signals that are our thoughts, etc.). GUC WS2022 8 What is a DNA “fingerprint”? qCertain variable regions in the human genome can generate a DNA profile of an individual, using samples from blood, bone, hair or other body tissues containing a nucleus with DNA. qIn criminal cases, this generally involves obtaining samples from crime-scene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions. GUC WS2022 9 What is a DNA “fingerprint”? DNA profiling depends on repeated sequences in the non- protein coding regions of the genome. These sequences, called variable number tandem repeats (VNTRs), are of particular interest in determining genetic identity. Every person has some VNTRs that were inherited from his or her mother and father. GUC WS2022 10 What is a DNA “fingerprint”? No person has VNTRs that are identical to those of either parent (since we receive one allele from each parent and they may not be the same). The individual’s VNTRs are a combination of repeats of those of the parents’ DNA regions in tandem. The uniqueness of an individual’s VNTRs provides the scientific marker of identity known as a DNA fingerprint. GUC WS2022 11  VNTRs DNA fingerprints produced by PCR are usually restricted to detecting the presence of microsatellites which are one- to six-nucleotide repeats dispersed throughout chromosomes. Because these repeated regions can occur in many locations within the DNA, the probes used to identify them VNTRs complement the DNA regions that Variable Number Tandem Repeat surround the specific microsatellite being (VNTR) analyzed. The small size of these repeated segments has resulted in the term short tandem repeat, or STR. GUC WS2022 12  VNTRs The FBI has chosen 13 unique STRs to test for DNA profiles contained within their Combined DNA Index System, or CODIS. In early 2015, the FBI announced that an additional seven loci would Variable Number Tandem Repeat be added to the CODIS Core (VNTR) sequences that are repeated effective January 1, 2017. multiple times and the number of repeats varies from person to person. GUC WS2022 13 Anatomy of a DNA Microsatellite A microsatellite is a variable number of repeated nucleotides (like TTA) that occur in specific locations in the genome. Using primers for the flanking regions on the ends of the microsatellite allows for amplification using PCR (from both directions). Individuals inherit a specific number of these repeats from their parents, but the number of repeats varies for unrelated individuals, forming a distinct pattern or DNA fingerprint. GUC WS2022 14  Human Short Tandem Repeat Patterns The FBI has chosen 13 unique STR regions (shown on human chromosomes) for analysis and comparison in its library of DNA fingerprints. DNA primers for the flanking regions of these sites are commonly found in PCR kits that amplify these sites. After DNA sequencing, the results indicate the number of repeats for each homologous chromosome at that site and constitute a DNA fingerprint for that individual. GUC WS2022 15 MCQs 1. DNA fingerprinting relies on identifying specific a. Coding sequenced b. Non-coding sequences c. Both a and b d. promoters GUC WS2022 16 MCQs 2. DNA fingerprinting relies on identifying microsatellites which are a. Repeated coding short DNA sequences b. Repeated non-coding short DNA sequences c. Repeated coding and non-coding short DNA sequences d. Non-repeated non-coding short DNA sequences GUC WS2022 17 MCQs 3. Which of the following is true regarding microsatellites a. Contain a variable number of repeated nucleotide sequences b. Are also called VNTRs c. Are also called STRs d. Contain repeated sequences that vary in number from person to person e. All of the above GUC WS2022 18 How is DNA typing performed? qThere are two main types of forensic DNA testing: Restriction fragment length polymorphism (RFLP) The polymerase chain reaction (PCR) GUC WS2022 19 Forensic DNA testing - RFLP Restriction enzymes recognize a short sequence of nucleotides and cut the DNA. Restriction enzymes are used to cut in the flanking sequences and the presence of VNTRs creates different lengths of DNA. The pattern of these cut fragments (when separated on a gel by electrophoresis) can be used to determine similarities and differences. GUC WS2022 20  Forensic DNA testing - RFLP Individual 1 has 3 cut sites in his DNA, while individual 2 has only 2. The banding patterns resulting from the electrophoretic separation shows the differences. GUC WS2022 21 Forensic DNA testing - RFLP qRFLP is not used as often for DNA testing as it once was because: RFLP testing requires larger samples than does PCR (several thousand cells are required to do RFLP analysis). The DNA must be intact and undegraded. Crime-scene evidence that is old or present only in small amounts is often unsuitable for RFLP testing. Warm, moist conditions may accelerate DNA degradation, rendering it unsuitable for RFLP testing in a relatively short period of time. GUC WS2022 22 Forensic DNA testing - PCR PCR-based testing: requires less DNA than RFLP testing is still effective if the sample is partially degraded. GUC WS2022 23 Forensic DNA testing - PCR PCR is used to amplify the DNA sample to an amount that can be analyzed (Revise the PCR steps from lecture 2 rDNA technology). Primers are designed to flank the regions of CODIS sites results in DNA amplification at specific STR sites. However, PCR-based tests are also extremely sensitive to contamination by foreign DNA at the crime scene and within the laboratory. GUC WS2022 24 Preparing a DNA Fingerprint Specimen DNA isolation PCR and collection quantification amplification. GUC WS2022 25 Preparing a DNA Fingerprint - specimen collection Sources of DNA at a crime scene: Dirty laundry A licked envelope A cigarette butt Tiny blood stains or a trace of saliva Anything else that might be a source for human cells left behind (cups, spoons, plates… etc.) GUC WS2022 26 Preparing a DNA Fingerprint - specimen collection qEvery crime scene is full of sources of possible contamination. qScrupulous attention to detail is required in collecting and preserving evidence. qTo protect the evidence, workers at a crime scene must take the following precautions: Wear disposable gloves and change them frequently. Use disposable instruments (such as tweezers and swabs). Avoid talking, sneezing, and coughing to prevent contamination with microdroplets of saliva. Avoid touching anything that might contain DNA (like their own body parts) while handling evidence. Air-dry evidence thoroughly before packaging. Microbes can contaminate a sample. Store evidence in specifically designed materials. 27 GUC WS2022 Preparing a DNA Fingerprint - specimen collection Enemies of evidence: Sunlight and high temperatures can degrade DNA. Bacteria, busy doing their natural work as decomposers, can contaminate a sample before or during collection. A small amount of moisture can damage and ruin a sample. qDNA fingerprinting is a comparative process. DNA from the crime scene must be compared with known DNA samples from the suspect. qThe ideal specimen used to compare evidence is 1 mL or more of fresh whole blood treated with an anticlotting agent called ethylenediaminetetraacetic acid (EDTA). qThis quantity of whole blood contains an adequate amount of leukocytes with nuclear DNA for testing. GUC WS2022 28 Critical Thinking Questions 3. While watching an episode of CSI: crime scene investigation, can you spot any errors in the procedure of preparation a DNA fingerprint? Usually the errors are due to contamination of DNA evidence during sample collection by foreign DNA by the investigators. GUC WS2022 29 Preparing a DNA Fingerprint – DNA extraction 1. Sample is treated with detergents that chemically that wash away the unwanted cellular material. 2. Adding protein- and RNA-digesting enzymes. 3. Precipitation in ethanol. 4. DNA quantification for accurate comparisons with other samples. There are commercially available tool kits (a set of reagents) for efficient and high-quality DNA extraction from difficult samples from crime scenes including dried blood, bone, and sexual assault samples, and swabs. GUC WS2022 30 MCQs 9. Which of the following is NOT a potential source of DNA for forensic analysis? a. Hair roots b. Red blood cells c. Epithelial cells in urine d. Muscle tissue Although blood is an excellent source of DNA, the DNA does not come from the red blood e. Saliva cells, as these cells have no nuclei. Rather, the DNA comes chiefly from white blood cells in the blood. GUC WS2022 31 Preparing a DNA Fingerprint – PCR and STRs analysis Primers are designed to flank the STRs to amplify those regions. After STRs are amplified by PCR, the alleles are separated and detected using capillary electrophoresis, which separates the lengths of amplified DNA, allowing the number of repeats in each of the two alleles on homologous chromosomes to be determined. An STR contains repeated numbers of a short (typically three- to four-nucleotide) DNA sequence. The number of repeats within an STR is referred to as an allele. GUC WS2022 32 Preparing a DNA Fingerprint – PCR and STRs analysis Example of an exclusion based on on STR from a suspect: At locus D5S818 (chromosome 5); § Person A has 9 repeats from his mother and 12 repeats from his father. § Person B has 8 repeats from both his parents. GUC WS2022 33 Preparing a DNA Fingerprint – PCR and STRs analysis For instance, the STR known as D7S820, found on chromosome 7, contains between 5 and 16 repeats of GATA. Therefore, there are 12 different alleles possible for the D7S820 STR sequence. An individual with D7S820 alleles 10 and 15, for example, would have inherited a copy of D7S820 with 10 GATA repeats from one parent and a copy of D7S820 with 15 GATA repeats from the other parent. There are 12 different alleles for this STR, creating 78 different possible genotypes, or pairs of alleles, for this one site. GUC WS2022 34 Preparing a DNA Fingerprint – PCR and STRs analysis The large number of variations for each STR provides the variability that is needed to distinguish different individuals using DNA fingerprints! GUC WS2022 35 MCQs 7. A genetic locus that is analyzed in forensic and paternity testing laboratories has four major alleles at this locus known as 1, 2, 3, and 4. How many different genotypes are possible for these four alleles? a. 8 The possible genotypes are (1,1), (1,2), (1,3), b. 10 (1,4), (2,2), (2,3), (2,4), (3,3), (3,4), and (4,4). c. 12 d. 16 e. 24 GUC WS2022 36 MCQs 5. The correct arrangement of the procedure to prepare a DNA fingerprint using PCR is a. Specimen collection – DNA extraction and quantification – STR amplification – STR analysis. b. DNA extraction and quantification – specimen collection – STR amplification – STR analysis. c. Specimen collection – DNA extraction and quantification – STR analysis - STR amplification. d. Specimen collection – STR amplification - DNA extraction and quantification – STR analysis. GUC WS2022 37 MCQs 6. The VNTR alleles analyzed in DNA fingerprinting experiments are highly variable from individual to individual. How do the many VNTR alleles at a single locus differ from each other? a. The number of protein coding genes b. The number of times a short DNA sequence is repeated c. The number of DNA methylation sites d. None of the above GUC WS2022 38 Putting DNA to Use – Case mockup Who Is Excluded by DNA Comparison? ü The DNA profiles shown below were taken from a crime scene. ü Knife stains A and B were found on the blade of a knife used in the crime, ü All stains were amplified for DNA analysis. ü Two individuals were tested for a match to the DNA profiles. Conclusions: Dorothy Smith CAN be excluded from contributing to the DNA found on the knife blade stains A and B. Jane Doe CANNOT be excluded from contributing to the DNA found on the knife blade stains A and B. GUC WS2022 39 DNA profiles for Paternity Testing For disputed paternity suits, it is possible to draw a few fetal cells from the fluid that surrounds the fetus (amniotic fluid) without harming the growing fetus. When cultured, these cells can be a source for DNA extraction and fingerprinting. When compared with the DNA of the suspected father, exclusion or inclusion of the individual Thanks to amniocentesis, it is possible to verify a can be determined. child’s parentage even before birth! GUC WS2022 40