DNA in CRIME SOLUTION

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DNA in CRIME SOLUTION : 

DNA in CRIME SOLUTION DR.IDABEL BERNABE-PAGULAYAN Former Executive Officer, ODDTS, NBI Forensic Chemist, Registered Criminologist Consultant: TF 211-DOJ; IATF-DILG; CHED;DDB Lecturer: PHILJA-Supreme Court; NFSTI

Slide 2: 

- Deoxyribonucleic Acid (de–ak–si–ri–bo–n(y)u–kle–ik) - is a chemical substance found in all cells of living organism whose composition have been passed on from parents to offsprings. It is called as the genetic or hereditary material. A person’s DNA is the SAME in every cell. It is contained in blood, semen, skin cells, tissue, organs, muscle, brain cells, bone, teeth, hair, saliva etc. - chemically, it is an acid and is composed of three sub-units, namely: 1. the phosphate group 2. a deoxyribose sugar, and 3. one of the four bases – Adenine (A), Thymine (T), Cytosine (C) and Guanine (G) What is DNA?

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FROM THE WHOLE TO THE (MICROSCOPIC) PARTS The There Each One The Genes are human is a nucleus chromosome chromosomes segments body nucleus contains 46 of every are filled of DNA contains inside chromosomes pair with that contain 100 each arranged is from tightly instructions trillion human in 23 each coiled to make cells cell pairs parent strands proteins of the building DNA blocks of life

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BIOLOGICAL EVIDENCE AMENABLE FOR DNA ANALYSIS; blood and bloodstains semen and seminal stains hair with follicle/root saliva and buccal cells tissues and skin cells organs bone marrow and bones teeth

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ADVANTAGES OF DNA ANALYSIS VS CONVENTIONAL SEROLOGICAL METHOD DNA is stable – it can be isolated from material that is months or even years old DNA can be recovered from a wide variety of biological resources like blood, semen, hair, saliva and bone DNA can be replicated in the laboratory from a small amount of initial material through the process of PCR (Polymerase Chain Reaction) DNA shows greater variability from one individual to the next

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LINE-UP of CASES where DNA ANALYSIS can be of help: Sexual assault cases like RAPE Murder Homicide Robbery Hit and run Extortion Paternity cases Identification of remains in mass disaster

Human Identity Testing : 

Human Identity Testing Forensic cases – matching suspect with evidence Paternity testing – identifying father Historical investigations Missing persons investigations Mass disasters – putting pieces back together Military DNA “dog tag” Criminal DNA databases Population genetic databases

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To avoid contamination of evidence that may contain DNA, always take the following precautions:   Wear gloves. Change them often. Use disposable instruments or clean them thoroughly before and after handling each sample. Avoid touching the area where you believe DNA may exist. Avoid talking, sneezing, and coughing over evidence. Avoid touching your face, nose, and mouth when collecting and packaging evidence Air-dry evidence thoroughly before packaging. Put evidence into new paper bags or envelopes, not into plastic bags. Do not use staples. IDENTIFYING DNA EVIDENCE

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IDENTIFYING DNA EVIDENCE

GUIDELINES FOR COLLECTING AND SUBMITTING DNA EVIDENCE : 

GUIDELINES FOR COLLECTING AND SUBMITTING DNA EVIDENCE DOCUMENTING, COLLECTING, PACKAGING and PRESERVING DNA EVIDENCE from CRIME SCENE If DNA evidence is not properly documented, collected, packaged and preserved, it will not meet the legal and scientific requirements for admissibility in a court of law. If DNA evidence is not properly documented, its origin can be questioned. If it is not properly collected, biological activity can be lost. If it is not properly packaged, contamination can occur. If it is not properly preserved, decomposition and deterioration can occur.

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DOCUMENTING DNA EVIDENCE Photograph the evidence before it is touched, moved or collected. Note the location and condition of the evidence. Note and sketch relationships of the evidence relative to the crime scene and other objects present. Note and sketch the condition of the biological evidence. COLLECTING DNA EVIDENCE COLLECTING KNOWN SAMPLES from VICTIM/SUSPECT Blood Only qualified medical personnel should collect blood samples from a person. Collect at least two 5-ml tubes of blood in purple-top tubes with EDTA as an anticoagulant for DNA analysis. Identify each tube with the date, time, subject’s name, location, collector’s name, case number, evidence number. Refrigerate, DO NOT FREEZE blood samples. Use cold packs, not dry ice during shipping. Pack liquid blood tube individually in Styrofoam or cylindrical tube containers with absorbent material surrounding the tube

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Bloodstain Prick the left ring finger of the subject with a blood lancet and stain a drop of blood in a clean sterile white cotton cloth or gauze. Air dry the stain for at least two hours out of direct sunlight without any heat source such as dryer. Label the stain with date, time, subject’s name, location, collector’s name, case number and evidence number. Pack in a clean white envelope and seal it. Submit to the laboratory as soon as possible. Saliva Use clean cotton swabs to collect saliva samples. Rub the inside surfaces of the cheeks and gums thoroughly. Air dry the swabs and place in a clean paper envelope with sealed corners. DO NOT use plastic containers. Identify each sample with the date, time, subject’s name, location, collector’s name, case number and evidence number. Saliva samples do not need to be refrigerated if properly dried. Submit to the laboratory as soon as possible.

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Hair Using a tweezer carefully pluck at least 10 strands of hair (head hair, armpit hair, beard or moustache), ensure the presence of root with sheath material. Wrap in a clean paper and place in an envelope with sealed corners. Note: DO NOT CUT the hair!!! Identify each sample with the date, time, subject’s name, location, collector’s name, case number and evidence number. Hair samples do not need to be refrigerated. Submit to the laboratory as soon as possible. COLLECTING BLOOD/BLOODSTAIN Liquid/Dried Blood Absorb suspected liquid blood onto a clean cotton or swab. Air dry the cloth or swab and pack in clean paper or an envelope with sealed corners. Do not use plastic containers. Absorb suspected dried blood onto a clean cotton cloth or swab moistened with distilled water. Air dry the cloth or swab and pack in clean paper or an envelope with sealed corners. Do not use plastic containers.

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2) Blood on Surfaces or Water Absorb suspected liquid blood or blood clots onto a clean cotton cloth or swab. Air dry the cloth or swab and pack in clean paper or an envelope with sealed corners. Do not use plastic containers. Collect suspected blood in water immediately to avoid further dilution. Place in a clean airtight container. Freeze the evidence and submit as soon as possible to the laboratory. Bloodstains Air dry suspected wet bloodstained garments. Wrap suspected dried bloodstained garments in clean paper. DO NOT place wet or dried garments in plastic or airtight containers. Place all debris or residue from the garments in clean paper or an envelope with sealed corners. Air dry small suspected wet bloodstained objects and submit the objects to the laboratory. Preserve bloodstain patterns. Avoid creating additional stain patterns during drying and packaging. Pack to prevent stain removal by abrasive action or packaging materials during shipping. Pack in clean paper. Do not use plastic containers.

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When possible cut a large sample of suspected bloodstains from immovable objects with a clean sharp instrument. Collect an unstained control sample. Pack to prevent stain removal by abrasive action. Pack in clean paper. Do not use plastic containers. Absorb suspected dried bloodstains on immovable objects onto a clean cotton cloth or swab moistened with distilled water. Air dry the cloth or swab and pack in clean paper or an envelope with sealed corners. Do not use plastic containers. COLLECTING SEMEN and SEMINAL STAINS Absorb suspected liquid semen onto a clean cotton cloth or swab. Air dry the cloth or swab and pack in clean paper or an envelope with sealed corners. Do not use plastic containers. Submit small suspected dry semen-stained objects to the laboratory. Pack to prevent stain removal. Pack in clean paper. Do not use plastic containers. When possible, cut a large sample of suspected semen stains from immovable objects with a clean sharp instrument. Collect an unstained control sample. Pack to prevent stain removal. Pack in clean paper. Do not use plastic containers.

Slide 16: 

Absorb suspected dried semen stains on immovable objects onto a clean cotton cloth or swab moistened with distilled water. Leave a portion of the cloth or swab unstained as a control. Air dry the swab or cloth and place in clean paper or an envelope with sealed corners. Do not use plastic containers. COLLECTING SEMINAL EVIDENCE from SEXUAL ASSAULT VICTIM(S) Sexual assault victim(s) should be medically examined by a medico-legal officer or city/municipal health officer using a sexual assault evidence kit to collect vaginal, oral and anal evidence. In the absence of a kit, collect vaginal and anal swabs (two each) by using sterile cotton swabs in long wooden stick as well as additional evidence like underwear and shorts worn by the victim at the time of the crime if available. DO NOT forget to collect reference samples from victims: - if living, collect saliva or buccal swabs - if deceased, collect blood, hair with root or muscle tissues with no preservatives like formalin or alcohol Refrigerate and submit the evidence as soon as possible to the laboratory.

Slide 17: 

COLLECTING SALIVA Absorb suspected liquid saliva onto a clean cotton cloth or swab. Leave a portion of the cloth unstained as a control. Air dry the cloth or swab and pack in a clean paper or an envelope with sealed corners. Do not use plastic containers. Submit suspected small, dry saliva-stained objects to the laboratory. Pack to prevent stain removal by abrasive action. Pack in clean paper or an envelope with sealed corners. Do not use plastic containers. When possible, cut a large sample of suspected saliva stains from immovable objects with a clean sharp instrument. Pack in a clean paper. Do not use plastic containers. Pick up cigarette butts with gloved hands or clean forceps. Do not submit ashes. Air dry and place the cigarette butts from the same location (ashtray) in clean paper or an envelope with sealed corners. Do not submit the ashtray unless latent print examinations is requested. Package the ashtray separately. Do not use plastic containers. Pick up chewing gum with gloved hands or clean forceps. Air dry and place in clean paper or an envelope. Do not use plastic containers. Pick up envelopes and stamps with gloved hands or clean forceps and place in a clean envelope. Do not use plastic containers.

Slide 18: 

COLLECTING HAIR Pick up hair carefully with clean forceps to prevent damaging the root tissue. Air dry hair mixed with suspected body fluids. Package each group of hair separately in clean paper or an envelope with sealed corners. Do not use plastic containers. Refrigerate and submit as soon as possible to the laboratory. COLLECTING TISSUES Pick up suspected tissues with gloved hands or clean forceps. Collect 1-2 cubic inches of red skeletal muscle. Place tissue samples in a clean, airtight plastic container without formalin/formaldehyde. Freeze the evidence, place in Styrofoam containers, and ship overnight on dry ice.

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SUBMITTING DNA EVIDENCE REQUESTING EVIDENCE EXAMINATIONS All requests for evidence examinations should be in writing and must contain the following information: The requesting and submitting contact person’s name, agency, address and telephone number. Description of the nature and the basic facts concerning the case (brief history of the case). The name(s) of and descriptive data about the individual(s) involved (subject, suspect, victim) and the agency-assigned case identification number. A list of the evidence being submitted herewith(enclosed) or under separate cover. State what types of examinations are requested. State where the laboratory report should be sent. Chain of Custody of Evidence

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PACKAGING AND TRANSPORTING EVIDENCE Take precautions to preserve the evidence. Place porous evidence in individual protective covering such as paper envelopes. Stabilize the evidence to avoid movement or friction during transport. Wrap and seal each item of evidence separately to avoid contamination. Place the evidence in a clean, dry and previously unused inner container. Seal the inner container with tamper-evidence tape. Affix EVIDENCE and appropriate BIOHAZARD labels to the inner container. Affix the evidence examination request and all case information between the inner and outer container. Place sealed inner contained in a clean, dry and previously unused outer container with clean packaging material. Completely seal the outer container so that opening of the container would be evident.

COLLECTION OF DNA EVIDENCE : 

COLLECTION OF DNA EVIDENCE

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DNA typing or profiling is a procedure wherein DNA extracted from the evidentiary sample as well as from the reference biological samples obtained from the victim and suspect are analyzed and processed to generate a particular pattern or profile for each samples. This profile is unique for each person except from identical twins. The patterns are compared either with that of a known individual to determine a match, or a set of possible relatives to determine consanguinity. In individual identification, the pattern obtained from the evidentiary sample is compared with that of a suspect. If the patterns are different, definitely it has not originated from the suspect. If it is SIMILAR, the probability that the evidentiary sample arose from the suspect and not from a random individual in the population is calculated using a formula based on well-accepted concepts of statistical probabilities and population genetics. HOW IS DNA TYPING DONE?

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In cases of determining consanguinity, DNA from the subject and his/her relatives are analyzed and compared. DNA fragments of an individual are contributed by his/her father and mother. Identification of mass disaster victims are done in the same way, the DNA of unidentified victim and relatives are analyzed and compared. In paternity cases, the DNA fragment contributed by the father should be observed in the alleged father. Then, the probability that the alleged father is the father of the child is calculated as a ratio between that of the alleged father and any random male in the population. There are several types of DNA tests that can be performed. The NBI currently employs the PCR-based testing using the Short Tandem Repeats (STR) systems like PE-ABI Profiler and Promega’s PowerPlex 16 and PowerPlex Y Systems of analysis.

STAGES OF ANALYSIS : 

STAGES OF ANALYSIS 1. EXTRACTION – to obtain the DNA material from the specimen. Two commonly used methods are Chelex, DNA IQ (rapid methods) and organic extraction. 2. QUANTITATION - to determine the amount of DNA material extracted from the sample. NBI make use of the QUANTIBLOT kit which is human specific and highly sensitive up to picogram level. 3. DNA AMPLIFICATION by PCR (Polymerase Chain Reaction) – to make many copies of specific DNA fragment. PCR is a synthesis reaction that is repeated for a number of cycles and results in exponential accumulation of the specific DNA fragment. The thermal cycler is the machine that does this PCR and is compared to a Xerox machine.

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PCR PROCESS 5’ 3’ 5’ 3’ Starting DNA Template 5’ 5’ 3’ 3’ Separate strands (denature) Reverse primer Forward primer 3’ 5’ 5’ 3’ Add primers (anneal) Make copies (extend primers) Repeat Cycle, Copying DNA Exponentially

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Thermal Cycling Parameters Time T E M P E R A T U R E 94 0C 94 0C 60 0C 720C 94 0C 94 0C 60 0C 60 0C 72 0C 72 0C Single Cycle Typically 25-35 cycles performed during PCR The denaturation time in the first cycle is Lengthened to 10 minutes when using AmpliTaq Gold to perform a “hot-start” PCR

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4. DNA TYPING/PROFILING – to determine the genotype of the donor/source of the DNA material. The NBI make use of the fully automated hi-tech equipment called ABI-PRISM 310 GENETIC ANALYZER. which allows simultaneous analysis of fifteen (15) Short Tandem Repeat loci plus Amelogenin for gender determination. Three (3) steps in PCR: DENATURATION – template DNA is made single-stranded by heat denaturation ANNEALING – temperature is lowered, annealing of primers to template will occur EXTENSION – temperature is raised again which favors specific annealing of primers and extension by DNA polymerase.

Slide 30: 

Tips for Avoiding Contamination Pre- and post-PCR sample processing areas should be physically separated Equipment, such as pipettors, and reagents for setting up PCR should be kept separate from other lab supplies, especially those used for analysis of PCR products Disposable gloves should be worn and changed frequently Reactions may also be set up in a laminar flow hood, if available Aerosol-resistant pipet tips should be used and changed on every new sample to prevent cross-contamination during liquid transfers Reagents should be carefully prepared to avoid the presence of any contaminating DNA or nucleases Ultraviolet irradiation of laboratory PCR set-up space when the area is not in use and cleaning workspaces and instruments with isopropanol and/or 10% bleach solutions help to ensure that extraneous DNA molecules are destroyed prior to DNA extraction or PCR set-up

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Steps in DNA Sample Processing Sample obtained from Crime scene or Paternity Investigation BIOLOGY DNA Extraction DNA Quantitation PCR Amplification of Multiple STR Markers TECHNOLOGY Separation and Detection of PCR products (STR Alleles) Sample Genotype Determination GENETICS Comparison of Sample Genotype to other Sample Results Generation of Case Report with Probability of Random Match If match occurs, comparison of DNA profile to population databases

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SHORT TANDEM REPEATS (STRs) Fluorescent Dye label AATG AATG AATG 7 repeats 8 repeats The repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another

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Commercially Available STR Kits

PATERNITY TESTING : 

PATERNITY TESTING A question could be raised as to who is the most likely father of a child whose mother had the misfortune of having sexual encounters with two men prior to the conception of the child. (Some real life situations include a married woman who had sex with her husband and then raped by another man prior to conception, a woman with simultaneous sexual relations with two men, etc.). The traditional tests include blood typing selected protein analysis, however, these tests are not definitive. They can only indicate whether the alleged father could possibly be the father but since many other persons in the population can have the same blood and protein type, these tests are only indicative. Biological (blood, hair or buccal swabs) samples from the child (C), the mother (M) and the alleged father/s (AF) are obtained. DNA is then extracted and the DNA patterns compared as in the following example:

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C A S E 1 C A S E 2

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In Case 1, the Child (C) has fragments b and e. It shares its b fragment with its mother (M) hence, fragment b is referred to as its maternal allele and its e fragment the paternal allele has been inherited from its father. Since fragment e is not present in the alleged father, then the alleged father (AF) could not possibly be the father of the child. In Case 2, the child again has two different parental alleles c and a. Fragment c is inherited from the mother and fragment a from its father. Hence, the alleged father is likely the father of the child because the fragments which is contributed by the father is also present in the alleged father.

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TYPES OF RESULTS 1. INCLUSION – means that the test may indicate that DNA found at the crime scene is that of the suspect. - in paternity testing, means that the alleged father could be the biological father of the child 2. EXCLUSION – when the results obtained from the suspect’s sample or known individual are not present in the results from the unknown crime scene sample. The test results show that the sample found at the scene does not belong to the suspect. - in paternity testing, means that the alleged father CANNOT be the biological father of the child. 3. INCONCLUSIVE RESULTS – include situation in which NO RESULTS were obtained from the sample because there was not enough DNA to test or in which results are obtained from an unknown sample from the crime scene but there are NO samples from known individuals or suspect to test for comparison

INCLUSION : 

INCLUSION

INCLUSION : 

INCLUSION

EXCLUSION : 

EXCLUSION