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Crime Scene and DNA Basicsfor Forensic AnalystsThis course is provided free of charge and ispart of a series designed to teach about DNAand forensic DNA use and analysis.Find this course live, online at:http://dna.gov/training/evidenceUpdated: October 8, 2008DNAINITIATIVEwww.DNA.gov

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Crime Scene and DNA Basics for Forensic AnalystsWelcome to the Crime Scene and DNA Basics for Forensic Analysts courseThis course provides information in the two lessons Evidence at theCrime Scene and History and Types of Forensic DNA Testing.The first lesson addresses the importance of documenting, protecting,and preserving the scene and what types of evidence can be found thereand methods used for its collection and preservation.The second lesson addresses the historical use and disadvantages ofrestriction fragment length polymorphisms (RFLP), the method andsequence of steps in which DNA profiles are developed, and theconcept of short tandem repeats (STR) testing and its advantages over earlier methods.DisclaimerThe opinions and points of view expressed in this training program represent a consensus of the authors anddo not necessarily reflect the official position of the U.S. Department of Justice.This project was developed by the National Forensic Science Technology Center under Award No.2004-DN-BX-0079 awarded by the National Institute of Justice, Office of Justice Programs.Evidence at the Crime Scene: IntroductionResponding to a crime scene is a critical step in the scientific investigationof a case. Unless the crime scene response is handled correctly, theinvestigation may be severely compromised. Investigators and crime scenespecialists are responsible for identifying, securing, collecting, andpreserving the evidence that is submitted to the crime laboratory. Theinvestigator's knowledge in crime scene documentation and the variety ofmethods for the collection and processing of all types of evidence is crucial.Additionally, many times the investigator must make timely decisionswhether to obtain written consent or a search warrant, so that the evidencewill be admissible and not subject to a motion to suppress.ObjectivesUpon successful completion of this unit of instruction, the student shall be able to: Understand the importance of protecting and preserving the scene to ensurethe integrity of the physical evidence within. Understand the different types of crime scenes and the types of evidenceencountered in these scenes. Understand the importance of scene documentation through photography, video recording, andsketching. Understand the different methods used for collection and preservation of evidence.Types of Evidence1/14

Crime Scene and DNA Basics for Forensic AnalystsEvidence can be divided into two categories: Testimonial - statements or the spoken word from the victim(s) orwitness(es). Physical - also referred to as real evidence, consists of tangible articles suchas hairs, fibers, latent fingerprints, and biological material.The concept known as the "Locard's Exchange Principle" states that every time someone enters anenvironment, something is added to and removed from it. The principle is sometimes stated as "every contactleaves a trace," and applies to contact between individuals as well as between individuals and a physicalenvironment. Law enforcement investigators are therefore taught to always assume that physical evidence isleft behind at every scene. This will be generally true, and the amount and nature of the evidence created willbe largely dependent on the circumstances of the crime.Examples include: Biological material - blood, semen or saliva Fibers Paint chips Glass Soil and vegetation Accelerants Fingerprints Hair Impression evidence – shoe prints, tire tracks or tool marks Fracture patterns – glass fragments or adhesive tape pieces NarcoticsOftentimes, evidence tells a story and helps an investigator re-create the crime sceneand establish the sequence of events. Physical evidence can corroborate statementsfrom the victim(s), witness(es) and/or suspect(s). If analyzed and interpreted properly,physical evidence is more reliable than testimonial evidence; testimonial evidence ismore subjective in nature. An individual's perception of events and memory of whathappened can be incomplete or inaccurate. Physical evidence is objective and whendocumented, collected, and preserved properly may be the only way to reliably place or link someone with acrime scene. Physical evidence is therefore often referred to as the "silent witness."There are three types of crime scenes: Oudoor Crime Scene Indoor Crime Scene ConveyanceOutdoor2/14

Crime Scene and DNA Basics for Forensic AnalystsAn outdoor crime scene is the most vulnerable to loss, contamination, and deleteriouschange of physical evidence in a relatively short period of time. Individuals withaccess to the scene can potentially alter, destroy or contaminate evidence. The risk isgreatest when investigators fail to secure the crime scene properly.Destruction or deterioration of evidence due to environmental conditions such as heat,cold, rain, snow and wind are problems associated with outdoor scenes. Evidence thatcannot be protected under these conditions should be collected expeditiously without compromising itsintegrity. Investigators who encounter a combination of an indoor and outdoor scene should give priority toprocessing the outdoor component.Nighttime outdoor crime scenes are especially problematic. Regardless of the quality of the light source usedto illuminate the scenes, the lack of sunlight can lead to investigators inadvertantly missing or destroyingevidence. Whenever possible, outdoor crime scenes should be held and secured until daylight for processing.IndoorCompared to an outdoor scene, evidence at an indoor scene is generally less susceptible to loss, contaminationand deleterious change. Indoor crime scenes are usually easier to secure and protect, and securing a scene canbe as simple as closing a door.The methods used by forensic laboratories have evolved so that very small amountsof biological material can produce a usable DNA profile. This, however, means thatthe potential for detecting DNA traces deposited by contamination at crime scenesbecomes a factor. Contamination of any crime scene can easily occur if properprecautions, such as limiting the number of people inside the scene, are not taken. Forexample, first responders, emergency medical personnel, patrol supervisors, crimescene investigators, and medical examiners are all potential sources of contaminationand/or loss of evidence.ConveyanceConveyance is defined as "something that serves as a means of transportation." Typesof crimes committed in conveyances include, but are not limited to: Vehicle Burglary Grand Theft Car Jacking Narcotics Violation Sexual Battery HomicideIt is important that the crime scene investigator recognize that physical evidence recovered from these scenesmay extend well beyond the conveyance itself. The flight path of the perpetrator may reveal evidenceimportant to the investigation. For example, impression evidence, such as shoe or footprints in soil, may befound leading away from the scene, and property removed from the conveyance may be deposited or droppedas the perpetrator flees the scene. Cigarette butts are sometimes found in and around the conveyance. Thenature of the crime may give the investigator an idea of the type of evidence present.To protect the scene against inclement weather and other factors that may contribute to evidence loss and/ordestruction, a conveyance such as a vehicle may be transported to the laboratory after proper documentation3/14

Crime Scene and DNA Basics for Forensic Analystshas been completed.Location & Collection of EvidenceItems of physical evidence are not always visible to the naked eye and may be easily overlooked. A deliberate,methodical, disciplined approach to collection and preservation of evidence is essential. One exception maybe if evidence integrity is at risk, and under those circumstances it is important that rapid decisions be made toprevent its degradation and/or loss.It is imperative that the investigator obtain as much information as possible regarding the circumstances of thecrime prior to entering the scene. Statements from witnesses, victims, or first responders can provide abroader understanding of the investigation. The investigator can develop an approach to the scene based onthis information and the nature of the crime. For example, at the scene of a burglary, attention may focus onthe point of entry. Fragments of wood, metal, or broken glass may be discovered, along with fingerprints,blood, and fibers from clothing deposited when the perpetrator forced entry.In the case of a violent crime such as a sexual assault, attention may be directed to the clothing and the personof the victim(s) and the suspect(s). An investigator might find body fluids, stains, torn clothing, fingerprints,fibers, hair, and other trace materials in the areas where the attack took place. Potential evidence such assaliva, bite marks, semen, hair, skin tissue under the finger nails, and other trace materials may be found onthe victim(s). Transferred evidence such as cosmetics, vaginal fluid, hair from the victim, and blood may alsobe found on the suspect.Once potential evidence is located and documented, the next step is to collect andpackage the items in a manner that prevents contamination, loss, and deleteriouschange.Biological evidence requires care to guard against the possibility of crosscontamination either by the investigator or by other biological specimens at the scene.Equipment is available to crime scene investigators which aide in the prevention ofcross contamination.Types of equipment include: Tyvek white paper body suit Paper mask that covers nose and mouth Eye protection Latex or Nitrile gloves Sleeve protectors Shoe covers Hair net4/14

Crime Scene and DNA Basics for Forensic AnalystsThe investigator should prioritize the order in which evidence is collected. Biologicalevidence, trace materials, and evidence of a fragile nature should be collected first.Collection methods used to gather and package this evidence vary. The use of analternate light source (ALS) or oblique lighting may be necessary. A sample detectedwith the ALS should be properly packaged with a notation alerting the analyst that itis a luminescent sample.Preservation of EvidenceFrom crime scene to forensic laboratory to courtroom, all evidence must beinventoried and secured to preserve its integrity. Evidence admissibility in court ispredicated upon an unbroken chain of custody. It is important to demonstrate that theevidence introduced at trial is the same evidence collected at the crime scene, and that access was controlledand documented.An understanding of the rules governing chain-of-custody is vital for an investigator. For example, in a sexualassault incident, the victim is typically transported to another location to have a sexual assault examinationperformed. Many jurisdictions have established teams to perform these examinations, and they go by severalnames, such as: Sexual Assault Victim Examination (S.A.V.E.), Sexual Assault Nurse Examiners (S.A.N.E),Sexual Assualt Response Team (S.A.R.T) . The examination involves the collection of the victim's clothing,hair samples, swabs for body fluids, and documentation of bruising and bitemarks. The materials collected arepackaged by the team members.Proper evidence packaging includes: Appropriate packaging and labeling of all items Each item properly sealed and marked Correct and consistent information recorded on label and procedural documentationThe evidence is turned over to the investigator for submission to a department's property and evidence section.A receipt documenting the transfer is obtained. Generally, submissions to the forensic laboratory are done ona request for analysis form, listing the evidence items, and a documented chain of custody. Each individualassuming custody of the evidence from collection through analysis signs the chain of custody document.Many departments have automated this process using an information management system, whereby alltransfers are securely done using barcodes. The chain of custody report will identify each individualcontributing to the analysis of the evidentiary materials.Once the analysis is complete, the evidence is either returned to the submitting agency or stored by thelaboratory. The chain of custody will document this disposition. All law enforcement reports, photographs,lab analysis reports, and chain of custody documents are kept in the case file, which can be made available tothe prosecution and is subject to discovery by defense counsel.Think of the chain of custody as a chain, if one link should be broken, the chain is broken, and the evidencecollected may be ruled as inadmissible.Helpful Hints to Safeguard the Chain of Custody:5/14

Crime Scene and DNA Basics for Forensic Analysts Limit the number of individuals handling evidence. Confirm that all names, identification numbers, and dates are listed on the chain of custodydocuments. Ensure that all evidence packaging is properly sealed and marked prior to submission. Obtain signed or otherwise secure receipts upon transfer of evidence.Collection TechniquesThe importance of avoiding cross contamination cannot be overemphasized. The investigator performing thecollection must ensure tools are clean or sterilized and that gloves are changed between handling each sample.Collection methods differ depending on the type of evidence and the substrate upon which it is found. It ispreferable to collect evidence in its original state. If the evidence is fragile or can easily be lost, the entireobject should be collected and packaged, if size and circumstances permit.Some laboratories recommend the submission of substrate controls. Substrate controls are clean samples ofthe collection materials or unstained portions of the material the biological evidence is deposited on. Thelaboratories can use these to troubleshoot contamination, Polymerase Chain Reaction (PCR) inhibition, orinterference with fluorescence.The investigator should consult the local forensic laboratory and refer to the department standard operatingprocedures regarding collection and preservation of biological evidence.Procedures for Evidence CollectionBlood & Other Body FluidsType of CollectionProcedureCuttingsRemoval of a section of the item containing the stain using a sterile or cleancutting device.Wet AbsorptionA sterile swab, gauze pad, or threads are slightly moistened with steriledistilled water. An effort should be made to concentrate the stain in alocalized portion of the swab or pad. For example, when using a swab, thestain should be concentrated on the tip. The collection medium isconcentrated into the stain and allowed to air dry. Some laboratoriesrecommend following the first moistened swabbing with a second dryswabbing to ensure thorough sample collection. Both swabs are retained andsubmitted for analysis.Scraping MethodUsing a clean razor blade or scalpel, the sample is scraped into a clean pieceof paper that can be folded and packaged in a paper envelope or otherappropriate packaging.Lifting with TapeFor dried blood stains on a non-absorbent surface, fingerprint lifting tapemay be placed over the stain and lifted off. The stain is transferred to theadhesive side of the tape, which may then be secured on a clear piece ofacetate for submission to the laboratory.Hair & Fiber CollectionType of CollectionProcedureVisual CollectionOn some surfaces, hairs and fibers can be seen with the naked eye. Usingclean forceps and trace paper, the sample can be removed from the surfaceand placed into a clean piece of paper that can be folded and packaged in apaper envelope or other appropriate packaging.6/14

Crime Scene and DNA Basics for Forensic AnalystsTape LiftingVacuuming MethodWater or methanol soluble tapes are available for the collection of trace hairand fiber evidence. The tape is applied to the location of the suspectedsample, removed, and packaged.The area where the suspected samples are located are vacuumed up andcaught in a filtered trap attached to the vacuum. These samples are packagedin clean trace paper for submission to the laboratory. Vacuuming is the leastdesirable collection method because there is a risk of cross contamination ifthe equipment is not properly cleaned between each use.Reference Sample CollectionReference samples should be collected from individuals who might be linked to the crime scene where DNAevidence is found. Reference samples can be used for elimination or comparative analysis. For example,buccal swab samples taken from the suspect and/or victim, a known source, should be compared to biologicalevidence found at the crime scene to eliminate or place them at the scene.Procedures for Reference Sample CollectionType of Collection ProcedureBuccal SwabSterile swabs or other buccal collection devices arerubbed against the inside cheek of the individual'smouth to collect epithelial cells for analysis.Liquid BloodGenerally collected in purple topped vacuum tubesSamplesthat contain the preservative ethylenediaminetetraacetic acid (EDTA).Packaging & StorageBiological evidence should be dried before packaging to minimize sample degradation. Packaging in paper ispreferred; however, some laboratories allow packaging in plastic if the sample is thoroughly dried.Liquid samples, such as water from a toilet bowl or pipes, should be properly documented and packaged insterile glass or plastic containers and refrigerated as soon as possible.Documentation - Chain of CustodyDocumentation of the scene begins with the first responder. Police officers are taught the importance of takingnotes from the time of arrival. The crime scene investigator documents the scene in the form of still and videophotography. Sketches are completed at the scene to illustrate relationships between articles of evidence noteasily depicted by photography. The following methods of crime scene documentation are used to provide anaccurate representation of the scene.7/14

Crime Scene and DNA Basics for Forensic AnalystsMethods of Crime Scene DocumentationType of DocumentationGuidelinesNote TakingIt is important that the responding officers note the condition of the scene asit existed upon their arrival. Note taking should be continuously updatedduring the course of the investigation.Investigator's notes might include such factors as: Victim and witness statements Who was present at the scene Lighting conditions Open doors and windows Odors Signs of activity such as food preparation Date and time indicators such as newspapers or mail General descriptions of the scene and surrounding areaPhotography and Videography The primary means of crime scene documentation is still photography.Police officers should have an understanding of the importance of keepingthe scene preserved, and not moving anything until it is photographed. Thephotographer must be able to testify that the photograph is a true andaccurate representation of the scene at the time the photograph was taken.Crime scene photographs should reveal a detailed, chronological story of thescene.SketchingPhotographs may not always depict spatial relationships between objects;sketches are used to supplement photographs. Sketches can more easilydepict the overall layout of the scene and the relationships between objects.Investigators usually complete hand-drawn, rough sketches while at thecrime scene. These sketches contain all the necessary information for theinvestigator to subsequently complete a finalized version.For courtroom presentation, hand drawn sketches may be converted using computerized programs such ascomputer aided design (CAD), which provide a dynamic, professional appearance.Types of sketches may include: Entire Scene - the complete scene with measurements Bird's-eye View – an overhead view of the scene Elevation Sketch Cross Projection Sketch Three Dimensional Sketch8/14

Crime Scene and DNA Basics for Forensic AnalystsHistory and Types of Forensic DNA Testing: IntroductionDNA testing is a relatively recent technological advance in the field of forensic biology. The technology has aremarkable power to discern genetic differences, pretty well to the point of individualization. Coupled withthe development of increasingly sensitive methods, DNA testing is now an essential part of the crimelaboratory's armamentarium in the investigation of crimes against the person.ObjectivesUpon successful completion of this unit of instruction, the student shallbe able to do the following: Understand the history of forensic science and the use ofrestriction fragment length polymorphisms (RFLP) Understand the method in which DNA profiles are developed,including the use of oligonucleotides as probes Understand the disadvantages of RFLP analysis Understand the concept of short tandem repeats (STR) testing,including multiplexing and the ability to label nucleotides withfluorescent tags Describe the history of forensic DNA testing Describe the advantages of STR typing over earlier methods Describe the sequence of steps involved in DNA typingDNA Typing by RFLP AnalysisDNA typing was introduced into forensic science in the mid-1980s, arising from discoveries made inbiomedical research. Ray White, an American geneticist at the University of Utah, identified regions of DNAthat did not code for proteins but were highly variable between individuals.Early research included the use of restriction enzymes to cut strands of DNA at specific locations and produceDNA fragments of defined lengths. White separated the fragments based on size, calling the variationsrestriction fragment length polymorphisms (RFLP). In 1980, White and colleagues described the firstpolymorphic RFLP marker and proposed methods for mapping the human genome based on RFLPtechnology.01The first forensic science applications of the technique arose from the work of Alec Jeffreys who found thatRFLP technology could be used to develop patterns of restricted DNA that were more or less specific to anindividual. At the time, his work focused on paternity testing. In 1985, the British police from West Midlandsapproached Jeffreys to assist them in a rape-homicide case. Jeffrey's work resulted in the release of awrongfully convicted man and the apprehension and conviction of the true perpetrator. Soon thereafter, RFLPDNA evidence contributed to the convictions of Tommy Lee Andrews in Florida and Timothy WilsonSpencer in Virginia.02The pieces of DNA cut by the restriction enzymes contain genes and non-coding DNA. The non-coding DNAincludes regions consisting of direct repeats of the same sequence of bases, referred to as tandem repeats. Thenumber of repeats of the sequence is genetically determined and, provided that the sequence is long enoughand is repeated a sufficient number of times, will affect the length of the restriction fragment. These regionsare called variable number of tandem repeats (VNTR) loci.9/14

Crime Scene and DNA Basics for Forensic AnalystsVariable Number of Tandem Repeats (VNTRs)Since each VNTR region consists of repeats of a specific sequence of bases, complementary oligonucleotidescan be synthesized. These oligonucleotides, when labeled with a marker such as P32 or a chemiluminescentcompound, are known as probes.There are five basic steps to developing DNA profiles using VNTRs:1. Extracting DNA2. Cutting DNA into fragments using restriction enzymes3. Separating the fragments based on size using gel electrophoresis4. Transferring the fragments to a nylon membrane (southern blotting), causing immobilization5. Locating and identifying the fragments by applying a solution containing the probe of interest, whichthen hybridizes to the immobilized DNA. Visualization of the fragments requires a lengthy exposureof the probe to a detection system and can add several days to the assay time.Example of 16-base pair tandem repeats in the D1S80 locus.The sequence is usually repeated between 14 and 40 times.ProbesProbes will bind specifically to complementary VNTR fragments. Unbound and non-specifically bound probeis removed using a washing process. The RFLP profile is then visualized by exposing the membrane to filmor through the use of equipment, such as the Kodak imaging station.Visit the Kodak website to read more about the KODAK Image Station 4000R Digital Imaging System.The technique originally developed by Jeffreys used multi-locus probes which could hybridize to severalVNTR sites.Alternatively, profiles can be developed by using a single probe process. The membrane containing theimmobilized fragments is treated with one probe and visualized. This single probe is then removed andanother probe is applied to the membrane. This was the most common method used in the United States, andthe process generally consisted of four to six probes. Single locus probe procedures are easier to interpret andallow for simplified court presentation.Disadvantages10/14

Crime Scene and DNA Basics for Forensic AnalystsWhile highly discriminatory, RFLP analysis of VNTRs has severaldrawbacks, including: The process is extremely laborious and time-consuming Radioactive probes pose health and disposal risks (althoughchemiluminescent technology eliminated this risk) A relatively large amount of sample is required to perform thetests The method requires high molecular weight, un-degraded DNA The use of yield gels is an essential, but time consuming, stepin the analysis not only to estimate the amount of DNArecovered but also to determine the suitability of the sample foranalysisRead more about yield gels in the DNA Extraction & Quantitation PDF file.RFLP allele assignment was performed by incorporating a reference sizing ladder in the electrophoresis andcomparing the fragments to the size markers. Various approaches were developed to deal with the intrinsicvariation in the method, including the use of windows or bins to express the range within which the truefragment size lay. Apart from the undesirability of having to explain why there was not a definitive sizeassigned to a fragment, the inherent continuous distribution of allelic states posed challenges for the nationalDNA database software (CODIS).Polymerase Chain Reaction (PCR)In 1983, Kary Mullis developed the technique known as the polymerase chain reaction (PCR),03 whichultimately revolutionized molecular biology, including forensic DNA analysis. Through PCR, forensic DNAanalysis essentially became more rapid and sensitive. The problems of analysis time, use of radioactivematerials, large sample size, presence of un-degraded high molecular weight DNA, and the need to deal withsizing variations were all dealt with by the various PCR techniques.Read more about Quantitative PCR in the DNA Extraction & Quantitation PDF file.One of the first forensic PCR tests was based on identification of human leukocyte antigen (HLA). The HLAmarkers are proteins of known sequence; the genes coding for each antigen can be identified. A PCR-basedassay for one of the HLA loci, DQ-Alpha, was developed in 1991 and was used in crime laboratories. The kit,developed by the Cetus Corporation and marketed by Roche, was simple to use and required minimalequipment.DQ-Alpha11/14

Crime Scene and DNA Basics for Forensic AnalystsDQ-Alpha has four main alleles, numbered 1 through 4. The DQ-Alpha1 and 4 alleles have sub-types (1.1, 1.2, 1.3 and 4.1, 4.2, 4.3). However,the strips could not recognize the specific 1.2 allele nor could theydistinguish between the 4.2 and 4.3 alleles.The concept was extended to incorporate a number of other loci to betyped concomitantly with DQ-Alpha, and marketed as Polymarker in1993. A kit containing reagent for both DQ-Alpha and Polymarkerbecame quite popular. The system was sensitive and easy to use buteven the combination kit did not afford the discrimination power ofRFLP.AmpFLPsAmpFLP (D1S80) visualized using silver stainingThe next phase in the development of DNA for use in forensic science returned to the conce

specialists are responsible for identifying, securing, collecting, and preserving the evidence that is submitted to the crime laboratory. The investigator's knowledge in crime scene documentation and the variety of methods for the collection and processing of all types of evidence is crucial.