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DNA The Indispensable Forensic Tool 1 DNA The Indispensable Forensic Tool 1

Introduction • Genetic information is carried in the form of DNA in all cellular Introduction • Genetic information is carried in the form of DNA in all cellular organisms. • DNA is the genetic code that imparts our individuality. 2

Introduction • Increasing knowledge of the human genome and improved detection technologies and the Introduction • Increasing knowledge of the human genome and improved detection technologies and the development of the polymerase chain reaction (PCR) technique have increased the sensitivity, speed and discrimination potential of DNA profiling. 3

Introduction • For identification of an individual, we can examine specific regions of DNA Introduction • For identification of an individual, we can examine specific regions of DNA as they vary from person to person, in order to create a DNA profile or fingerprint of the individual in question. • There is an extremely low probability that another person has the same DNA profile for the given set of regions. 4

Introduction • DNA as a tool forensic identification has broad applications, including: – identification Introduction • DNA as a tool forensic identification has broad applications, including: – identification of potential suspects or subjects involved in a crime or who may have contributed to a crime scene stain – exoneration of wrongly accused persons – identifying family relationships or establishing paternity – identifying victims of crime, war, catastrophe or other death investigations 5

Objectives • Understand the principles of Forensic DNA profiling • Have an understanding of Objectives • Understand the principles of Forensic DNA profiling • Have an understanding of the range of profiling methods • Know the method of sample processing and sample limitations • Understand the significance of DNA profiling results and the limitations of the data • Know the methods used in DNA extraction and Quantization • Know the components of a Genetic analyzer • Be familiar with DNA databases available and their applications • Understand the applications of Mitochondrial DNA 6

Classical Genetics 1 Children resemble their parents. 2 Genes come in pairs. 3 Genes Classical Genetics 1 Children resemble their parents. 2 Genes come in pairs. 3 Genes don't blend. 4 Some genes are dominant. 5 Genetic inheritance follows rules. 6 Genes are real things. 7 All cells arise from pre-existing cells. 8 Sex cells have one set of chromosomes; body cells have two. 7

Classical Genetics 9 Specialized chromosomes determine gender. 10 Chromosomes carry genes. 11 Genes get Classical Genetics 9 Specialized chromosomes determine gender. 10 Chromosomes carry genes. 11 Genes get shuffled when chromosomes 11 exchange pieces. 12 Evolution begins with the inheritance of gene variations. 13 Mendelian laws apply to human beings. 14 Mendelian genetics cannot fully explain human health and behavior. 8

Individual Characteristics 15 DNA and proteins are key molecules of the cell nucleus. 16 Individual Characteristics 15 DNA and proteins are key molecules of the cell nucleus. 16 One gene makes one protein. 17 A gene is made of DNA. 18 Bacteria and viruses have DNA too. 19 The DNA molecule is shaped like a twisted ladder. 20 A half DNA ladder is a template for copying the whole. 9

Molecules of Genetics 21 RNA is an intermediary between DNA and protein. 22 DNA Molecules of Genetics 21 RNA is an intermediary between DNA and protein. 22 DNA words are three letters long. 23 A gene is a discrete sequence of DNA nucleotides. 24 The RNA message is sometimes edited. 25 Some viruses store genetic information in RNA. 10

Molecules of Genetics 26 RNA was the first genetic molecule. 27 Mutations are changes Molecules of Genetics 26 RNA was the first genetic molecule. 27 Mutations are changes in genetic information. 28 Some types of mutations are automatically repaired. 11

Command & Control of Genetics 29 DNA is packaged in a chromosome. 30 Higher Command & Control of Genetics 29 DNA is packaged in a chromosome. 30 Higher cells incorporate an ancient chromosome. 31 Some DNA does not encode protein. 32 Some DNA can jump. 33 Genes can be turned on and off. 12

Command & Control of Genetics 34 Genes can be moved between species. 35 DNA Command & Control of Genetics 34 Genes can be moved between species. 35 DNA responds to signals from outside the cell. 36 Different genes are active in different kinds of cells. 37 Master genes control basic body plans. 13

Command & Control of Genetics 38 Development balances cell growth and death. 39 A Command & Control of Genetics 38 Development balances cell growth and death. 39 A genome is an entire set of genes. 40 Living things share common genes. 41 DNA is only the beginning for understanding the human genome. 14

Principles of Heredity DNA 15 Principles of Heredity DNA 15

Principles of Heredity • Genes are the basic unit of heredity. • Each gene Principles of Heredity • Genes are the basic unit of heredity. • Each gene by itself or with others controls the development of a specific characteristic in the new individual. • Genes are located on chromosomes. • Human cells contain 46 chromosomes which are mated in 23 pairs. The only case in which this is not so is human reproductive cells which have 23 chromosomes. During the fertilization of the egg, the reproductive cells combine to form the zygote which will then have 46 again. 16

Principles of Heredity • The egg cell always contains the X chromosome, but the Principles of Heredity • The egg cell always contains the X chromosome, but the sperm cell may have either the X or a Y chromosome. The combining of an XX will develop into a female, and XY will develop into a male. • Just like our chromosomes, the genes they carry also come in pairs. When the chromosomes pair up, so do the genes that they carry (eye color with eye color, and hair color with hair color) • Alternative forms of genes that influence a given characteristic and are aligned with one another on a chromosome pair are known as alleles. 17

Principles of Heredity • An example of these allele genes is human blood types Principles of Heredity • An example of these allele genes is human blood types which are categorized with the A-B-O system. • For example, a gene pair consisting of similar genes is called homozygous (AA, BB) • A pair that consists of two different genes is said to be heterozygous (AO) • When there is a heterozygous pair, the dominant gene is the one that will show. The A and B genes are said to be dominant and the O is recessive. 18

Principles of Heredity • A pair of allele genes together make up what is Principles of Heredity • A pair of allele genes together make up what is called the genotype (which is the genetic make up) Phenotypes are someone’s physical characteristics that we would be able to see. • Genotypes can be determined by studying someone’s family history. • If the genotypes of both parents are known, the genotypes of their possible offspring can be somewhat predicted. 19

Principles of Heredity • A simple way of predicting genotypes is the Punnett square. Principles of Heredity • A simple way of predicting genotypes is the Punnett square. • Although genotyping is useful for studying the transmission of genes it isn’t directly related to criminal investigations. • It is more useful during paternity testing which would be disputed in civil, not criminal court. 20

Packaging of Bio Evidence • Before the collection of biological evidence begins, it is Packaging of Bio Evidence • Before the collection of biological evidence begins, it is important that it be photographed and recorded on sketches. • Wearing disposable latex gloves while handling the evidence is required. • Clothing from victim and suspect with blood evidence must be collected. • The packaging of biological evidence in plastic or airtight containers must be avoided because the accumulation of residual moisture could contribute to the growth of DNA-destroying bacteria and fungi. 21

Packaging of Bio Evidence • Each stained article should be packaged separately in a Packaging of Bio Evidence • Each stained article should be packaged separately in a paper bag or in a well-ventilated box. • Dried blood is best removed from a surface by using a sterile cotton swab lightly moistened with distilled water that is air dried before being placed in a swab box, then a paper or manila envelope. • All biological evidence should be refrigerated or stored in a cool location until delivery to the laboratory. • Standard/reference DNA specimens must also be collected, such as blood or the buccal swab 22 (swabbing the mouth and cheek).

DNA in a Nutshell • DNA is constructed as a very large molecule made DNA in a Nutshell • DNA is constructed as a very large molecule made by linking a series of repeating units called nucleotides. • A nucleotide is composed of a sugar, a phosphorouscontaining group, and a nitrogen-containing molecule called a base. • Four types of bases are associated with the DNA structure: adenine (A), guanine (G), cytosine (C), and thymine (T). • As a result, adenine pairs with thymine and guanine pairs with cytosine. • This concept is known as base pairing. 23

DNA in a Nutshell • Each group of three nucleotides in a DNA sequence DNA in a Nutshell • Each group of three nucleotides in a DNA sequence codes for a particular amino acid. – Example: G-A-G codes for the amino acid glutamine, while C-G-T codes for alanine. – If a nucleotide is “changed”, for example a T is substituted for A and G-A-G becomes G-T-G, the “wrong” amino acid is placed in the protein – This is the basis for many diseases and health issues. 24

How nucleotides can be linked to form a DNA strand. S designates the sugar How nucleotides can be linked to form a DNA strand. S designates the sugar component, which is joined with phosphate groups (P) to form the backbone of DNA. Projecting from the backbone are four bases: A, adenine; G, guanine; T, 25 thymine; and C, cytosine.

DNA in a Nutshell • DNA duplicates itself prior to cell division. • DNA DNA in a Nutshell • DNA duplicates itself prior to cell division. • DNA replication begins with the unwinding of the DNA strands of the double helix. • The ability to multiply small bits of DNA now means that sample size is no longer a limitation in characterizing DNA recovered at a crime scene. • All humans have the same type of repeats in DNA, but there is tremendous variation in the number of repeats each of us have. 26

A representation of a DNA double helix. Notice how bases G and C pair A representation of a DNA double helix. Notice how bases G and C pair with each other, as do bases A and T. This is the only arrangement in which two DNA strands can align with each other in a double-helix configuration. 27

(a) A string of amino acids composes one of the protein chains of hemoglobin. (a) A string of amino acids composes one of the protein chains of hemoglobin. (b) Substitution of just one amino acid for another in the protein chain results in sickle-cell hemoglobin. 28

Replication of DNA. The strands of the original DNA molecule are separated, and two Replication of DNA. The strands of the original DNA molecule are separated, and two new strands are assembled. 29

DNA in a Nutshell • Typically, a core sequence consists of 15 to 35 DNA in a Nutshell • Typically, a core sequence consists of 15 to 35 bases in length and repeats itself up to a thousand times. • The key to understanding DNA typing lies in the knowledge that numerous possibilities exist for the number of times a particular sequence of base letters can repeat itself on a DNA strand. 30

DNA Profiling By extracting DNA and digesting it with a restriction enzyme that cut DNA Profiling By extracting DNA and digesting it with a restriction enzyme that cut the DNA outside but not within the repeat block, the size of each block of repeats could be determined by electrophoresis, and the term restriction fragment length polymorphism (RFLP) was applied. 31

DNA Profiling The first application of RFLP analysis was in the study of genetic DNA Profiling The first application of RFLP analysis was in the study of genetic diseases. Many RFLP sequences were mapped to a specific location on a chromosome. The gene associated with a genetic disease, i. e. , Huntington’s disease, could be located by studying the transmission of many RFLP sequences through the generations of a family. 32

The DNA RFLP typing process. 33 The DNA RFLP typing process. 33

DNA Profiling Forensic serology was utilizing protein and enzyme genetics for the purpose of DNA Profiling Forensic serology was utilizing protein and enzyme genetics for the purpose of identification. The source of a body fluid or tissue could be narrowed down by determining the ABO type or by studying variants in a few enzymes These were separated by electrophoresis and visualized using an enzymatic activity -based color reaction. 34

DNA Profiling Most of the markers of forensic interest were located on blood cells DNA Profiling Most of the markers of forensic interest were located on blood cells and were therefore not useful in characterizing saliva, semen or vaginal secretions. The ABO types were present in these body fluids, but only in “secretors” who make up about 80% of the population. The fragile nature of proteins also leads to loss of enzymatic activity and detectability after short periods of time in adverse environmental conditions. 35

DNA Profiling 1985, Sir Alex Jeffreys was credited with the first practical forensic application DNA Profiling 1985, Sir Alex Jeffreys was credited with the first practical forensic application of human DNA analysis. Jeffries used the RFLP method to identify the semen donor in a serial rapehomicide case in Britain. This first use of forensic DNA profiling is described in Joseph Wambaugh's The Blooding. 36

DNA Profiling In 1986, two companies in the US began offering commercial DNA testing. DNA Profiling In 1986, two companies in the US began offering commercial DNA testing. Lifecodes and Cellmark developed RFLP methods using 2 different restriction enzymes. Due to the use of different restriction enzymes, data generated by the two companies could not be inter-compared. The FBI went online in 1988 with a method using the restriction enzyme Hae III which has become fairly standardized among state and local forensic laboratories 37

Restriction Fragment Length Polymorphism The RFLP method identifies variation in the number of repeats Restriction Fragment Length Polymorphism The RFLP method identifies variation in the number of repeats in tandem repeat tracts. After size fragmentation by gel electrophoresis, DNA is transferred to a nylon membrane by the Southern blot method. 38

Restriction Fragment Length Polymorphism Southern Blot 39 Restriction Fragment Length Polymorphism Southern Blot 39

Clean Technique • Similar to what Microbiology uses. • Prevents cross-contamination • Surface areas Clean Technique • Similar to what Microbiology uses. • Prevents cross-contamination • Surface areas where samples are processed should be cleaned with a fresh 10% bleach solution or appropriate disinfectant cleaner. • All instruments used to process forensic samples (e. g. , forceps, scissors, scalpel & razor blades, bone cutting equipment, pipettes' and metal probes) must be cleaned. 40

Clean Technique Samples should always be placed on clean surfaces and in sterile tubes. Clean Technique Samples should always be placed on clean surfaces and in sterile tubes. Waste that may contain amplified DNA should be disposed of with great care. Contamination of extraction areas with amplified product is the most difficult kind of contamination to eradicate after it has occurred. Finally the foot traffic in the lab where analysis is being conducted should be limited 41

Sampling Questioned samples should be processed separately from reference standards, by completing the analysis Sampling Questioned samples should be processed separately from reference standards, by completing the analysis of questioned samples at a different time in a different location than reference standards. Eliminates the possibility of cross-contamination or sample switching 42

Sample Size & Sampling Size • The preservation & conservation of evidence is important Sample Size & Sampling Size • The preservation & conservation of evidence is important to forensic DNA quality assurance. • The most effective challenge to a DNA test is reanalysis by a second independent laboratory. • Before testing the DNA analyst must decide whethere is sufficient sample for one or more tests. (When the quantity of sample is sufficient, a portion of the sample is set aside and preserved for possible future re-analysis). 43

Degraded DNA If samples are thought to contain partially degraded DNA, more sample may Degraded DNA If samples are thought to contain partially degraded DNA, more sample may be needed to obtain useful results. Environmental conditions before, during and after the deposition of a stain and even in collection, packaging and storage greatly affect its quality. 44

Degraded DNA • If a drop of blood falls on a cloth and dries Degraded DNA • If a drop of blood falls on a cloth and dries quickly, the DNA will be well-preserved. • If the drop of blood falls on cloth, which remains damp for any appreciable period of time, microorganisms may degrade the DNA in white blood cells (remember red blood cells don’t contain DNA). • The surface upon which the body fluid is deposited is important. The DNA in human body fluid cells will be degraded rapidly on items (rich in microorganisms) i. e. , : foliage soil carpeting any warm and moist surface 45

Degraded DNA • Most agencies have evidence submission manuals that include handling; the FBI Degraded DNA • Most agencies have evidence submission manuals that include handling; the FBI Handbook can be found at the following website: http: //www. fbi. gov/hq/lab/handbook/intro. htm 46

Relevance of the Evidence The relevance of any evidence to a particular case depends Relevance of the Evidence The relevance of any evidence to a particular case depends on the close interactions between the DNA analyst, the crime scene investigator, the case investigator and the prosecutor. 47

Relevance of the Evidence • The crime scene investigator has a brief window of Relevance of the Evidence • The crime scene investigator has a brief window of opportunity to collect evidence that may be relevant to the crime being investigated. The investigator will collect a broad range of evidence samples. • The crime scene investigator or responding officer must document unique information relating to the state of the scene and evidence collected. 48

Relevance of the Evidence After preliminary investigation, the case investigator may be able to Relevance of the Evidence After preliminary investigation, the case investigator may be able to provide information about the offense, such as any prior relationship of the victims and suspects, statements to police, medical reports, and how various evidentiary items relate to the crime. 49

Relevance of the Evidence The prosecutor brings knowledge of defense theories, pertinent case questions, Relevance of the Evidence The prosecutor brings knowledge of defense theories, pertinent case questions, legal standards of proof and an overview of information about the case gleaned from many different sources. The DNA analyst contributes knowledge of the capabilities and limitations of various DNA testing methods. The analyst knows what items are most likely to produce profiles and what interpretations might be made regarding those profiles. The analyst can use DNA to determine the source of a biological sample, but not when or under what circumstances it was deposited. 50

Relevance of the Evidence The most beneficial reason for allowing the DNA analyst access Relevance of the Evidence The most beneficial reason for allowing the DNA analyst access to certain aspects of the case is in enhancing his/her ability to select and test evidence in an order consistent with its likelihood to yield results and its probative quality. Because stains are not always easy to see, crime scenarios may help lead the analyst to the best area for testing and save time for the analyst, contributor and courts. 51

Relevance of the Evidence Not uncommon to find blood from multiple individuals on the Relevance of the Evidence Not uncommon to find blood from multiple individuals on the clothing of violent perpetrators. Lacking evidence to connect the suspect to the injured victim, the DNA analyst might perform an exhaustive analysis of blood on the victim’s clothing or other items from the scene, as the suspect may have been injured during the event. 52

Relevance of the Evidence It is not uncommon for a perpetrator to injure him/herself Relevance of the Evidence It is not uncommon for a perpetrator to injure him/herself during a knife attack, particularly when one hand is used to control the victim while the other holds the knife. Most physical struggles will result in the transfer of some trace DNA evidence from subject to victim. 53

Choosing the Technique Currently there are three main varieties of PCR-based forensic DNA typing. Choosing the Technique Currently there are three main varieties of PCR-based forensic DNA typing. The major technique used in most crime labs is the STR multiplex which relies on nuclear DNA and is applicable to all nucleated specimen types capable of yielding at least one nanogram of DNA. Samples might involve saliva, blood, semen, vaginal secretions, body tissues, spongy bone, and growth phase hairs with roots or sheaths. 54

Choosing the Technique A common evidence type not amenable to nuclear STR testing is Choosing the Technique A common evidence type not amenable to nuclear STR testing is hair shafts. A hair root may contain a tissue tag or nucleated cells that will give STR results, but not the shaft. The only DNA test capable of providing results on this material is mitochondrial testing. (FBI started using in 1996) mt. DNA testing is also the method of choice for badly degraded samples such as old skeletal remains. mt. DNA is inherited through the maternal line. Any maternally related individual or the children of a female individual could serve as reference standards. 55

Choosing the Technique In a mixture of male and female DNA where the female Choosing the Technique In a mixture of male and female DNA where the female component is expected to greatly exceed the male component, Y-chromosome STRs are a technique of choice. Based on Y-chromosome STR loci, the test can indicate the presence of a male to corroborate a story or yield a profile of a specified male. This test is almost exclusively used on mixtures of male and female DNA. Where semen does not contain spermatozoa or the mixture consists of male saliva and female secretions, nuclear STRs can discern an interpretable mixture up to about a 1: 10 ratio of male to 56 female DNA.

Comparison of Techniques The US Department of Defense employs mt. DNA typing through its Comparison of Techniques The US Department of Defense employs mt. DNA typing through its Defense Prisoner of War/Missing Personnel Office. http: //www. dtic. mil/dpmo/family/dnatyping. htm 57

Organic Extraction • Once the sample is chosen the DNA must be extracted from Organic Extraction • Once the sample is chosen the DNA must be extracted from the sample before electrophoresis can be conducted. 58

Organic Extraction Organic extraction is appropriate for most forensic specimens including: • Blood & Organic Extraction Organic extraction is appropriate for most forensic specimens including: • Blood & blood stains • Epithelial cells • Saliva • Vomit • • Urine Feces Sweat Semen (differential extraction method) • Hair • Tissue • Bone 59

Organic Extraction • Organic extraction uses and steps: – a digest buffer (SDS) to Organic Extraction • Organic extraction uses and steps: – a digest buffer (SDS) to denature proteins – serine (pro-K) to cleave the proteins into smaller fragments. Pro-K digestion helps lyse the cells and solubulizes components while denaturing the protein. – alcohol, provides an alkaline environment and precipitates the RNA and protein which separates it from aqueous DNA, Phenol and chloroform. – wash sample with buffer (TE) – filter to remove leftover extraction reagents and contaminants. 60

Organic Extraction • The amount of sample used is based on the sample type, Organic Extraction • The amount of sample used is based on the sample type, concentration, substrate and expected condition. • Blood stains may be obtained from a cutting of the fabric on which they were deposited except when found on blue denim. The dye in denim is a PCR inhibitor, so swabbing a bloodstain on jeans is the best method of recovery. Cotton swabs should be used for DNA extraction. 61

Chelex Extraction This method is appropriate for many forensic specimens including: • Blood & Chelex Extraction This method is appropriate for many forensic specimens including: • Blood & blood stains • Epithelial cells • Saliva • Hair • Semen (differential extraction method) 62

Chelex Extraction • It is highly selective for heavy metals and divalent cations versus Chelex Extraction • It is highly selective for heavy metals and divalent cations versus monovalent cations. . • The basic nature of Chelex resin keeps extracted DNA in a single stranded state available for concentration and/or quantization. 63

Chelex Extraction Blood • Samples are first extracted in DI water or saline to Chelex Extraction Blood • Samples are first extracted in DI water or saline to help free cells from the substrate and to lyse any red blood cells. The supernatant, which may contain heme, can be discarded. Washing steps help remove any residual protein. Cells are then incubated with Chelex and lysed (broken apart) by boiling. 64

Chelex Extraction • Epithelial Cells • Chelex extraction is a rapid method for extraction Chelex Extraction • Epithelial Cells • Chelex extraction is a rapid method for extraction of DNA from epithelial cells found on items such as cigarette butts, envelope flaps, stamps, buccal swabs, beverage containers and swabbings or cuttings from other textiles or hard surfaces. • Usually exposed to the environment 65

Chelex Extraction • Hairs that just fall out are in telogen or resting phase Chelex Extraction • Hairs that just fall out are in telogen or resting phase and do not possess nucleated root cells or a tissue tag and will not provide sufficient DNA for typing. • Hairs in the growth or cessation stages (anagen or catagen) may have nuclear DNA in their roots. • Hairs, forcibly removed, may or may not have tissue tags. 66

Chelex Extraction • It is very important to clean the hair thoroughly to remove Chelex Extraction • It is very important to clean the hair thoroughly to remove any foreign body fluids or other materials. Retain the hair shaft as a control. • If nuclear DNA testing fails, the shaft may be sent for mt. DNA analysis. Approximately a 1 cm (2. 54 cm=1 inch) length of hair, obtained form the root end is necessary for analysis. Hairs mounted on slides can also be recovered for analysis. 67

Chelex Extraction Sperm • Mixtures containing spermatozoa provide a unique opportunity to separate the Chelex Extraction Sperm • Mixtures containing spermatozoa provide a unique opportunity to separate the DNA profiles of the contributors. • After pelleting and washing the sperm, a second extraction employing DTT lyses the sperm cells to release their DNA. 68

Organic Extraction • Organic extraction of stains or swabs either known or suspected to Organic Extraction • Organic extraction of stains or swabs either known or suspected to contain sperm calls for a slightly altered procedure. The most commonly encountered example would be vaginal swabs from a positive sexual assault case. 69

Chelex Differential Extraction • As with organic differential extraction methods, this procedure allows for Chelex Differential Extraction • As with organic differential extraction methods, this procedure allows for differential extraction of DNA from stains containing sperm using Chelex resin. • This sperm digest is then mixed with 5% Chelex. After boiling with Chelex resin to remove ions and inhibitors, the two fractions of DNA are then ready to be concentrated and or quantitated. 70

DNA Extraction Kit • Most use either modified glass or magnetic beads which in DNA Extraction Kit • Most use either modified glass or magnetic beads which in the presence of salts which promote binding between DNA and the specialized glass particles. • Some kits are optimized for extraction from specific substrates, such as blood or feces. • Overall, these proprietary kits offer rapid extraction, but may require additional washing in order to isolate samples appropriate for PCR. 71

Yield Gel Extraction Yield Gel • Quantization of DNA with ethidium bromide visualization of Yield Gel Extraction Yield Gel • Quantization of DNA with ethidium bromide visualization of DNA bands in an electrophoretic submarine gel is used to assess the total amount of total DNA present (from any source) and the relative size (condition) of the DNA fragments. This technique helps to determine a ball-park range of DNA quantities, preventing under- or overloading of the slot blot 72

Slot Blot Extraction Slot Blot for Human DNA Quantization • Analysis to determine how Slot Blot Extraction Slot Blot for Human DNA Quantization • Analysis to determine how much human DNA is present. Aliquots of extracted DNA are fixed to a positively charged nylon membrane along with a serial dilution of known human DNA standards. The limits of detection are between 10 and 0. 15 ng. Aliquots of DNA samples having DNA quantities outside of this range cannot be accurately measured. 73

Slot Blot Extraction • Slot Blot Human DNA Gel Plate 74 Slot Blot Extraction • Slot Blot Human DNA Gel Plate 74

Fluorometric or Luminometric Assessment • DNA intercalators in the form of dyes or excitable Fluorometric or Luminometric Assessment • DNA intercalators in the form of dyes or excitable enzymatic systems can be used to label DNA for detection by spectrophotometers, fluoremeters or luminometers. Common research protocols. Forensic DNA analysis tends not to use this type of quantitation, as it is labor intensive and not as reliable as other techniques. 75

Spectroscopic Methods • Once the DNA is isolated and extracted, the amount of recovered Spectroscopic Methods • Once the DNA is isolated and extracted, the amount of recovered DNA can be estimated using spectroscopic methods. • Nucleic acids have UV-Vis absorption characteristics in the 240 -280 nm range due to the chemical nature of the purine and pyrimidine bases. 76

PCR DNA Quantitization • Real Time PCR Method (Taq. Man®)for Amplifiable Human DNA Quantization PCR DNA Quantitization • Real Time PCR Method (Taq. Man®)for Amplifiable Human DNA Quantization The yield gel measures the amount and condition of total DNA, and the slot blot measures the amount of isolated higher primate DNA, the RT PCR system measures the amount of amplifiable human DNA 77

DNA Amplification • Amplification of extracted and quantitated DNA samples is a process where DNA Amplification • Amplification of extracted and quantitated DNA samples is a process where DNA is copied to generate sufficient sample for analysis. Amplification using PCR is a method of chemically copying DNA. • In forensic DNA analysis is multiplex PCR short tandem repeats. 78

DNA Amplification • For animated descriptions of PCR see the following websites: • http: DNA Amplification • For animated descriptions of PCR see the following websites: • http: //vector. cshl. org/Shockwave/pcran whole. html • http: //www. people. virginia. edu/~rjh 9 u/ pcranim. html • http: //allserv. rug. ac. be/~avierstr/principl es/pcr. html 79

The Genetic Amplifier • The underlying premise by which capillary electrophoresis operates is that The Genetic Amplifier • The underlying premise by which capillary electrophoresis operates is that electrical species (negatively charged DNA) suspended in an electrolyte will migrate according to an applied electrical current. DNA fragments should travel away from a negatively charged electrode (cathode) and toward a positively charged electrode (anode). CE is easily automated and reproducible, fast and with high separation efficiency, with a small sample size requirement. 80

Databases • One of goals of CODIS (the FBI’s Combined DNA Index System) is Databases • One of goals of CODIS (the FBI’s Combined DNA Index System) is the creation of a database of states’ offenders’ profiles. This would aid in solving crimes where there are no specific suspects. 81

Databases • The national DNA identification index was authorized by the DNA Identification Act Databases • The national DNA identification index was authorized by the DNA Identification Act of 1994 and has been in operation since October, 1998. • Another important aspect of CODIS is its use as a population statistics database, for research and protocol development and for quality control if personally identifiable information is removed. 82

Databases • CODIS is a computer software program developed by the FBI that maintains Databases • CODIS is a computer software program developed by the FBI that maintains local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and profiles of missing persons. 83

Databases • Likelihood ratios (LR) & match probabilities are widely used statistical measurements used Databases • Likelihood ratios (LR) & match probabilities are widely used statistical measurements used in assessing or explaining the significance of DNA evidence • A powerful means of testing assumptions. • Match probability – is the forensic sample & the subject or suspect sample from the same person & the match probability that they came from different parties. MP is another presentation of forensic calculations, calculated from the frequencies of DNA markers in a database. 84

Databases • As of April 2003, all states & the US Army & FBI Databases • As of April 2003, all states & the US Army & FBI participated in National Data Index System (NDIS) except for MS & RI. In the US, some states hold voluntary samples, often provided for exclusion in investigations, but the NDIS at the FBI consists only of convicted offenders. • CODIS is a coordinated system of local, state and national databases, which enables crime labs to exchange and compare profiles electronically. • http: //www. fbi. gov/hq/lab/codis/index 1. htm 85

Significance • The Evaluation of Forensic DNA Evidence, is available online from the National Significance • The Evaluation of Forensic DNA Evidence, is available online from the National Academies Press at http: //www. nap. edu/catalog/5141. html. Many labs employ software to aid in appropriately crunching the numbers. • PCR-based analyses has facilitated typing of minute amounts of evidence, allowing for more matches to be made between standards and crime scene evidence and in cold hits. 86

Databases • Occasionally or in the event a body is badly decomposed and parental Databases • Occasionally or in the event a body is badly decomposed and parental lineage is unavailable, secondary standards known to belong to the individual in question may be used in casework, such as make-up applicators, toothbrushes, jewelry, and other personal items that might hold traces of the person’s biological material. 87

mt. DNA • Mitochondrial DNA (mt. DNA) is a useful tool in the event mt. DNA • Mitochondrial DNA (mt. DNA) is a useful tool in the event that available samples are degraded and incapable of providing nuclear DNA for traditional forensic DNA analysis. mt. DNA is a circular DNA molecule found in the mitochondria of a cell. While only one copy of nuclear DNA exists in the cell nucleus , a typical cell may have hundreds of mitochondria. 88

mt. DNA • Because they are present in the female gamete at conception, mt. mt. DNA • Because they are present in the female gamete at conception, mt. DNA is maternallylinked. • 99. 99% of mammalian mt. DNA is inherited from the mother. • The other sect of mt. DNA is incorporated as the sperm carries approximately 100 mitochondria on its tail region as opposed to the ~100, 000 found in the oocyte (egg). 89

mt. DNA • The US Armed Forces Institute of Pathology has outlined a DNA mt. DNA • The US Armed Forces Institute of Pathology has outlined a DNA Outreach Program to insure proper identification of fallen servicemen. • Trial courts in some states have yet to demonstrate admissibility of mt. DNA evidence in criminal cases, but with increasing technology and general awareness, it is expected that mt. DNA analysis will rapidly join n. DNA analysis by PCR and RFLP or STR as an accepted tool in forensic identification. 90

mt. DNA • Mitochondrial DNA typing does not approach STR analysis in its discrimination mt. DNA • Mitochondrial DNA typing does not approach STR analysis in its discrimination power and thus is best reserved for samples, such as hair, for which STR analysis may not be possible. • Forensic analysis of mt. DNA is more rigorous, time consuming, and costly when compared to nuclear DNA analysis. 91

mt. DNA • Also, all individuals of the same maternal lineage will be indistinguishable mt. DNA • Also, all individuals of the same maternal lineage will be indistinguishable by mt. DNA analysis. • Two regions of mt. DNA have been found to be highly variable and a procedure known as sequencing is used to determine the order of base pairs. 92

mt. DNA Tomb of the Unknown Soldier, Arlington, VA President Reagan presided over the mt. DNA Tomb of the Unknown Soldier, Arlington, VA President Reagan presided over the funeral, and presented the Medal of Honor to the Vietnam Unknown. The President also acted as next of kin by accepting the interment flag at the end of the ceremony. The remains of the Vietnam Unknown were exhumed May 14, 1998. Based on mt. DNA testing, Department of Defense scientists identified the remains as those of AF 1 Lt. Michael Joseph Blassie, who was shot down near An Loc, VN, in 1972. The identification was announced on June 30, 1998 and on July 10, his remains arrived home to his family in St. Louis, Missouri; he was reinterred at Jefferson Barracks National Cemetery. The crypt, which once held the remains of the Vietnam Unknown, has been replaced. The original inscription of "Vietnam" and the dates of the conflict has been changed to 93 "Honoring and Keeping Faith with America's Missing Servicemen.