Forensic Genetics Module 6 PDF

27/10/2024, 17:15 Module 6: Page 1: Module Overview: PHA6855-Forensic Genetics Module 6: Page 1: Module Overview ufl.instructure.com/courses/514169/pages/module-6-page-1-module-overview Module 6: DNA Profiling & Forensic...

27/10/2024, 17:15 Module 6: Page 1: Module Overview: PHA6855-Forensic Genetics Module 6: Page 1: Module Overview ufl.instructure.com/courses/514169/pages/module-6-page-1-module-overview Module 6: DNA Profiling & Forensic Investigation Page 1 Module Overview Introduction Now we will look to the use of DNA analysis as a tool in forensic practice...more on this is a moment. This course, as mentioned at the start of the term, is designed as an introduction to genetics and intended to be user friendly for non-geneticists, yet still be useful to those who eat, sleep, drink, and socialize genes. Within the wider program of University of Florida courses there are courses specifically detailing Forensic Analysis of DNA, so this course is designed to address a headline of necessity, while providing a grounding to the forensic aspects of DNA profiling. The Discussion Board for this course remains a great way of tailoring the content to fit the class. From module 1 you will have hopefully explored the web and decided and agreed with your classmates on a couple of good websites to support your studies. These keep changing but if you have no websites to consult let us know and we will try to help out! It may also be worth revisiting topics such as genomic mapping, recombination, and population genetics as they are an essential backdrop that we will not repeat here. Within the field of forensic DNA analysis, the primary focus for the collection of evidence might be summarized by some as convicting the guilty and freeing the innocent. Criminals ultimately leave traces behind: biological evidence, fibers, modus operandi in repeated crimes, financial records, cell phone traces, and so on. The task of the forensic scientist is to collect and evaluate evidence to determine its usefulness in a robust judicial system. Of course, there may be other reasons than crime to use such evidence, including identification of individuals after a mass disaster or in paternity testing. DNA is one way of https://ufl.instructure.com/courses/514169/pages/module-6-page-1-module-overview 1/2 27/10/2024, 17:15 Module 6: Page 1: Module Overview: PHA6855-Forensic Genetics attempting to stamp individuality on the scene - "this individual was here because they left a trace"; "this person must be the father". There may be other reason as well, for example, "these remains are not human". Before DNA, and now alongside it, there was blood group analysis, fingerprint comparison, and other forms of "profiling" of an individual such as graphology and psychological profiling. Where DNA scores significantly is its complete reliance on statistics from the very outset of the study of genetics. So DNA does not claim to give absolute answers, but answers with a degree of confidence and reliability unlike any other forensic discipline. Objectives By the completion of this module the student should: Appreciate that an individual's DNA is believed to be unique Realize what identical twins means at a DNA level! Be able to review the history of DNA typing Understand the uses of DNA testing Understand the pitfalls of DNA testing Have a general overview of the technology involved https://ufl.instructure.com/courses/514169/pages/module-6-page-1-module-overview 2/2 27/10/2024, 17:17 Module 6: Page 2: The Concept of Individuality: PHA6855-Forensic Genetics Module 6: Page 2: The Concept of Individuality ufl.instructure.com/courses/514169/pages/module-6-page-2-the-concept-of-individuality Module 6: DNA Profiling & Forensic Investigation Page 2 The Concept of Individuality The Concept of Individuality To some this may be an obvious place to begin, but the concept of individuality is a surprisingly Western (and modern) one at a philosophical level. Think back in history, and in some places today, how important family vengeance was, and how guilt passed from one generation to the next. The importance of individuality is thus a product, not just of biology, but also of culture. But having said that, we acknowledge that we are individuals; each of us is unique. We know that identical twins are genetically identical, but we would also concede that they are two individual people. to this, identity and individuality may be considered slightly different concepts. We are all different. In the past, fingerprint evidence and other less reputable means of determining differences allowed crime investigation to focus on a single individual and confirm their proximity to a crime scene. The discovery of DNA structure, and more importantly, the development of readily usable technology, has revolutionized identity matching. Perhaps at some point DNA will not be enough or will require additional investigation. This is because, as we have alluded to in previous modules, DNA is inherited from father and mother and during the life of an individual our somatic DNA is subject to recombination and mutation. In the case of the genes, coding for immunoglobulins in B lymphocytes and T cell receptors of T lymphocytes, recombination results in hyper-variability, allowing differences in DNA sequence even between otherwise identical twins. Whether this property is useful, how widespread, and if we would wish to use it is another matter. https://ufl.instructure.com/courses/514169/pages/module-6-page-2-the-concept-of-individuality 1/2 27/10/2024, 17:17 Module 6: Page 2: The Concept of Individuality: PHA6855-Forensic Genetics We have 23 pairs of chromosomes. Between individuals we have about 99.5% similarity; that is, we are only about 0.5% different. That may not seem a lot, but at approximately 2% different and you are a chimpanzee Links to an external site.. So in forensic practice, we put a huge emphasis on this tiny proportion where we differ. Of our full complement of DNA, only about 4% actually codes for anything and the greatest variability is often in non-coding regions. For many years, these regions were referred to as "junk DNA", but are increasingly termed "filler DNA", or simply, "non-coding DNA" in forensic circles. In contrast to traditional genetic mapping, forensic DNA testing is not about gene transcription, but is much more centered around variable, non-coding areas of repetitive sequences that represent about a quarter of the genome. Fine, so if individuality can be ascertained from exhaustive searching of DNA; what use are fingerprints then? Maybe that is a question best addressed on the discussion board but for starters: In fingerprinting, we are brought up to believe everyone is unique, no two fingerprints are identical (including identical twins). Just like snowflakes. The problem is that fingerprint matching preceded modern day statistical analysis and has arguably never been robustly validated. A small number of points are compared. If they match, then proof of identity is presumed - but with what statistical certainty? This has been addressed in the controversial 2016 United States PCAST report Links to an external site.. This report is required reading for nearly every forensic science practitioner in the U.S and was especially critical of those "comparison" fields of forensic science. A major issue...No statistics - so instead we rely on expert opinion regarding the evidence. How do we know it is believable? How can its scientific validity be tested? But we are not here to dismiss other techniques, but instead to flag the concern that they appear to have less statistical validity than DNA testing and that they seem to be regarded as a means of proving identity, rather than the more scientific approach of disproof. But as in any evidence scenario, several sources of valid data are more valuable than a single source, no matter how scientific we might wish it to be. What is uniqueness? It would appear that maybe we cannot prove it, but we can have probabilities so infinitesimal that to all intents and purposes we have established uniqueness. This is the practical approach adopted by agencies such as the FBI and others, although the debate continues. There are a couple of caveats though. Calculations depend on (i) the type of DNA assay (ii) the number of different tests (iii) the degree of relatedness (ask yourself what the effect would be if humans had multiple monozygotic pregnancies!). Finally, and we will return to this, finding DNA at a scene is not confirmation of guilt. Source attribution Links to an external site. may lead us to accept an individual as the originator of the sample, but it does not prove complicity in a crime. https://ufl.instructure.com/courses/514169/pages/module-6-page-2-the-concept-of-individuality 2/2 27/10/2024, 17:17 Module 6: Page 3: A Historical Perspective: PHA6855-Forensic Genetics Module 6: Page 3: A Historical Perspective ufl.instructure.com/courses/514169/pages/module-6-page-3-a-historical-perspective Module 6: DNA Profiling & Forensic Investigation Page 3 A Historical Perspective A Historical Perspective It was in the 1940s that DNA was implicated as the carrier of genetic uniqueness, and 1953 when Watson and Crick figured that DNA was packaged as a double helix. We see approximately ten years or so between these two cardinal discoveries. In the late 1970s it was realized that mapping the genome could be aided by utilizing enzymes which cut DNA at restricted sequence specific sites. As individuals differ in where these enzymes cut, thus giving different fragment sizes, the restriction fragment length polymorphism (RFLP) was born! In 1984 Alec Jeffreys in Leicester, England used scaled up RFLP technology to profile individuals' DNA. Three years later, in 1987, Colin Pitchfork entered the history books with the dubious distinction of being betrayed by his own DNA. This is the first criminal case reported to have used DNA evidence to assist in a conviction. Just over a year later, polymerase chain reaction (PCR) Links to an external site., discovered by American biochemist Kary Mullis, finally emerged and made DNA technology available to any laboratory and allowed massive scaling up of effort and output. Genome mapping and sequencing followed in hot pursuit. By the late 1990s cloning became a reality and Dolly the sheep Links to an external site. was born. During this time the forensic community had moved from the classical Jeffreys' approach, looking https://ufl.instructure.com/courses/514169/pages/module-6-page-3-a-historical-perspective 1/3 27/10/2024, 17:17 Module 6: Page 3: A Historical Perspective: PHA6855-Forensic Genetics at multiple loci with large amounts of DNA being required and Southern blot analysis, to single locus testing and the use of PCR to study tiny amounts of source DNA, that may even be quite seriously degraded. Further refinement came with exploitation of the stability of mitochondrial DNA in historical or decomposed tissues to identify maternal identity. Mitochondrial DNA is almost entirely inherited as a single haplotype from the mother as it is present in the oocyte. The study of two hypervariable regions allows lineage tracing and identification of remains between maternally unrelated sources. By analogy, the study of variable regions of the Y chromosome facilitated studies of paternal lineage. An emerging profiling technique in forensic DNA laboratories is the use of Next Generation Sequencing (NGS). NGS was originally referred to as massively-parallel sequencing, because it allowed for decoding of multiple DNA strands simultaneously in contrast to the single strand Sanger style sequencing Links to an external site. performed by capillary electrophoresis (CE). Moreover, the addition of targeted Single Nucleotide Polymorphism (SNP) arrays has spawned an entirely new avenue for forensic identification in the field of Investigative Genetic Genealogy (IGG) Links to an external site.. Criminal investigations that have sat "cold" for years, have now been solved in recent years using this new ancestral-based technology. One example is that of Seattle Washington's Green River killer Links to an external site.. And finally, before we move on, not just humans can be DNA profiled. Potatoes, elephants, strawberries, whatever can all be profiled as an aid in breeding programs, conservation projects, and even for use in forensic practice. Should we test everyone? If everyone from a population was DNA typed, any sample collected should, in theory, be attributable. Any crime scene, for example a rape, where DNA evidence is collected should lead to rapid identification and apprehension of the guilty party. True or false? Again the argument hinges on the distinction between excluding or including an individual. What should we collect? DNA and retain the sample indefinitely, or the result of the DNA profiling? If we have DNA from everyone, can we then use it to look for gene-disease associations? Are an individuals' rights to privacy protected if their ancestry SNP profile in a database is used to identify a familial relative using IGG? How available should the information be to law enforcers, courts, insurance companies, or government? What happens if I discover inadvertently that I am not my child's genetic parent? What are the long-term implications of knowing? https://ufl.instructure.com/courses/514169/pages/module-6-page-3-a-historical-perspective 2/3 27/10/2024, 17:17 Module 6: Page 3: A Historical Perspective: PHA6855-Forensic Genetics You may already have raised and discussed these issues in relation to Assignment 6, if not, this should be more food for thought and discussion now. https://ufl.instructure.com/courses/514169/pages/module-6-page-3-a-historical-perspective 3/3 27/10/2024, 17:17 Module 6: Page 4: Some Technical and Other Pitfalls: PHA6855-Forensic Genetics Module 6: Page 4: Some Technical and Other Pitfalls ufl.instructure.com/courses/514169/pages/module-6-page-4-some-technical-and-other-pitfalls Module 6: DNA Profiling & Forensic Investigation Page 4 Some Technical and Other Pitfalls Some Technical and Other Pitfalls Before we briefly visit the methodology of DNA testing, there are some vital points to consider. The optimal place to learn the practicalities of DNA testing is in an operational laboratory or in an academic laboratory specializing in teaching quality assurance and control. The best place is not by distance learning in the comfort of your own home or workplace! However, the great advantage of distance education is the opportunity to reflect, discuss, and think the unthinkable. Many of the potential pitfalls are of course well known to you. We will mention some in no particular order and encourage you to extend the list and discussion on the course Discussion Board. Contamination As in every example where you are seeking to gather evidence, the sampling procedures used and the degree of technical skill, verified by good quality assurance protocols, must be of the highest standards. The potential for contamination occurs at nearly every stage of laboratory analysis: sampling of the evidence, extraction of DNA from the substrate, quantification, amplification, and setup of the sample for an NGS or CE run. This is why standard operating procedures (SOPs), good laboratory practices (GLP), and audit and quality assurance schemes are all essential for a modern scientific laboratory. A further important point to emphasize, is that collection of evidence in the field, if improperly performed, ruins everything that follows, no matter how good the science is. Some use the term, "garbage in-garbage out", other variations exist... Protocols must start at the https://ufl.instructure.com/courses/514169/pages/module-6-page-4-some-technical-and-other-pitfalls 1/3 27/10/2024, 17:17 Module 6: Page 4: Some Technical and Other Pitfalls: PHA6855-Forensic Genetics scene of a crime and evidence gathering. Straying from this goal may have the collateral effect of discrediting the laboratory and could potentially limit the use of the evidence in a court of law. Degradation Warm, humid conditions, bacterial contamination from soil, gastric contents, exposure to the air, decomposition, fire, and chemicals may all cause DNA to degrade. For the classical approach of RFLP analysis, large amounts of DNA in good condition are required. Any of the factors above may degrade the DNA to small, unusable fragments. PCR can deal more readily with small fragments, and mitochondrial DNA may survive longer in a useful form. Tooth pulp may be a useful repository for DNA if all else fails. After collection of a sample, it is important to stop further degradation as quickly as possible. Degraded DNA often results in partial resolution of a DNA result or even a failed test. Particularly in PCR assays this is worth remembering. If a PCR assay gives a positive result or a negative one there is a danger of a failed assay being mistaken for a negative result. Validation of DNA quality is always a useful check as well as optimal design of PCR strategies. The majority of chemistries used to quantify and amplify DNA in forensic laboratories have incorporated measures to assess situations of possible DNA degradation and inhibition due to external agents. This allows the scientist to take preventative measures to combat these issues in order to obtain the best outcomes. Mixtures In a sexual assault, the majority of DNA collected may be from the victim, especially if there is bleeding. If the assailant was reported to have worn a condom, has had a vasectomy, or is otherwise azospermic (too few sperm), there may be very little of the assailant's DNA to deposited. Approaches developed to analyze such samples must take into consideration that the assailant's DNA may be a minority of the sample and care must be taken to maximize its usefulness. Find out what techniques can be employed to try to overcome such difficulties and share your findings with your colleagues on the Discussion Board for this module. You may also wish to speculate on the Discussion Board whether may be other concerns inherent with analysis of very small amounts of DNA from sperm cells, which originate as a result of meiotic division and are, therefore, haploid. Approaches have been developed to try to deduce which alleles in a mixed sample come from the same source on the basis of the strength of signal the different alleles produce when they are detected. The theory being that if you have four alleles at a locus, two of which give a very strong signal of equivalent strength and two of which give a much weaker signal of equivalent strength the two stronger alleles came from one individual and the two weaker alleles from another individual. This may seem plausible if we surmise that the DNA from one of the contributors was in excess of the other in the sample analyzed, but what other alternatives might have given rise to this result? https://ufl.instructure.com/courses/514169/pages/module-6-page-4-some-technical-and-other-pitfalls 2/3 27/10/2024, 17:17 Module 6: Page 4: Some Technical and Other Pitfalls: PHA6855-Forensic Genetics Consider the scenario when we have 5 peaks - how many different individuals might have contributed to the sample? Think about all the scenarios where we have individuals who are heterozygous for alleles, homozygous for alleles, and the situation where individuals share alleles in common. Remember we may not always have a distinction in the peak height that might provide a clue to the allele allocation. Mixture analysis is complex and should not be considered definitive. Often, the mixture in its entirety must be considered when formulating determinations on the number of contributors (NOC) along with the presence of major, minor, and contributions foreign to those reasonably assumed to be present. Over the past decade, technological advancements using statistical algorithms have been employed to assist in these aspects of mixture deconvolution. These will be discussed later in this course. https://ufl.instructure.com/courses/514169/pages/module-6-page-4-some-technical-and-other-pitfalls 3/3 27/10/2024, 17:18 Module 6: Page 5: Some Technical and Other Pitfalls - Continued: PHA6855-Forensic Genetics Module 6: Page 5: Some Technical and Other Pitfalls - Continued ufl.instructure.com/courses/514169/pages/module-6-page-5-some-technical-and-other-pitfalls-continued Module 6: DNA Profiling & Forensic Investigation Page 5 Some Technical and Other Pitfalls - Continued Some Technical and Other Pitfalls - Continued "Innocent" presence of DNA Let's say I visit your home (which you will be relieved to learn is not very likely), you may offer me some hospitality. I may use your bathroom. I may shed some hairs and/or touch some items while washing. Now suppose your residence is burglarized and your home transforms to a crime scene. Samples are obtained of hairs, swabs of "touched" surfaces, and so on. The DNA analysis is performed on the items collected. If my DNA is present does that make me a suspect? Well, yes maybe - but hopefully I have a cast iron alibi. It wasn't me - honest! My DNA may easily be at a scene for perfectly explainable reasons. DNA is fairly stable given reasonable environmental conditions and so its presence in space cannot be linked to its presence in time - that is the time of a crime. The ability to detect increasingly smaller traces of DNA by techniques such as LCN (Low Copy Number) DNA analysis should ring alarm bells with the competent scientist. Beware the temptation to attach greater significance to the fact that a profile might be detected – in actual fact it wasn't me there at all; perhaps someone else had borrowed my coat. The presence of my DNA does not even confirm my presence at the scene. It is simply an observation to be reconciled to every other piece of data. By contrast if my fingerprints were found at the scene at least we can be sure I was physically there, if you believe the fingerprint analysis that is... https://ufl.instructure.com/courses/514169/pages/module-6-page-5-some-technical-and-other-pitfalls-continued 1/3 27/10/2024, 17:18 Module 6: Page 5: Some Technical and Other Pitfalls - Continued: PHA6855-Forensic Genetics Quality assurance Although it is not typically talked about when referring to forensic laboratories in regular conversation, the importance of quality assurance and control cannot be understated. Suffice is to say here that going through the motions is not enough. There must be a translation of procedure into verifiable practice. There is sometimes an interesting debate within my laboratory staff (not in the clinical laboratories but the research laboratories) how the relative quality and skill of their molecular genetic work compares to a "jobbing" forensic profiling or sequencing laboratory. The answer is, of course, that in general the "non-academic" laboratory has much more stringent standards and procedures. The forensic laboratory cannot afford a false positive later corrected, or a false negative, and there is no scope for asking for a better sample because the freezer broke down. A solid quality assurance program is essential to any modern forensic laboratory and will surely have individuals (Quality Managers/Officers) whose entire focus is maintaining the quality system. Prosecutor's fallacy It's not within the scope of this module to cover the area of DNA interpretation and statistics in depth, but we have already stressed how important statistics are in presentation of DNA evidence. Remember that we have moved away from the term DNA "fingerprinting" to "profiling" or "typing" because the former has connotations of exactness that we (incorrectly) associate with conventional fingerprinting. The level of proof we require will vary. Identification of lineage in an anthropological survey is less stringent than the identification of an alleged rapist. In practice, a number of assays are performed that lead to a degree of unlikeliness that anyone other than the suspect is the source of the DNA. But it is only a degree of probability, a confidence limit. If I develop a DNA profile from an evidentiary item and calculate that the probability of a DNA match with someone other than the suspect is 1 in 1 million, what am I saying? Let's suppose there are not reasons locally, such as inbreeding and an identical twin, to complicate matters, we can say confidently that we cannot exclude the suspect with a probability of being right 999,999 out of 1,000,000 times. Of course, a further set of polymorphic markers may just prove that one time in a million and we can go on and exclude involvement. It is apparent that the fewer tests we use (the fewer polymorphic markers we look at) the more likely we are to fail to exclude an innocent party as a possible source of the DNA. The prosecutor's fallacy is to transpose the unlikeliness of being able to exclude guilt (1 in 1 million) as being the same as the probability of guilt and there is, therefore, 999,999 chances in 1,000,000 that the suspect is guilty as charged! We had better mention the defender's fallacy as well, if there is a one in a million chance of this not being the suspect's DNA then in a country with a population of 300 million, 299 other people will match this DNA profile. Therefore, the accused has a 299 in 300 chance https://ufl.instructure.com/courses/514169/pages/module-6-page-5-some-technical-and-other-pitfalls-continued 2/3 27/10/2024, 17:18 Module 6: Page 5: Some Technical and Other Pitfalls - Continued: PHA6855-Forensic Genetics of being innocent! Confused? It sure can be. This example reveals the importance of the scientist presenting their results in an unbiased and appropriate manner in legal proceedings. https://ufl.instructure.com/courses/514169/pages/module-6-page-5-some-technical-and-other-pitfalls-continued 3/3 27/10/2024, 17:18 Module 6: Page 6: DNA Extraction and Polymerase Chain reaction (PCR): PHA6855-Forensic Genetics Module 6: Page 6: DNA Extraction and Polymerase Chain reaction (PCR) ufl.instructure.com/courses/514169/pages/module-6-page-6-dna-extraction-and-polymerase-chain- reaction-pcr Module 6: DNA Profiling & Forensic Investigation Page 6 DNA Extraction and Polymerase Chain reaction (PCR) DNA Extraction and Polymerase Chain reaction (PCR) High-grade DNA material is not usually available in forensic investigations and so extraction is a critical phase of the DNA analysis procedure to ensure there is maximal return of quality and quantity of the DNA extract. In addition, the end product must be free from contaminant chemical impurities that inhibit PCR reactions. Details (procedures manuals) for extraction methodologies are readily available online from commercial manufacturers of biochemical and molecular biology products, public forensic labs, and other sources. https://ufl.instructure.com/courses/514169/pages/module-6-page-6-dna-extraction-and-polymerase-chain-reaction-pcr 1/3 27/10/2024, 17:18 Module 6: Page 6: DNA Extraction and Polymerase Chain reaction (PCR): PHA6855-Forensic Genetics Polymerase chain reaction (PCR) is an invaluable technique, because it can be used (i) on small samples (ii) small fragments, for example those obtained from degraded DNA (iii) much of the process can be automated (iv) it is rapid (v) it does not require use of radioactive probes. The key is to design oligonuleotide primers, which we commonly refer to as primers, that correspond to genomic nuclear or mitochondrial DNA. Under appropriate conditions, these anneal to their complementary sequence in the sample DNA, which has taken up the single stranded form. The addition of a polymerase enzyme produces a new strand of DNA at the site of interest: think back to the process of DNA replication and you will see the similarities. The fascinating bit to PCR is that the enzyme is thermostable. The heat-resistant polymerase commonly used for PCR is derived from the bacterium Thermus Aquaticus (known as Taq Polymerase) which are found in hot springs; originally discovered in Yellowstone Nation Park in the Pacific Northwest of the United States. Using Taq Polymerase, we can heat the sample to separate the new strands from the original template in order to get the single stranded form again and on cooling, the strands recombine, but because there is still lots of primer, these will anneal to their complementary sequence and we can again generate new strands as the enzyme has not been denatured by heat. Cycle after cycle of this process results in an exponential increase in the target sequence quite specifically. Hence, the name polymerase chain reaction, or PCR. Restriction Fragment Length Polymorphism (RFLP) It was the Restriction Length Fragment Polymorphism (RFLP) technique that was the original method of DNA analysis applied to forensic casework back in the 1980s and it was this method that was dubbed "DNA fingerprinting". With RFLP analysis more DNA is required than for the techniques currently employed and the DNA must have a larger fragment size. DNA aliquots are digested with restriction enzymes that cut only at specific known sites in the DNA strand. When DNA from different individuals is digested, polymorphisms result in different sizes after cutting with the restriction enzyme. These can be separated by electrophoresis on a gel according to size. The gel is made of polyacrylamide which acts like a sieve by virtue of pores which form in the gel as it "sets" or crosslinks. When a potential difference is applied across the gel the negatively charged DNA molecules migrate through the gel from the cathode (negative electrode) to the https://ufl.instructure.com/courses/514169/pages/module-6-page-6-dna-extraction-and-polymerase-chain-reaction-pcr 2/3 27/10/2024, 17:18 Module 6: Page 6: DNA Extraction and Polymerase Chain reaction (PCR): PHA6855-Forensic Genetics anode (positive electrode). The smaller the DNA fragment the easier it is for it to "wiggle" its way through the gel. Larger fragments are retained in the gel for longer and, hence, the fragments will travel through the gel at a rate that is inversely proportional to their length. After separation the entire genomic DNA, comprising different sized fragments, is present on the gel so the whole thing is an unsightly smear of DNA! What we need to do is be able to identify and highlight the variable regions we are interested in and then visualize them to record the presence of bands which will be unique to the individual if sufficient different sites are studied. Visualization again relies on the property of DNA when denatured to lose its double strand conformation on heating but regain it on cooling. Instead of interposing a primer on cooling the trick for RFLP analysis is to have a short probe made from the target sequence, labeled with radioactivity or some other marker. This binds to the denatured single stranded DNA fragments and can be detected by detecting the radioactivity or fluorescence. Because the probe binds the digested genomic DNA wherever it migrated in the gel the size of visualized fragments can be measured and different samples compared. https://ufl.instructure.com/courses/514169/pages/module-6-page-6-dna-extraction-and-polymerase-chain-reaction-pcr 3/3 27/10/2024, 17:19 Module 6: Page 7: So What Strategy Should We Use?: PHA6855-Forensic Genetics Module 6: Page 7: So What Strategy Should We Use? ufl.instructure.com/courses/514169/pages/module-6-page-7-so-what-strategy-should-we-use Module 6: DNA Profiling & Forensic Investigation Page 7 So What Strategy Should We Use? So What Strategy Should We Use? Up to 15 sites of extremely variable DNA can be studied by RFLP analysis. Some of the limitations of classic RFLP analysis were analysis time, DNA quantity required, and quality of the sample, but the results can be extremely accurate and thorough, allowing really impressive statistical significance for the time. The original work of Jeffreys relied upon a probe that bound to a number of different loci. Newer applications have tended towards single locus probes that increase the stringency of hybridization reactions. Use of four single locus probes can give very good DNA profiles that are technically repeatable, but of course the downside is that the power of discrimination (that is the ability to exclude samples as being the same, or to confirm uniqueness) is reduced. STR Profiling The use of short tandem repeats (STR) as a target exploits using markers which are highly variable. STRs are preferred to larger polymorphisms (variable number tandem repeats VNTRs) because of the attractiveness of being able to automate STR analysis and use PCR on degraded DNA. STRs are based on loci with tetranucleotide repeats with nigh on 90 different alleles at some of the loci tested, for example the D21S11 locus1. The different alleles differ in the number of times the particular tetranucleotide being analyzed is repeated at the locus, which may vary from a few to dozens of times. Because of the small size of the STR loci and extreme variability PCR analysis is an ideal approach and indeed several different loci may be studied in the same PCR reaction. This is referred to as multiplexing. If fluorescent labeled nucleotides are used, the results can be read by a Genetic analyzer, allowing greater throughput and standardization. Traditional DNA sequencers utilized vertical polyacrylamide slab gels to separate the PCR products on the basis of their size, as described previously. However, since their introduction in the mid-90s, forensic laboratories have the shift to separate the fragments by capillary electrophoresis (CE). Rather than having a large slab gel of polyacrylamide, capillary electrophoresis uses fine capillaries of 50-100μm diameter which are packed with a viscous polymer through which the DNA fragments migrate. The polymer serves the same purpose as the polyacrylamide "sieve", but the capillary electrophoresis system https://ufl.instructure.com/courses/514169/pages/module-6-page-7-so-what-strategy-should-we-use 1/3 27/10/2024, 17:19 Module 6: Page 7: So What Strategy Should We Use?: PHA6855-Forensic Genetics has the advantage in being able to separate a sample in minutes rather than hours, because higher voltages can be used in the separation. Minute amounts of sample are needed for the analysis, which makes it preferable when samples are in short supply and increases the possibility for reanalyzing samples if need be. The system can be fully automated (injecting the sample, separating the fragments and fragment detection) and there is no risk of cross-contamination between lanes of the gel or a need to track lanes for that matter. The first CE instruments introduced had a single capillary and would process one sample at a time. Although more time consuming than modern-day CEs, the ease of detection was an acceptable trade-off from that of the slab-style instrumentation. Applied Biosystems 3500 Genetic Analyzer -image credit to Thermofisher.com However, capillary arrays have now been developed such as the Applied Biosystems 3500Genetic Analyzer, 8 capillary system shown which has 8 individual capillary tubes combine in an array. If four or five STR loci are examined this gives a high level of discrimination between samples and individuals, but the number of loci studied can be increased depending on background variability (for example, related to ethnicity) and the importance of exclusion in the case being examined. The more loci that are used to generate a DNA profile the less likely it is to get an adventitious match. In the United States, the Federal Bureau of Investigation (FBI) CODIS database formerly required typing 13 STR loci which were referred to as "the core 13"; however, in 2017 moved to requiring an expanded 20 loci for DNA profiles entered into the US national database, which is known as NDIS (National DNA Index System). The software for management of the database and profile comparisons is called CODIS (COmbined DNA Index System) and is used for NDIS as well as databases at U.S. state (SDIS) and local (LDIS) crime lab levels (and also in some select non-U.S. countries). Commercially https://ufl.instructure.com/courses/514169/pages/module-6-page-7-so-what-strategy-should-we-use 2/3 27/10/2024, 17:19 Module 6: Page 7: So What Strategy Should We Use?: PHA6855-Forensic Genetics available amplification "kits" are used that include the required 20 loci. The UK National DNA Databasestores profiles produced from commercially available kits encompassing 16 loci, Scotland uses 21 loci. The likelihood of obtaining a match from two unrelated individuals by chance when comparing two full profiles is presented in Court as being in the neighborhood of 1 in 1 billion or greater. The electropherogram below depicts appearance of a modern STR profile. Positive Control 9948 - Qiagen Investigator 24plex kit, Analyzed using Life Technologies Genemapper ID-X software https://ufl.instructure.com/courses/514169/pages/module-6-page-7-so-what-strategy-should-we-use 3/3