DNA profile from sexual and violent criminals automatically entered into database. DNA samples from crime scenes collected and entered into database. Match! Offender Index Forensic Index Victim / Perpetuator / Accomplice Identified DNA FORENSICS Andy Cheng, Mike Garofalo, Jon Keung, Bo Li, Jenny Nguyen, Jeff Wharton Introduction: DNA forensics? An increase in the research of STR has led to the discovery of a number of STR on the Y-chromosome. These “Y-STR” are important because they can help track lineage better than autosomal STR due to the fact that the Y-chromosome is solely paternally inherited. While both RFLP and STR are very common and very effective methods of DNA forensics, RFLP is more widespread and preferable to STR. This is due to the fact that RFLP is more sensitive to mutations and can therefore give more conclusive results. A difference of one or two repeats is very difficult to see on a gel, while one mutation on a genome at a recognition site for an enzyme will yield a very different band on the gel. DNA Forensics a form of forensics that harness the minute differences that exist between individual’s genetic material in order to distinguish, identify, and match subjects. The differences (polymorphisms) between the genetic materials can be examined and used in objective ways in fields such as archeology, crime forensics, identification of species, and even agricultural monitoring. The field was greatly advanced by the advent of DNA-based identification techniques. There exist several powerful techniques, each relies on a specific characteristic of DNA sequence, but usually involve laboratory examination of the sequence or length of unique regions of DNA. The techniques that are commonly used are Restriction Length Polymorphisms (RFLPs), Short Tandem Repeats (STRs), and analysis of mitochondrial DNA and the Y-chromosome. Each of the above approach carries its own set of advantages and limitations. DNA based forensics relies on differences between DNA samples. These differences can be inherited or obtained from random mutation events, but this only occurs rarely. Polymorphisms without a selectional pressure occurs at a fixed rate. Forensics utilizes these frequencies to calculate the probability of an individual having a certain set of polymorphisms. As DNA-based forensic techniques improve and become more efficient, the costs and implications of using these techniques must be considered. Cost and Efficiency Table 1. Restriction enzyme and the core recognition sequene associated. Any change in the core sequen will result in large decrease in efficiency of enzyme. Cost: - Cost of processing of DNA is approximately $50.00 per sample ~ $2.5 million/year/state (Michigan State’s House Fiscal Agency). - Private samples can be sent in and processed at company called Genex Diagnostics™ for approximately $180.00 per sample - RFLP is the most common – techniques have become much cheaper lately – hybridization solutions composed of cost-effective reagents (7% SDS, 10% PEG, and phosphate buffer) Efficiency: - Efficiency of DNA forensics analysis continuously increasing and is now much easier. - 90% of analys is done by computers with little room for human error - With longer autoradiographic exposures, as little as 20-100 ng of genomic DNA is sufficient for analysis Reliability: -Very reliable – DNA comparisons nearly foolproof -Reliability increases with more probes used and matched between samples -RFLP is more reliable because tests can be much more variable Figure 1. Electrophoresis gel of RFLPs analysis. The different bands are the different lengthed fragment generated after digestion with restriction enzyme. Figure 2. Outline of RFLPs procedure. DNA is first extracted and digested by a set of restriction enzymes. Fragments are separated by electrophoresis and Southern blott performed as methods of detection. Results can be compared to other samples that have also undergone the same reactions, with the same mixture of restriction enzymes. Detection can also be done via electrophoresis (not shown). A large amount of sample needed. DNA Forensic Techniques Restriction Length Polymorphisms (RFLPs). RFLPs are differences in the length of DNA fragments after digestion with restriction endonucleases. These differences in length are caused by variations, or polymorphisms in the DNA sequences in the human genome. While most of the human genome is identical from individual to individual, about 3 million single nucleotide polymorphisms (SNPs) exist, the genome itself being approximately 3 billion base pairs. Restriction enzymes cleave double stranded DNA by hydrolyzing the backbone between the deoxyribose sugar and the phosphate at the 5’ phosphate and the 3’-OH. These enzymes bind, loosely at first, to the DNA and move along it until it comes in contact with the recognition sequence. The tighter binding with the recognition sequence then leads to a conformational change that allows the enzyme to cleave the DNA. Since there are a number of polymorphisms in the genome it happens that some of these variations would occur at the recognition sequences for restriction enzymes. If an enzyme would normal recognize a sequence such as GAATTC (for EcoR1), it would inhibit the enzyme’s ability to cleave the DNA, or conversely a non-cleavage sequence could be changed to a recognition sequence, causing the enzyme to cut the DNA. These different cleavage sites lead to differences in the length of the DNA segments after digestion. (An example of a RFLPs gel is seen in Figure 1). When the digested DNA is run with gel electrophoresis, there can be a detectible difference in the bands from two individuals. This information is useful in criminal investigations as well as paternity tests. Limitations of RFLP A large sample size is required to run a RFLP gel. The technique detects the length of cut DNA. DNA that is heavily damaged cannot be used. If only a small sample size is available, RFLP may still be usable if coupled with PCR. PCR can amplify a small sample into a larger one. However, contamination of sample may cause amplification other than the desired DNA. CODIS Short Tandem Repeat Analysis (STRs) Another method of DNA identification makes use of DNA repeats. There are two classes of repeats Variable Number Tandem Repeats (VNTRs) with a core sequence of 20-9 bps and Short tandem repeats (STRs) with a core sequence of 2-5 bps. STRs are short segments of DNA that are repeated a number of times in a sequence. Across the population, different individuals have different numbers of these repeats. The lengths of DNA of STR loci are usually smaller than RFLP fragments and hence are better for using on damaged DNA. When a DNA polymerase encounters a repeated sequence it has a tendency to stutter or slip a little, leading to increases and decreases in the number of repeats, which is a source of polymorphisms. By determining the sequences that flank the STR, and amplifying them via PCR (polymerase chain reaction), the differences in length can be detected via gel electrophoresis. An individual with an increased number of repeats will have a longer segment of DNA, and therefore a higher band on the gel. In addition to running size exclusion gel electrophoresis, the STR can be identified with sequencing. While the price of DNA sequencing has gone down dramatically, it is still more expensive than running a gel, and it also takes longer. The Federal Bureau of Investigation (FBI) uses a system that combines computerized matching algorithms and forensic science to solve violent crimes. The system is called the COmbined DNA Index System or CODIS. Since it’s inception as a small pilot program in 1990 the CODIS system has grown to include over 3 million samples and is now relied upon to solve crimes nationwide. It has become an invaluable tool, assisting in more than 33 thousand investigations. CODIS is divided into three tiers customized to the local, state, and national level. The DNA Identification Act of 1994 established CODIS as the national DNA database for logging the DNA collected from violent crimes. All states are required by law to participate in the program. The CODIS system contains two main databases referred to as the Forensic and Offender Index. The Offender Index contains DNA samples from individuals convicted of sexual and violent crimes. The Forensic Index contains DNA samples from the crime scenes of unsolved crimes. Matches between the Forensic and Offender Index can give officers leads, identify the perpetuator, and make a stronger case against serial offenders. Figure 3. Sequencing of STRs. Since STRs are small in size, sequencing can be applied to STR analysis as well as separation by electrophoresis. Picture shows sequencing result of a CCAACC repeat region. Creating the DNA Profile A DNA Profile is generated by analyzing the number of tetranucleotide single tandem repeats (STRs) in 13 loci of the human genome. The odds that two persons would contain the same number of repeats at each locus are approximately one in one billion. The STR loci are isolated and amplified using PCR. Exhaustive studies have shown an absence of false positives and negatives while using the system (Moretti et al 2001). However, the system does have limitations when DNA evidence from two or more persons becomes intermixed. A recent study showed that a three person intermixture could be construed as only 2 persons 3% of the time, whereas a 4 person genetic mixture could be construed as a 2 or 3 person mixture 70% of the time (Paoletti et al 2005). Contamination and sample degradation can also inhibit the ability to make a genetic profile. Despite these limitations the CODIS system has been used to aide and solve thousands of crimes. The ability to identify a biological sample from a crime scene to known convicted criminals has proven to be an effective tool of law enforcement. Hammer, Michael, Alan J. Redd. “Forensic Applications of Y-Chromosome STRs and SNPs” Deparment of Justice. Jan 2006 http://22.214.171.124/search?q=cache:hBWiApYKTT4J:www.ncjrs.gov/pdffiles1/nij/grants/211979.pdf+Y-chromosome+forensics%22&hl=en&gl=us&ct=clnk&cd=1 “Blackett Family DNA Activity 2” The Biology Project: University of Arizona. 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