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Ward's World+MGH Forensic Biology

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2 nonhuman samples such as botanical, fungal, entomological, or zoological specimens, which can also be used to link victims and suspects with each other or to the crime scene. Forensic biology may also be applied to homeland security, in which crimes may include weapons of mass destruction such as pathological microbes. The forensic biologist may be able not only to identify the microbe (such as anthrax) but to type the microbial DNA and link the strain of the microbe to a strain pro- duced at a certain location or to the original progenitor strain. The establishment of a United States national DNA database, the Combined DNA Index System or CODIS, has facilitated the ability to compare DNA profiles from unknown biological crime scene evidence to DNA databases of known convicted crimi- nals ("cold hits") or to DNA left at other crime scenes, resulting in the ability to link cases. Evidence Detection and Screening Detection starts with evaluating the investigative information to understand the nature of the case and samples. Visual examina- tion of stains with alternative light sources (such as ultraviolet or infrared light and lasers) and chemical enhancement reagents may be performed. Forensic biologists can determine the nature of the biological stain (for example, blood, semen, or saliva), and whether it is human through presumptive and confirma- tory tests. These tests consist of analytical procedures including microscopy, chemical tests, and immunological assays. DNA Extraction Following the detection and screening of samples from a crime scene, DNA must be extracted. Among the methods used are the following: 1. Organic extraction. This method consists of lysis of the cells in a detergent-based buffer followed by one or more rounds of purification using an organic phase separation and concentration using column centrifugation or ethanol precipitation. 2. Chelex resin extraction. This method utilizes a fast, simple extraction of small amounts of sample in the presence of a chelating resin. It results in a somewhat crude extract but is usually adequate for PCR amplification of the forensic genetic loci. 3. Solid-phase extraction. These methods utilize a mem- brane that act as a capture device for the DNA. Samples are spotted onto the membranes, and the subsequent washes remove the impurities. 4. Silica-based extraction. Nucleic acids are first adsorbed to the silica in the presence of chaotropic salts (salts that weaken hydrophobic associations). Polysaccharides and proteins do not adsorb and are removed. Next, following a wash in low salt, pure nucleic acids are released. This method has been automated using robotic stations and is being used in several laboratories. DNA Quantification Assessing the quantity and quality of the sample is the next step. Several methods are used in crime laboratories. These in- clude agarose gel electrophoresis in the presence of quantifica- tion standards (samples with known quantities of DNA), known as yield gel electrophoresis; slot blot hybridization, using known DNA standards immobilized on a membrane followed by hybridization to a human/higher primate-specific DNA probe; homogeneous plate assays, using a DNA fluorescent dye and scanning in a plate reader; and more recently, real- time detection using quantitative PCR (QPCR). Real-time QPCR has several advantages over the other methods in that it is extremely accurate and sensitive over a broad dynamic range, and it occurs in a closed-tube system, reducing the potential for carryover contamination. Using this technique, a forensic biologist can monitor and quantify the accumulation of PCR products during log phase amplification. DNA Amplification PCR is a fast in-vitro DNA synthesis process that can provide up to a billion (109) copies of a given target sequence. Specific DNA markers can be targeted for duplication by a DNA polymerase. Primers are designed to hybridize to the specific markers along the length of the DNA template during the cycling of tempera- tures. In the thermal cycle, DNA strands are separated, primers bind to the template, and then a special DNA polymerase that is heat-stable is used to copy and amplify the genetic markers. Through a process of 28–32 heating and cooling cycles, the DNA is increased so that it can be analyzed. The thermal cyclers contain many sample wells, permitting the amplification of multiple samples simultaneously; as many as 96 samples may be amplified in under 3 h. Multiplex PCR allows several different loci to be simultaneously amplified in a single tube. This enables typing from a single ali- quot of the extracted genomic DNA, reducing sample consump- tion. Recently the ability to analyze as many as 15 autosomal short tandem repeats (STRs) simultaneously has been reported using DNA from a very small amount of degraded sample. Forensic Biology (continued) + ward ' s science

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