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miniPCR bio™ Electrophoresis Forensics Lab. Wrongfully Convicted? Instructor's and Student's Guide Version: 1.1 - Release February 2022 - © 2022 by miniPCR bio™ Student's Guide P./12 Generating STR profiles - Forensic DNA analysis starts with a biological sample, such as a hair follicle or blood stain. Scientists break open the cells and extract the DNA for analysis (Figure 5). Luckily, forensic scientists only need a tiny amount of DNA for analysis. For example, crimes have been solved using DNA from a single eyelash or trace saliva on a cigarette butt. The key is a technique called polymerase chain reaction (PCR). PCR allows scientists to make many copies of specific regions of DNA, such as regions with a variable number of STRs. Biological sample Genomic DNA STR DNA DNA extraction PCR Electrophoresis Statistical analysis STR STR profile STR location STR 1 5, 8 12, 13 11, 12 12, 12 12, 18 12, 18 12, 18 12, 12 12, 12 11, 12 12, 12 12, 13 14, 14 5, 8 5, 8 STR 2 STR 3 STR 4 STR 5 Evidence genotype Suspect 1 genotype Suspect 2 genotype Result Match No match Calculate random match probability Figure 5. Forensic DNA analysis workflow DNA is extracted from a biological sample. Then, PCR is used to make many copies of just the locations in the genome with STRs that forensic scientists want to analyze. Electrophoresis techniques separate the copied STR DNA by length and allow forensic scientists to determine the STR genotypes. If there is a DNA match, then forensic scientists perform statistical calculations to determine the strength of the DNA evidence and the likelihood of getting a random match. Recall that the vast majority of DNA sequences are the same in all humans and that forensic analysis focuses only on regions of DNA that tend to differ between people, like STRs. PCR allows forensic scientists to take a sample like blood that contains all of an individual's DNA and then copy just the 20 different STR locations they are interested in. After PCR, electrophoresis techniques determine the number of repeats present at each STR region (Figure 5). Gel electrophoresis allows scientists to separate a mixture of DNA fragments based on length. Scientists use gels made of agarose, a polysaccharide extracted from seaweed, that forms a web-like mesh. An electric field causes the negatively charged DNA fragments to migrate through the gel towards the positive electrode. Smaller DNA fragments move easily through the agarose mesh and migrate quickly through the gel. Longer DNA fragments get caught in the agarose mesh and thus migrate more slowly through the gel. Gel electrophoresis reveals "bands" of DNA fragments, with the shorter pieces of DNA being located further along the gel (Figure 6). By including a mixture of DNA fragments of known sizes, referred to as a DNA ladder, it is possible to calculate the size of unknown DNA fragments present in the samples.

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