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ONLINE LAB #8 BIO 101 PROF. SHIN (adapted from HHMI Biointeractive: www.biointeractive.org) Introduction: DNA fingerprinting is a technique that is currently used to identify sources of DNA in a number of contexts. Forensic scientists use this technique to identify the source of DNA at crime and disaster scenes. DNA fingerprinting is also used in paternity cases as a means of definitively identifying the father of a newborn child. Scientists also use DNA fingerprinting to determine the degree of relatedness between different species in evolutionary studies. The uses for DNA fingerprinting are widespread. In this week’s lab, we will be utilizing a particular form of DNA fingerprinting, called STR analysis in order to solve a crime! Background: While most of the genome is identical among individuals of the same species, differences do exist. DNA profiling takes advantage of these differences. Variations occur throughout the genome, and in particular, in regions of noncoding DNA, which is DNA that is not transcribed and translated into a protein. Variations in noncoding regions are less likely to affect an individual’s phenotype, and therefore changes in these regions are less likely to be eliminated by natural selection. DNA profiling uses a category of DNA variations called short tandem repeats. STRs are comprised of units of bases, typically two to five bases long, that repeat multiple times. The repeat units are found at different locations, or loci, throughout the genome. Every STR has multiple alleles, or variants, each defined by the number of repeat units present or by the length of the sequence. They are surrounded by nonvariable segments of DNA known as flanking regions. For example, the STR allele in Figure 1 could be designated as “6” because the repeat unit (GATA) repeats six times, or as 70 bp (where bp stands for base pairs) because it is 70 bp in length, including the flanking regions. A different allele of this same STR would have a different number of GATA repeat units but the same flanking regions. Flanking regions are important because knowing their sequences enables geneticists to isolate the STR using polymerase chain reaction, or PCR, amplification. Figure 1. Each one of the rectangles above represents a repeat unit. In this example, the STR is comprised of six repeats of the four-base unit GATA. On either side of the STR is a flanking region of DNA. If you were to write out the STR sequence in Figure 1, it would be GATAGATAGATAGATAGATAGATA. For STRs with many repeat units, writing out the sequence can get very unwieldy, so geneticists use shorthand. The repeat unit is placed in brackets with a subscript indicating the number of times it repeats. The shorthand for the STR in the example above would be [GATA]6. The difference STRs will differ slightly in length. These differences can be resolved using a technique called gel electrophoresis. In this process, a sample containing mixed fragment sizes of DNA is forced through a gel matrix that contains numerous molecular-sized pores. The gel matrix is made from an extract from algae called agarose. Once DNA is loaded onto a gel, the DNA must be forced to move. This is accomplished by the application of an electrical current to the DNA. DNA, which is negatively charged (remember the negatively charged phosphate groups on the nucleotides?), will move toward the positive node of the electrophoresis chamber and away from the negative node of the electrophoresis chamber. Once a mixture of differently sized DNA fragments begins moving through a gel matrix, the smaller fragments travel more easily than the larger fragments through the pores of the gel. This results in the smaller fragments moving faster than the larger ones, and separation of these fragments is accomplished. A quick video showing the process of gel electrophoresis can be found here. Because individuals are so unique genetically, scientists are able to utilize the differences in our DNA to create unique profiles, or DNA fingerprints. They can use these DNA fingerprints to match crime scene samples with the individuals that left them! Exercise: (1) Download the student worksheet. (2) Use the following link to launch the HHMI Biointeractive Exercise: CSI Wildlife. https://media.hhmi.org/biointeractive/click/elephants/dna/ (3) Solve the crime!