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Unformatted text preview: BI/CH 368 Laboratory Experiment 1 Gift Horse? Verification of Equine Maternity via PCR-RFLP Analysis Case Background Your uncle wants you to invest with him in a potentially valuable colt for sale on eBay. Rhettador Zs parents were both renowned show horses, and his dam (i.e., mother) has produced several valuable foals. The seller has forwarded proof of lineage (Figure 1), and at a low asking price of $10,000, your uncle is convinced that you can easily triple your investment. However, you are concerned that the colt may be an imposter. Your uncle has sent you hair samples from both the colt and Scarlett OHara, the presumed dam. (He obtained the mothers hair sample by bribing the ferrier at the breeders farm, since the colt is no longer stabled there. The sire is at a stud farm with tight security, so a DNA sample cannot be obtained from him. ) With the two hair samples in hand, your goal is to use DNA analysis to verify the maternity of the eBay colt. You will analyze mitochondrial DNA (mtDNA), which has several characteristics that make it ideal for this experiment. First, it accumulates mutations 5-10 times faster than nuclear DNA. Furthermore, unlike nuclear DNA, mtDNA can be amplified from hair shafts, not just roots. Because it is present in high copy number (several hundred genomes per cell), it is typically much easier to amplify than nuclear DNA. Finally, it is maternally inherited, so if the eBay horse is in fact Rhettador Z, his mtDNA should be an exact match to that of Scarlett OHara. Figure 1. With his impressive lineage, Rhettador Z is worth far more than the low, low asking price. Jean-Paul Greenbriar 555 Champions Way, Tallahassee, FL 32306 04/20/2010 BI/CH 368 Laboratory Experiment 1 Spring 2012 2 Experimental Background The original methodology for DNA profiling was based on "restriction fragment length polymorphisms" (RFLPs, pronounced "riflips"). In this procedure, genomic DNA is digested with a restriction enzyme, a molecule that acts as a pair of molecular scissors, catalyzing the double- stranded cleavage of DNA within a specific short nucleotide sequence. For RFLP analysis, a restriction enzyme that cleaves within a polymorphic site (one that varies between individuals) is used. In theory, the resulting restriction fragments, which should differ in size between individuals, can be separated via agarose gel electrophoresis. In practice, the number of fragments produced by restriction nuclease digestion of mammalian genomic DNA is so large that a smear appears on agarose gels, rather than individual bands. Therefore, Southern blotting is used to characterize a specific polymorphic site in a complex genome. Restriction fragments are separated by size on an agarose gel and then transferred to a nitrocellulose membrane, which is incubated with a radioactive probe complementary to the sequence of interest. The resulting band(s) can then be detected by X-Ray film....
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This note was uploaded on 03/31/2012 for the course CHEM 360 taught by Professor Millard during the Spring '12 term at Colby.
- Spring '12