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lab_16_DNA_fingerprinting

lab_16_DNA_fingerprinting - Miramar College Biology 205...

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Lab Exercise 16: DNA Fingerprinting Page 1 of 5 Miramar College Biology 205, Microbiology Lab Exercise 16: DNA Fingerprinting Background DNA fingerprinting requires the use of several biotechnological techniques, and can vary among protocols. One way to make a DNA fingerprint involves the use of Restriction Enzyme Digestion , Polymerase Chain Reaction (PCR) and Agarose Gel Electrophoresis (Figure 1). In order to understand DNA fingerprinting in its entirety, it is important to first understand each of these constitutive parts. Figure 1: Diagrammatic representation of the DNA fingerprinting to be done during this lab experiment. Restriction Enzyme discovery led to the awarding of the Nobel Prize to Daniel Nathans, Werner Arber and Hamilton Smith in 1978. These restriction endonucleases, as they were first called, are part of an innate bacterial defense system against viral attack which cuts apart ( digests ) viral DNA into smaller pieces and each is named for the bacterial species from which it was first isolated. For instance Eco RI (note italics) was isolated from Escherichia coli and has a recognition site of GAATTC and its complement (Figure 2). Along any given piece of DNA wherever this specific sequence of bases is found, Eco RI will digest (or cut) the DNA into two pieces. In vivo , this leads to non-functional viral DNA that is incapable of causing a viral infection. In vitro , these enzymes can be used to identify and cleave molecules of DNA at these specific restriction sites . Because DNA from any two individuals is unique, the digestion of every individual’s DNA (be it a bacterial or human cell) will result in a unique set of restriction fragments (pieces of DNA produced by digestion with a given restriction enzyme). These restriction fragments can be compared between individuals to identify restriction fragment length polymorphisms (RFLPs) . Although the name sounds complicated, it is descriptive; polymorphisms (poly- many; -morphos- shapes) of different lengths are created when DNA is digested by a given restriction enzyme. These RFLPs compose one type of DNA fingerprint . Figure 2: Recognition site of Eco RI is shaded; the actual cut in the DNA strand is indicated by the placement of the arrows. Restriction digest results in two DNA molecules with complementary single-stranded “sticky” ends. The discussion above describes the digestion of a single molecule of DNA with a single restriction enzyme. This is a situation that, practically speaking, is worthless- as even large molecules of DNA require the use of an electron micrograph to visualize them. In order to easily see the RFLPs that result from a restriction digest, it is necessary to start the process with millions of identical copies of DNA. That’s where PCR comes into play.
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Lab Exercise 16: DNA Fingerprinting Page 2 of 5 Polymerase chain reaction (PCR) as a technique was considered so important to the field of molecular biology that its discoverer, Kary Mullis, was awarded the Nobel Prize in 1983. Fundamentally, PCR is a process whereby a researcher may easily and inexpensively produce millions of copies of DNA from a single template molecule.
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