7_DNA fingerprinting

7_DNA fingerprinting - Lab Exercise DNA fingerprinting or...

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Lab Exercise: DNA fingerprinting or Restriction Fragment Length Polymorphism (RFLP) analysis OBJECTIVES 1. Understand the basis of DNA fingerprinting technology, including restriction enzyme digestion, polymerase chain reaction, and agarose gel electrophoresis. 2. Use the above techniques to identify the source of a simulated microbial outbreak from different isolated microbial DNA samples. BACKGROUND DNA fingerprinting requires the use of several biotechnological techniques, and can vary among different DNA fingerprinting 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 as 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 are named for the bacterial species from which they were 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, RI will cleave (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
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different lengths are created when DNA is digested by a given restriction enzyme. These RFLPs are the basis of 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 molecule 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
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This note was uploaded on 12/23/2009 for the course BIO 205 taught by Professor Murphy during the Fall '09 term at Miramar College.

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7_DNA fingerprinting - Lab Exercise DNA fingerprinting or...

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