1-PrelabInformation2011

1-PrelabInformation2011 - Prelab information for Genome...

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Prelab information for Genome Analysis I Lab Manipulating nucleic acids with enzymes The key to studying genetics at the molecular level is to be able to manipulate nucleic acids (DNA and RNA). However, nucleic acids are too small to work with directly. Fortunately, there are naturally occuring enzymes that can be used to modify DNA and RNA in several useful ways. We will be using three kinds of enzymes during lab: restriction enzymes, RNase, and DNase. Restriction enzymes are produced by many bacteria for defense against invading DNA (viruses for example). These enzymes work by ‘recognizing’ and binding to specific DNA sequences and then cutting the DNA at a very specific location within that sequence. Because of the way these enzymes work, the DNA of invading viruses will be cut but the bacterial DNA will remain intact. One restriction enzyme commonly used by geneticists is called Eco R I, the first such enzyme isolated from the bacteria Escherichia coli . The recognition sequence of Eco R I is 5 -GAATTC- 3’ and it will always cut DNA between the G and the first A. Restriction enzymes must cut both strands of DNA for them to be effective, thus the recognition sequence must be a palindrome (note that the complimentary sequence of 5’ -GAATTC- 3’ is also 5’ -GAATTC- 3’). This allows one enzyme to cut the phosphodiester bonds of both strands of DNA. If the following DNA molecule was treated with Eco R I, 5’ -CCATGCTAGAATTCACACGGGT- 3’ 3’ -GGTACGATCTTAAGTGTGCCCA- 5’ two fragments would result (note that the DNA remains double stranded): 5’ -CCATGCTAG AATTCACACGGGT- 3’ 3’ -GGTACGATCTTAA GTGTGCCCA- 5’ Two other kinds of enzymes commonly used to manipulate nucleic acids are RNase and DNase. These enzymes degrade RNA and DNA respectively by cleaving long chains of nucleic acids into individual nucleotides. These enzymes are used when one wants to remove either RNA or DNA from a mixture containing both types of nucleic acids. Learning Objectives Explain the natural function of restriction enzymes and how they work. Describe the unique attribute of restriction enzyme recognition sequences that allows them to cut both strands of a double-stranded DNA sequence. Given a DNA sequence and the recognition sequence of a restriction enzyme, draw the resulting DNA fragments. Explain the function and use of RNase and DNase.
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Prelab Information for Genome Analysis II Lab Gel electrophoresis Clumps of DNA like you saw in the onion DNA extraction lab provide no information about the nature of the DNA, however there are specific techniques that allow us to see differences among DNA samples. Gel electrophoresis is the standard way of visualizing DNA by separating DNA molecules according to their relative lengths (number of base pairs). Electrophoresis involves loading DNA fragments into an agarose gel slab, which has been placed into a chamber filled with a conductive buffer solution. An electrical current is passed between wire electrodes at each end of the chamber.
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