412U4 - Restriction Endonucleases (REs): (REs): catalyze a...

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Unformatted text preview: Restriction Endonucleases (REs): (REs): catalyze a ds cut in dsDNA at a specific pallindromic sequence Some REs make staggered cuts, producing sticky ends. Others make blunt cuts, yielding blunt ends. Restriction Endonucleases (REs) Biological Function: degrade foreign DNA, protect bacterium from phage infection 1. Discovered after the observation that phage that grow in one strain of E coli can not grow in others ( restricted growth). The cause of the restriction was identified: REs that cut up phage DNA. 2. Recognition of phage DNA vs own DNA: methylation pattern; a RE will not cut at its recognition sequence if that sequence is methylated at specific locations. Each strain has a methylase that methylates its DNA so that it will not be cut by its own RE (a given strains RE and methylase both recognize the same sequence). 3. The phage that can grow in a given strain of E coli have their RE sites methylated in the same way as the DNA in that strain is. Applications for REs Production of Recombinant DNA : many of the REs make staggered cuts, producing sticky ends. These ends on one molecule base pair with those on another molecule. Applications for REs: RFLP Restiction Fragment Length Polymorphism (RFLP): to detect genetic diseases, identify criminals (DNA fingerprinting), etc a. Chromosomal DNA is cleaved with an RE. b. resulting fragments are separated by electrophoresis: smaller fragments move faster RFLP c. DNA fragments are melted with NaOH and fragments are transferred to nitrocellulose paper (Southern blotting) where they are held in place (in the same pattern as on the electrophoresis gel) d. then a probe ( a ssDNA complimentary to the sequence of interest) is added. The probe only binds at the location of the one (or two, etc) band(s) of interest. The probe must have a detectable tag, such as radioactivity (or a colored group: chromophore) e. autoradiograph: lay a piece of x-ray film over the paper, radioactivity exposes the film. Inheritance of RFLPs according to the rules of Mendelian genetics. REs: Constructing a restriction map of DNA A restriction map of a DNA molecule shows all sites that will be cut by REs Figure 5-40 Restriction map for the 5243-bp circular DNA of SV40. Page 104 REs: Constructing a restriction map of DNA Treat different samples of the DNA with one RE, another RE, and a mixture of the 2 REs. Electrophorese each sample in a different lane and estimate fragment sizes in each lane (say we got the following fragments, for RE1: 3.1kb, 3.8kb, and 4.6kb; for RE2: 1.8kb, 4.4kb, and, 5.3kb; for RE1 + RE2: 1.6kb, 1.8kb, 2.2kb, 2.8kb, 3.1kb) Identify end fragments: these will be fragments that are the same size in the double digest (RE1+ RE2) as they are in a single digest (RE1 or RE2). (In this case, the 1.8kb and 3.1 kb fragments are ends.) REs: Constructing a restriction map of DNA Work in from each end: is there a fragment remaining...
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This note was uploaded on 04/22/2011 for the course BIOCHEM 412 taught by Professor Thomas during the Spring '11 term at Tennessee Martin.

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412U4 - Restriction Endonucleases (REs): (REs): catalyze a...

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