Lecture 26Outline of Last Lecture I. TranslationA. Eukaryotic TranslationB. Prokaryotic TranslationII. Chain InitiationIII. Chain ElongationA. Aminoacyl SiteB. Peptidyl SiteIV. Chain Termination Outline of Current Lecture I. DNA AnalysisA. Gene CloningII. NucleasesA. EndonucleasesB. ExonucleasesC. Restriction EndonucleasesIII. DNA PlasmidsA. Plasmid PropertiesCurrent Lecture This lecture moves onto the topic of DNA analysis and recombinant DNA. The focus comes from asking how an individual gene is able to be studied. This is hard to comprehend when the human genome is around 5,000,000,000 base pairs. A gene represents only 1/1,000,000 of the genome. There are two ways to analyze a gene in detail. One method is using PCR to amplify the gene in question. The other method, and the one focused on in this lecture, is gene cloning. Gene cloning refers to the process of isolating a specific gene in a form that is able to be replicated in abundance in a host vector. One thing needed for cloning is a type of nuclease. Nucleases degrade nucleic acids through enzymatic action. There are different types of nucleases. One type are exonucleases. These enzymes degrade nucleic acids at either the 5’ or 3’ end. Endonucleases are another type. These enzymes also degrade nucleic acids, butthey cleave at specific internal sequences. The final type are restriction endonucleases. These cut DNA at specific sequences, by hydrolyzing the phosphodiester backbone of DNA. The specific sequence targeted is often palindromic in nature. Palindromes are sequences that read the same forwards and backwards. In this case, both strands of DNA are considered. Palindromic DNA sequences have the following properties: one strand going 5’ to 3’ reads the same, in the same place, as the other strand read 5’ to 3’. Examples of restriction endonucleases include HaeIII, which is a 4 base-pair cutter and EcoRI, which is a 6 base-pair cutter. The frequency of these cuts is determined by where on the sequence is recognized. When restriction endonucleases are cleave DNA, two types of ends can be BCHM 307 1nd Editionformed. The first type is a blunt end, which does not leave any overhang. The other type is a sticky end, which has 2-4 base pairs overhanging. The DNA fragments can be rejoined together after cleavage using DNA ligase, with the help of ATP. It can rejoin DNA in the same place it was cut, or the DNA can be rearranged. There needs to be a host for the DNA, called a vector. Bacteriophage used to be the vector of choice, but the more common one today is plasmids. Plasmids are circular molecules of DNA that exist in bacteria. They are extremely small molecules. Plasmids carry their own origin of replication. They often carry antibiotic resistant genes, making them easy to manipulate and easy to identify. There are many copies of plasmids within a cell. They have multi-cloning sites, with many restriction enzyme
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