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BIOL 3110
Peter Cheung

Oct 8, 2009 DNA Sequencing -In order to understand the basic structure of DNA it is important to understand the prevalent primary and secondary structure of it. -Protein is the main component of DNA both in terms of incorporation and in the construction of the molecule; the first protein to be sequenced is insulin. -Dideoxy-nucleotide chain termination method is one of the oldest ways to sequence a protein, but it is considered old and inefficient compared to modern ways of sequencing (7.12 Singer & Burk) -The L isoform of amino acids are the ones that are used in proteins, not D. -In DNA replication there needs to be a template strand to indicate the specificity of which amino acid to insert along with other chemicals. -If you want to stop the synthesis process you can use ddATP since it is missing 3’OH, because there in no OH to attach the next nucleotide to. -When reading the sequence from a gel, you start at the bottom and move upwards, each of the nucleotides will occupy a different lane. How to Get a ss Template -The first step is to find an organism that can make a ss form of DNA and then infect with a bacteria phage (M-13 in this case) -The phage will exist in a ss form but will reproduce in a ds replicative form, the resulting progeny are also in a ss form. -Use a universal primer that can then bond to the ds replicative form of the phage and have it create an recombinant molecule (universal primers are just sequenced oligotides). -The phage will create a hybrid onto the primer and replicate the sequence of your choosing and then revert back to the ss form. ***r
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