DNA_and_Spectrophotometry

DNA_and_Spectrophotometry - A Look at DNA and...

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Unformatted text preview: A Look at DNA and Spectrophotometry Bases - Pyrimidines Note the difference between Cytosine and the other two pyrimidines (indicated by the arrows!). U and T are missing a double bond and consequently the Nitrogen has a H attached. Why is this important? Changes the H bonding from acceptor (on C) to donor (U, T) thus affects the base complimentarity (what the base binds to!) Bases - Purines Note the differences between these bases. Why do you think A can pair with T but not with C? Could A bind with U? Nucleosides = Sugar + Base Note which Nitrogen on the base is bound to the sugar. Are these RIBOnucleosides or DEOXYribonucleosides ? Also note the difference in naming between the purine and pyrimidine nucleoside. What do you think the nucleoside form of thymine would be called? Guanine? What did I tell you adenosine does in the brain and how is it associated with Caffeine? What is the structure of caffeine? Nucleotides = Base + sugar + phosphate Nucleotides will have either 1, 2, or 3 phosphates (mono, di, or tri) DNA Structure From Alberts et al, Molecular Biology of the Cell 5 th ed. Base Pairing Note!! Opposite orientation of the strands and of the bases (ie the T is “upside down”). From Alberts et al, Molecular Biology of the Cell 5 th ed. Diphenylamine (DPA) Under heat and in acid, DPA will bind to 2 deoxypentoses – like deoxyribose! But remember it will bind only to deoxyriboses attached to purines! BUT – we want to use this to QUANTITATE DNA in an unknown solution. In order to do this we need to make a STANDARD CURVE. What to use for a standard though??? We could use a known sample of DNA but failing that we could use a deoxynucleoside – like deoxyadenosine (dA)! Standards Standards have known amounts of a specific substance that will give a positive reaction with the assay – in this case, the standard is deoxyadenosine and it will react with DPA to give a blue colour. When we are trying to quantitate an unknown (determine how much), we use a series of known amounts of our standards. More molecules of our standard will result in a more intense reaction - in our case, more blue colour. DPA +dA When DPA is added to our dA standard, and then heated, a blue colour will form ( ) - more molecules more blue colour. When light of a...
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This note was uploaded on 12/16/2010 for the course BIOL 1010 taught by Professor Anafi/quinlan during the Spring '08 term at York University.

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DNA_and_Spectrophotometry - A Look at DNA and...

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