03-12-08 - 3/12/08 SANGER SYSTEM Polymerases Arthur...

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3/12/08 SANGER SYSTEM Polymerases - Arthur Kornberg polymerase I - Tom Kornberg polymerase II and III - Polymerase I - Klenon Polymerase (poly II) o no 5’ to 3’ exonuclease function - “size to sequence” Sanger - “size fact” B.J. Davis Fred Sanger - Fractionation - took random distributions and made order from random - first random distribution was Homochromatography - RNase T1 – enzyme that cleaves RNA after G P - there are 3 cuts resulting in 4 sections - the only section that doesn’t have a “G” is the last section at the 3’ end (AAU) - you can tell a T1 fingerprint 3’ end because there is no “G” - now you have to separate the 4 fragments - Sanger developed 2D fractionation for this o spot the bottom of the paper with capillaries o watch the spots o have another flat paper or solution/gel that’s spread on glass plate which is positively charged with DEAE (2 nd dimension) o put original paper on plate and fractionate it by size o then use X-ray to see spots Side Note: RNAse Panc cleaves at U P and C P
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- compare the fractionate plate to intensities of strip - unlabeled RNA degraded randomly slowly so that you would have at least one molecule of each size - the plate is positively charged and so nucleic acids will stick to it - one allogonucleotide with 3 phosphates and another with 4 and a third with 5 o these allogonucleotides will separate by size because the 4 N P will push the 3N P up and the 5 N P will push the 4 N P up and so on - T1 Fingerprint measure of purity and can tell what the 3’ end is “+ / –” System - Sanger then set up the “+ / –” system
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- insert a primer and then make pieces of the plasmid of all different sizes - if you make the piece of the plasmid a non-essential sequence – you can insert anything here using the “+ / –” system
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This note was uploaded on 08/21/2008 for the course BIOCHEM 301 taught by Professor Vanes during the Spring '08 term at Rutgers.

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03-12-08 - 3/12/08 SANGER SYSTEM Polymerases Arthur...

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