ELEMENTS OF BIOL CHEM
ELEMENTS OF BIOL CHEM BICH 303
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This 11 page Class Notes was uploaded by Sylvester Mante on Wednesday October 21, 2015. The Class Notes belongs to BICH 303 at Texas A&M University taught by Timothy Devarenne in Fall. Since its upload, it has received 30 views. For similar materials see /class/225849/bich-303-texas-a-m-university in Biochemistry at Texas A&M University.
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Date Created: 10/21/15
Why does RNA have uracil and DNA has thymine s commonly thought that evolutionarily RNA was the original hereditary molecule NH RNA is not stable so DNA evolved to store hereditary information N l Thus DNA needs to be a stable structure N cytosine can spontaneously deaminate to uracil thymine is stable N l NH H N l x I 0ll 1110 AN l I l H L rucil C lusinc it would cause a change in the base pairing CG changed to UA base pairing mutation deamination repication C G G gt U G G Arg CGG UGG Trp both uracil and thymine can base pair to adenine thus since thymine is stable it is used in DNA to prevent mutations Nucleic Acid Techniques What are the techniques used to work with DNA How is DNA cloned plasmid vectors restriction enzymes How is DNA produced in the lab polymerase chain reaction PCR How is the sequence of DNA determined DNA Gel Electrophoresis Electrophoresis movement of charged particles in an electrical field through a gel matrix Gel matrix agarose a polysaccharide of galactose from red algae In SDS PAGE SDS gives all proteins in a sample an overall charge Proteins move toward anode of electric field What is the charge of DNA due to phosphate in nucleotide DNA in a sample will move toward anode DNA in sample is separated by size large size moves slower w nnnnnnnnn rrrrr an small size moves quicker DNA Gel Electrophoresis How to visualize DNA after gel electrophoresis Ethidium Bromide EtBr A small molecule that stains nucleic acids by intercalating between strands of DNA double helix Under ultraviolet light EtBr will fluoresce a redorange color How to clone a gene Steps 1 Isolate piece of DNA with gene of interest many methods for this 2 cut DNA with restriction enzyme 3 ligate into plasmid vector 4 express gene on plasmid vector in bacteria to produce protein Restriction endonuclease an enzyme that hydrolyzes breaks cuts the phosphate ester bond between nucleotides cut DNA at specific sites cut sites are palindromic read the same left to right and right to left Named after organism from which they were isolated Restriction endonuclease EcoRI isolated from bacteria Escherichia coli E coli recognizes sequence G A A T T C 5 ATGCTGGAATTCGAGACG3 3 TAGGACCTTAATGCTCTGC5 EmRIliyr mlvsis breaks DNA smmu bumen G and Imscs What s wrong with this 7 Cutting DNA with restriction enzyme produces sticky ends overhanging ends DNA ligase enzyme that links two pieces of DNA Plasmid Vectors Plasmid a small circular DNA molecule capable of self replication contains site for insertion of genes for cloning Cut plasmid with restriction enzyme to produce sticky ends gt Cut gene with restriction enzyme to produce sticky ends Add cut plasmid cut gene and DNA ligase Plasmid Vectors Plasmid can be put into bacteria to produce large amounts of the gene of interest Gene in plasmid can also be expressed as protein to obtain large amounts of protein for further studies such as enzyme assays or o It m ngr u Singlr pmn my structure identi cation protein let bacteria a nilli39cc L39ndslo make more Plasm39di cmhufhvwigll DNA a v Url p y Foirigu DNA letkbactetria put plasmid in E ma e pro eln bacteria purify 4 mos ammcale rhomson Polymerase Chain Reaction PCR Method for amplification of small amounts of DNA into large amounts of DNA exponential obtain millions to hundreds of millions of DNA DNA polymerase must be able to remain active after being heated to 95 C 203 F Utilizes DNA polymerases from bacteria that live in hot springs Thermus aquaticus Taq polymerase first found in the hot springs and geysers of Yellowstone Nat l Park 50 80 C122 176 F subsequently found near deepsea thermal vents over 100 C 212 C Polymerase Chain Reaction PCR Kary Mullis inventor of PCR came up with idea to use DNA polymerase from Thermus aquaticus 1983 awarded Nobel Prize in Chemistry 1993 Polymerase Chain Reaction PCR 2 cool to 55 C allow primers to Requires m DNA polymerase Taq lbwquot I primer short 15 30 bases of DNA used to 39 base for Taq polymerase to synthesize l law39shrlri39nrzagw l new DNA template desired DNA to be amplified n r I m Nucleotides dATP dTTP dCTP dGTP r f II I I I cycle I I 1 heat to 95 C separates DNA l i strands 39 I bind to i v thermal cycler complementary sequence v 1 l 1 I 3 heat to 70 C optimal temperature I I for Taq polymerase l l l r I III i i quota r r l kr llll i 7 2006 Brookse Cule mumson
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