BIO 344 1st Edition Lecture 3Outline of Last Lecture I. DNA Topology and TopoisomeraseII. Replicationa. Initiationb. DnaA, DnaB, DnaCc. Polymerases and primersd. Processivity III. Problems with Replicationa. Okazaki Fragmentsb. Catenationc. Completing endsi. telomeresOutline of Current Lecture I.DNA Polymerizationa. Chemistry of deoxy and ribonucleoside triphosphatesII.Fidelityb. ProofreadingIII. ChemotherapeuticsIV. Dideoxyribonucleoside triphosphateV. “Seeing DNA”—Technologya. Radioactive labelingb. Fluorescent labelingVI. DNA Sequencinga. Sanger Methodb. Gel ElectrophoresisThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.c. PCRd. NextGenCurrent LectureDNA Polymerization- Always utilized the 3’ hydroxyl endo Needs the –OH to bind -OH is the nucleophile that attacks the 5’ phosphate to make a new bind- Occurs at the active site of DNA polymerase Pyrophosphate is released- Overall synthesis is 5’3’o Template strand= original DNA strand currently serving as the template for replicationo Primer strand= growing strand that is synthesized during replicationo Nontemplate strand= complementary strand to the template strand- Incoming nucleotides positioned at the active site snuglyo Only base pairs with canonical geometry are tolerated at the active site For example, if two purines are at active site, the 5’ phosphate will not be positioned properly for attack by the 3’ hydroxyl- How is making RNA avoided?o Differentiation between ribonucleoside triphospahte and deoxyribonucleoside triphosphate Ribonucleoside triphosphate has a 3’ and a 2’ hydroxyl which creates steric hindrance at the active site- How does a ribonucleoside not use a deoxyribonucleoside?o Functional groups and hydrogen bonding stabilize the 2’ hydroxylFidelity- Polymerization3’-5’ exonucleotide proofreadingstrand directed mismatch repairo Combined 1 in 10^9 errors per nucleotide- Proof readingo Exonuclease= cuts ends of DNA where there are incorrect nucleotides Wrong base pair creates distortion and exonuclease recognizes ito Endonuclease= cuts incorrect nucleotides within the DNADNA Polymerases are targets for Chemotherapeutics- Example: acyclovir= antiviral for Herpes treatmento Mimics deoxynucleoside (has one hydroxyl group and similar in structure)o Herpes virus has a kinase that phosphorylates the –OH (like in DNA polymerization) and once phosphorylated, toxic to herpes replication- Example: AZT= reverse transcriptase inhibitor for HIV treatmento Works similarly to acyclovir in polymerizationDideoxyribonucleoside triphosphates- Even though there is no hydroxyl, DNA polymerase will recognize it, but will cause replication to stopo No –OH to attack the phosphate of the next deoxyribonucleoside triphosphate“Seeing DNA”—Technology- Radioactively labeled DNAo 32P (alpha phosphate of triphosphate) Creates a black spot on x-ray film to mark location, but there is no differentiation between marked nucleotides- Fluorescently labeled DNAo Fluorescein couples to DNA and allows DNA to be seeno Can label different nucleotides with different colors by coupling with different fluorescents to determine location and identity of nucleotidesDNA Sequencing- Sanger invented DNA sequencingo DNA polymerase needs a primer for template strando Add in deoxyribonucleotides a small amount of dideoxyribonucleotides, which when incorporated terminated further growth at that site- this created different sites at which growth is terminated, creating small sequences that can then be run through a gel to obtain DNA sequence- Gel Electrophoresiso DNA is negatively charged, so it moves through the gel to the positive endo Small molecules move fastero Sequence can be read from the bottom upo While running the gel, if labeled with fluorescents, can shine a light through a window and see the different colored bands pass to obtain the DNA sequence easily- PCR= polymerase chain reactiono Starts from only one DNA genome Heat to separate strands Hybridize primers Add DNA polymerase and nucleotides: dATP, dGTP, dTTP, dCTP DNA synthesis from primerso These steps are repeated to continually double DNA copies to amplify DNAo DNA polymerase used needs to handle the heat without denaturing Use a DNA polymerase from a thermophillic organism- An organism that lives in a hot temperature- NextGen Sequencingo In Sanger, DNA was fragmented, clones individually into plasmids, separated and purified, then sequenced and assembled into contiguous fragments Takes only hours but yields less than 1,000 base pair readso In NextGen, DNA is handled in bulk—no cloning Takes weeks but yields 30 billion base pair readso Method of NextGen Take DNA sample, fragment (bombard with ultrasound), ligate ends of fragments with known sequences, put on a slide, DNA binds and populates cell surface, creates bridge amplification (cluster growth), and use fluorescent labels to sequence by using a
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