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BIOL 309
David Spafford

BIOL 208 FINAL EXAM REVIEW MODULE 2 Principles forces holding strands of DNA together - Hydrogen bonds - Hydrophobic interactions - Phosphate backbone - Stacking interactions Denaturation: separation of two strands of nucleic acid that only H bonds holding strands together are disrupted and other bonds not affected Treatments that lead to denaturation: 1. Low salt Removes or reduces counter-ions on phosphate backbone Two (-) strands repel Commonly used 2. High Temperature Thermal energy break H bonds but not covalent linkages of backbone Commonly used 3. High pH Changes charge on H-bond groups -> disrupt Commonly used 4. Formaldehyde Urea Base pair with bases or H-bond groups Formaldehyde binds irreversibly Urea is reversible Sometimes used 5. Hydrophobic Reduce hydrophobic effect of bases -> contribute to DNA stability Sometimes used Hypochromic Shift: shift from denatured state back to double strand Nucleic Acid Synthesis - Substrates are dNTPs - Incoming dNTP added to 3' OH group of last nucleotide of daughter strand - All strands extended in 5' to 3' direction - DNA polymerases require primer - source of 3' OH group - RNA polymerases do not need primer but synthesize ribose nucleotide strands in 5' to 3' direction using ribonucleotide triphosphates (NTPs) MODULE 3 Enzymes included in cloning: - DNA to be cloned fragmented with restriction enzymes - Vector linearized with restriction enzymes (simplest case) - Two DNAs joined using T4 DNA ligase and introduced into host cells (transformation) Hybridization reactions generated by in vitro DNA synthesis reactions would require: - DNA template (denatured) - Primer - dNTPs (one modified, fluorescent, radioactive, tagged) - Buffer containing MgCl2 - DNA polymerase Polymerase Chain Reaction (PCR) - Alternate way to recover specific genomic fragment - In vitro DNA synthesis reaction MODULE 4 Extraction and Purification of Nucleic Acids 1. Collect tissue containing nucleic acid of interest Use fresh material Or material frozen quickly -> maintain quantity and limit degradation 2. Homogenize tissue and lyse cells Collect cells and resuspend in extraction buffer Extraction Buffer Reagents: EDTA: eliminates divalent cations -> destabilizes outer membrane Inhibits DNase -> degrades DNA SDS: solubilizes lipids in membrane Specialized Treatment to Lyse Cell Types: Bacterial Cells: lysozome Animal or Plant Tissue: Mild detergent Use cellulose and pectonase to break cell wall Yeast: glass beads Sound wave -> low pitch breaks it open 3. Isolate desired nucleic acid away from other cellular components Purification Steps: RNA Removal: treatment with ribonuclease (RNase) Heat stable enzyme -> easy to ensure free of DNase DNA Removal: treatment of RNase-free DNase Protein Removal: Proteinase K non-specific protease not inactivated by SDS or EDTA Alcohol Precipitation Removal of residual phenol Allow DNA or RNA to be concentrated and resuspend in solution Plasmid Purification Caesium chloride gradients - high salt solution creates density gradient Ethidium bromde - added to visualize DNA and aids separation of genomic vs plasmid DNAs Alkaline Denaturation (Plasmid Purification) Column Purification Size-Selection Chromatography Affinity Chromatography 4. Quantify recovery and quality of nucleic acid Gel Electrophoresis Movement of charged particle in response to electric field Agarose: separate DNA fragments of approx. 200 bp Polyacrylamide: separate DNA fragments of 1-1000 bp Polyacrylamide used for methods that require high resolution including: - DNA sequencing - Primer extension Rate of migration in electric field depends on: - Charge on fragment - Strength of field - Viscosity of gel - Presence of bound molecule - Conformation of DNA/RNA Other uses of electrophoresis: - Analysis of proteins - First step in blotting experiment involving hybridization - Sequencing MODULE 5 Type I Restriction Enzymes: recognize specific site but move undefined distance before cutting DNA - Require 5' phosphate, 3' hydroxyl, and MgCl2 as cofactors Type II Restriction Enzymes: specific restriction sites and cut within sequence or defined distance for it - Requires Mg++ as cofactor - EDTA can be added to stop Ligation - Joining of two fragments (ligation) catalyzed by ligase - Most common type of ligase = bacteriophage T4 also T4 DNA ligase Removal of 5' phosphate group prevents ligation If DNA concentration too low... Get no inserts If DNA concentration too high... Get multimers Other Modifications to Ligation Reactions: - To eliminate vector religating to itself, without an insert: Treat vector with alkaline phosphatase which removes 5' phosphate groups - To eliminate insertion of multiple inserts: Phosphatase insert - To clone insert into vector in specific orientation: Digest vector with two diff. enzymes so cannot ligate without insert w/ compatible ends - Addition of new restriction sites to ends of fragment Fill-in ends with DNA polymerase Make ends blunt by 3'-5' exonuclease Linkers MODULE 6 Other Types of Vectors: Expression Vectors - Recover protein product it encodes - Resulting proteins are "recombinant" because not recovered from native host Transcriptional Fusion Vectors for Bacterial Hosts - Used to express coding sequence under control of different promoter - Construct introduced back into host organism Translational Fusion Vectors - Localization - Better translation and transcription systems Version One: - Insert fused to vector's promoter and translational signals - Insert fused in-frame with ATG of vector's sequence Vers
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