BIOL-L 211 Lecture 10 Outline of Last Lecture I. DNA PolymeraseII. Sliding Clamp LoaderIII. Finishing ReplicationIV. E. Coli Holoenzyme and ReplisomeOutline of Current Lecture I. Polymerase Chain ReactionII. Agarose Gel ElectrophoresisIII. Restriction EndonucleasesCurrent LecturePolymerase Chain Reaction and DNA Sequencing and Restriction EndonucleasesI. Polymerase Chain Reaction (PCR)A. Definition: one of the most widely used laboratory techniques used to copy genes using a method similar, but not identical to natural DNA replication1. Note: Performed outside of cell (i.e. in a lab)B. PCR Cycle: the completion of the three steps below (each cycle doubles amount of DNA)1. Denaturing: hydrogen bonds between base pairs are broken via heating (all at once, yielding no replication forks and two separate single DNA strands)2. Annealing: DNA primers (NOT RNA) are added to both DNA strands3. Extension: DNA polymerase recognizes the primer:template junction and extends the sequence in the 5' to 3' direction (in one continuous stretch with no leading/lagging strands)C. "Ingredients"These 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.1. DNA template (can be genomic or from plasmid)2. Set of DNA primers (synthetic in order to emphasize certain regions)3. Modified DNA polymerase (can tolerate high heat, commits fewer errors, and proceeds without the help of a sliding clamp)4. Solution with nucleotides and appropriate salt mixtureD. Note: be able to compare/contrast PCR with natural DNA ReplicationII. Agarose Gel ElectrophoresisA. Definition: process that separates DNA fragments by relative size using electrical chargeB. Procedure:1. Agarose gel produced (from compounds including ethidium bromide)2. Chamber prepared with gel and salt buffer3. Combined DNA and heavy dye are put into well4. Electric current is allowed to flow through the gela. DNA pieces travel (because they are negatively charged), and the smallest pieces go the farthest because they encounter the least resistance5. Results viewed with UV light boxC. Applications:1. Forensics: PCR2. Medical Diagnostic Tests3. Study extinct animals (as in the very first lecture)III. Restriction EndonucleasesA. Definition: Enzymes that are used for DNA analysis1. Analogous to scissors2. Cut DNA at restriction sites3. Found in bacteria and archaeaB. EcoRI1. Specific endonuclease found in E. Coli2. Recognizes and cuts 5'-GAATTC-3'3. Fragments can then go through Agarose gel electrophoresisC. Applications1. Diagnostic Tests2. DNA
View Full Document
Unlocking...