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Lecture 10

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Biology 2581B
Susanne Kohalmi

Genetics Lecture 10 Notes Restriction Enzymes:  Restriction enzymes are double stranded DNA endonucleases  Some have blunt ends, some have 3’ sticky ends, some have 5’ sticky ends 1. What do they do 2. Types of ends n 3. Calculation of average fragment size: 4 - The n is the length of recognition size Gel Electrophoresis:  Need to analyze DNA after it has been cut with restriction enzyme 1. Basic procedure - Run them on agarose or polyacrylamide gel 2. Basis of separation – size - Smaller DNA fragments run through faster than larger fragments DNA amplification 1 – molecular cloning: 1. How a plasmid vector works - Amplify DNA in bacteria or a bacteriophage - Deal with a plasmid vector that has an F factor and a selectable marker 2. Ligation - Cut plasma DNA with restriction enzyme - Add ligase to get them to come together to form chimeric molecules with plasma DNA and human DNA? 3. Transformation 4. Selection - Select for bacteria that have taken up the plasma DNA – will get a colony of bacteria that have taken up this plasmid DNA amplification 2: PCR: 1. Primers 2. Thermocycling 3. Chain reaction *Fig 9.16 Hybridization: 1. Principle of complementarity 2. Hydrogen bonding = driving force for complementarity 3. Labeled probe - Can label nucleic acids – can use this protocol to clone genes 4. Southern/ Northern blots Reverse transcription:  How mRNA is copied into single stranded complementary DNA  Making a cDNA copy of mRNA  Can isolate mRNA from particular cells  Anneal a primer to that mRNA – generally oligo-DT? o Use 3’ end to get single stranded DNA copy  Now you have a cDNA-mRNA hybrid – now have single stranded copy of the cDNA  Put the cDNA into a vector (plasma or phage vector)  Initiate synthesis of DNA and get complementary DNA clone? The cDNA clone: 1. Conversion of single stranded cDNA into double stranded DNA 2. Insertion into a plasmid vector Sanger DNA sequencing: 1. Basic procedure 2. Role of dideoxy nucleotides - Dideoxy nucleotides allow you to determine the sequence of DNA 3. Reading a sequencing gel History of Recombinant DNA:  Determined that DNA was heritable material in 1940s  The 1950s – discovery of structure of DNA o Biochemistry of DNA replication started  1960s – genetic code, restriction enzymes, analysis of plasmid & phage  Late 60s, early 70s – isolated the DNA for the lac operon of E.coli – not a general method o Isolate the specific DNA segment for a specific gene  Early 1970s – using restriction enzymes & a plasmid vector the first chimeric DNA molecule was created o Establishing a general method for cloning genes o First genes cloned in the 1970s were ones already characterized extensively at the biochemical level – hemoglobin and immunoglobulins – allowed them to isolate the biochemical DNA that encodes these proteins Hemophilia A:  On X chromosome – disease causes no blood clotting (bleed to death)  Victoria doesn’t exhibit the phenotype because she has a wild type allele on her
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