Handout for Lecture 18: Recombinant DNA (Nov. 8, 2005)OutlineI. Overview of CloningPlasmids are Extrachromosomal Self-Replicating DNA moleculesII. Restriction Endonucleases (enzymes that cleave DNA at specific sites)I. Overview of CloningII. Restriction EndonucleasesMBB 694:407 & 115:511Handout for Lecture 18: Recombinant DNA (Nov. 8, 2005)Dr. Marty NemerofWaksman [email protected]“I’m having my DNA fumigated!”—Carrie Fisher, actress and writer, in response to her father’s (singer Eddie Fisher) tell-all autobiography describing his extramarital afairs with numerous Hollywood starlets.OutlineI. Overview of CloningA. General procedure for cloning a DNA fragment into a plasmid vectorB. Features of plasmid vectorsII. Restriction EndonucleasesA. Discovery—basis of host-induced restrictionB. Types of restriction endonucleasesC. Type II restriction endonucleases1. Recognition sequences and palindromes2. Sticky versus blunt ends3. Separating restriction fragments4. Restriction mappingIII. LigationA. An example of a sticky end ligationB. DNA ligase mechanismIV. Cloning VectorsA. Bacterial Plasmids1. Essential structural features: extrachromosomal, circular, autonomously replicating2. Advantages & limitations3. Expression plasmids4. Fusion protein plasmidsB. Bacteriophage lambda ()1. Essential structural features: 50-Kb linear or circular forms, large dispensible regions, cosends2. Advantages & limitationsV. cDNA Libraries1VI. What You Need to Know for Exam #3VII. Annotated Version of p. 16 (Cot Curve Analysis) of Handout for Lectures 16 & 17 VIII. Reading listA. Biochemistry, Garrett and Grisham1. Chapter 10 (pp. 330-334)—3rd edition or Chapter 11 (pp. 350-353)—2nd edition2. Chapter 12—3rd edition or Chapter 13—2nd editionB. Molecular Biology of the Gene, Watson et al, 5th Edition1. Chapter 20I. Overview of Cloning2insertBacterial Transformation= Introduction of plasmid DNA into E. coliPlasmids are Extrachromosomal Self-Replicating DNA moleculesFeatures of plasmids:3(Fig. 20-7, Watson 5th ed.)1) Replication origin (ORI)—allows the plasmid to be replicated by the host.(usually a “high copy number” ORI 500 plasmids/bacterium)2) Selectable marker(s)—such as the ampicillin resistance gene that permits for selection of the plasmid3) Cloning site(s)—allows for the insertion of exogeneous DNA.Q: What Makes the Insertion of DNA into Plasmid Vectors Possible?A: Restriction EndonucleasesII. Restriction Endonucleases (enzymes that cleave DNA at specific sites)Restriction-Modification System of Bacteria4Part I: RestrictionBacteria produce restriction enzymes that digest foreign (e.g., viral DNA)Why are Type II Restriction Endonucleases so Useful?1) They recognize palindromic sequences.5Part II: ModificationBacteria methylate their DNA to protect it from digestion2) They cut at the site of recognition.Examples of Type II Restriction Endonucleases6(Fig. 20-4, Watson 5th ed.)The Random Probability of Encountering a Restriction Site Depends on the Length of theRecognition Sequence: 4-base cutter Sau3A: GATC6-base cutter BamHI: GGATCCAgarose Gel Analysis of Digested DNA Fragments7Two Types of Gels Used for Separating DNA1) Agarose Gels 2) Polyacrylamide Gels—Large pores —Small pores—0.5-20 kb DNA fragments —0.1-1 kb DNA fragments8(Fig. 20-1, Watson 5th ed.)Restriction Mapping Problem #11. How big is the plasmid?2. How many times was the plasmid cut? Fragment sizes?3. Do the band sizes add up the total plasmid length?(Are the fragments in equal stochiometric amounts?)4. In double digests, which band(s) gets cut by the other enzyme? (It will tell you where the sites are relative to one another.)945 4EcoR I Sma IMW Uncut3EcoR I+Sma I216 KbRestriction Mapping Problem #2A 6-Kb plasmid is cut with various restriction enzymes (Hind III, Pst I, BamH I, Hind III-Pst I, Hind III-BamH I, and Pst I-BamH I). Based on the agarose gel analysis of each digest, indicate the positions of the PstI, BamH I sites (the position of the Hind III site is given). 10Hind IIIPst IBamH IHindIII-Pst IHind III-BamH IPst I-BamH IMWUncut12346 Kb556 kb/01234Hind III1. How big is the plasmid?2. How many times was the plasmid cut? Fragment sizes?3. Do the band sizes add up the total plasmid length?(Are the fragments in equal stochiometric amounts?)4. In double digests, which band(s) gets cut by the other enzyme? (It will tell you where the sites are relative to one another.)III. LigationA. An Example of a Cohesive (“Skicky”) End Ligation11*DNA ligase can also religate (reseal) a cut plasmid (ligate without the insert)!B. DNA Ligase MechanismDNA ligase catalyzes the formation of a phosphodiester bond between a 3’ hydroxyl at the end of one DNA strand and the 5’ phosphate at the end of another strand.12(Fig. 12.3, G&G 3rd ed.)IV. Cloning VectorsA) Bacterial PlasmidsAn Expression-Cloning Vector13(Fig. 12.17, G&G 3rd ed.)(Fig. 12.3, G&G 3rd ed.)An Expression Vector for Making a LacZ Fusion ProteinLac Z can be replaced with: Affinity Ligand1) Glutathione-S-transferase (GST) Glutathione2) Maltose-binding protein (MBP) StarchLimitations of Plasmids1) Can’t ligate large inserts into them (<10 kb)2) Low efficiency of transformation3) Large colony size14N CLac Z—Protein X (Fig. 12.17, G&G 3rd ed.) Ligate gene of interest into any of these sitesGene Xcan’t screen many potential recombinants per plate (<5,000 colonies/plate)B) Bacteriophage Lambda ()—Very Useful for Cloning Large Inserts (25 kb)Lambda Virus Infection Results in Plaques15How to Use  Virus to Make a Genomic DNA Library:cos sequences at ends oflambda genome are neededfor packaging the DNAinto empty virus heads16Thus only recombinants with 20-25 kb inserts will produce plaquesAdvantages of  virus vectors1) Accepts large inserts (25 kb)2) Efficient infection3) Very small plaque size can screen many recombinants per plate (~50,000 plaques/plate)Disdvantages of  virus vectors1) Not as easy to working with as plasmidsWhat if you want to screen a library and 1) determine the coding sequence of the insert or2) express the gene?Problem intronsSolution Clone mRNAs (no introns)V. cDNA Libraries (converting mRNA into “complementary DNA”)17VI. What You Need to Know for Exam #3I. Overview of Cloning1. Know the essential features found on plasmid vectorsII. Restriction Endonucleases1. Know how the “restriction-modification” system of bacteria works.2.