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Chapter 12

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Department
Biochemistry
Course
BIOCHEM 2B03
Professor
Margaret Fahnestock
Semester
Fall

Description
Biochem 2B03 Jasmyn Lee Part 1: Molecular Components of Cells Chapter 12: Recombinant DNA: Cloning and Creation of Chimeric Genes DNA Manipulation 12.1 What Does it Mean “To Clone”  We now routinely use cloned DNA for industrial and experimental purposes  The technology we use is based on the basic molecular mechanisms of DNA replication and repair that exist in cells  Cloning 101 1. Select a host – E. coli is a common choice 2. Make a DNA copy of the mRNA using reverse transcription 3. Amplify the DNA copy using PCR 4. Digest the DNA with a restriction enzyme 5. Digest a cloning vector (plasmid) with a restriction enzyme 6. Insert the DNA into the plasmid – by DNA ligation 7. Introduce the recombinant plasmid into E. coli cells – transformation 8. Confirm that the inserted DNA has the right sequence – by DNA sequencing E. coli as a host  Extensively studied and fairly well understood  Reproduce very rapidly (20 minutes per generation)  Easy to grow in a liquid medium or on an agar plate  Contains a single chromosome  Many kinds of E. coli for different purposes o Note – in 2L06 you will use different strains in term 1 and 2 (page 65 2L06) Plasmids  Plasmids are circular extra chromosomal DNA  Many kinds of plasmids for different purposes o 2L06 uses pET28b  Plasmids for cloning require three key elements: 1. Origin of replication – permits autonomous replication 2. A selectable marker (antibiotic resistance gene) 3. “Multiple Cloning Site” with many restriction sites – so that you can insert the DNA of interest  Nuclease cleavage at a restriction site linearizes (opens up) the circular plasmid – foreign DNA fragment can be inserted  Ends of linearized plasmid and ends of fragment have corresponding sticky ends so that they are able to be joined together to create a recombinant plasmid  Recombinant Plasmids/Chimeric Constructs/Chimeric Plasmids– hybrid DNA molecules consisting of plasmid DNA sequences plus inserted DNA elements (inserts) Reverse Transcription  Reverse transcriptase – produced by eukaryotic “retroviruses” o Eg/ HIV, common cold o Telomerase is one type of reverse transcriptase  RNA-dependent DNA polymerases  Use RNA template rather than DNA  Synthesize DNA in the same 5’ to 3’ direction  Can’t do first bond synthesis – need a primer  Eukaryotic mRNA have 3’ poly A tails  “Primer”: 15 deoxythymidines (dT15)  Mix dT15 and the mRNA  Add reverse transcriptase and dNTPs (dATP, dCTP, dGTP and dTTP)  After this reaction you get double-stranded RNA-DNA hybrid  Degrade the mRNA by NaOH/ribonuclease 1 Biochem 2B03 Jasmyn Lee  You are left with a molecule of ssDNA  The product is called “cDNA” (complementary DNA)  There are alternative ways to design primer if the mRNA sequence is known  Note: you will design primers to amplify folA by PCR in 2L06  Mix unique primer and the mRNA  Add reverse transcriptase and dNTPs (dATP, dCTP, dGTP and dTTP)  Enzyme generates a ssDNA copy of the RNA  Degrade the mRNA by NaOH/ribonuclease 12.4 What Is the Polymerase Chain Reaction? (PCR) Polymerase Chain Reaction – versatile means of amplifying any DNA sequence  Take advantage of: o Synthetic DNA – design oligonucleotide primers  Designed to be complementary to the two 3’ ends of the specific DNA segment to be amplified  Added in excess amounts of >1000x o Thermotolerant DNA polymerases (Taq, Pfu, Vent, etc)  Problem: o Lack of or inefficient 3’5’ exonuclease activity o Means that PCR is generally error prone *important to sequence clones PCR with duplex DNA as template  Cycles o Denature o Anneal o Extend PCR with single stranded cDNA as template 2 Biochem 2B03 Jasmyn Lee  Boundaries of primers determine boundaries of amplified DNA o You can add extra nucleotides at 5’ end o Means you can add useful sequences at ends – restriction sites or tags  Other applications of PCR o DNA sequencing o Gene SOEing – Splicing by Overlap Extension o Site-directed mutagenesis  More “potent” versions of the lactase enzyme 3 Biochem 2B03 Jasmyn Lee Restriction Digestion of DNA  Image your duplex from PCR is made to have two identical restriction sites, one each at one end of the DNA  You can digest it with a restriction enzyme (EcoRI in this case)  Then purify it by gel electrophoresis DNA Separation by Electrophoresis  DNA molecules can be separated by gel electrophoresis o The gel – agarose or polyacrylamide – is macromolecular mesh  When a mixture of DNA is added to wells and voltage is applied, a large DNA fragment migrates slower than a small one  Agarose Gel of Restriction Digests o DNA fragments are visualized by staining with chromophores (such as ethidium bromide) that bind DNA o Usually a mixture of DNA molecules of known sizes are used as the markers o We can also cut bands out of these gels to purify them 4 Biochem 2B03 Jasmyn Lee Making a Recombinant Plasmid  Digest both the plasmid and your DNA with a restriction enzyme and purify them  Mix them together  Add DNA ligase o Covalently links adjacent 3’-OH and 54PO groups  Incubate at lower temperature  In this example, a single restriction enzyme is used – insert can be ligated into the vector in two orientations  Vector can also be religated 5 Biochem 2B03 Jasmyn Lee Directional Cloning  DNA must be placed downstream of a promoter to insert a particular DNA (gene) in a vector so that its gene product is synthesized o Promoter – nucleotide sequence lying upstream of a gene; controls expression of a gene o RNAP molecules bind specifically at promoters and initiate transcription of adjacent genes, copying template DNA into RNA products  Use different restriction sites at the two ends of your gene – creates different overhang/sticky ends  To insert foreign DNA in a particular orientation  This can be done by making two cleavages with two different restriction enzymes  Construct foreign DNA with same two restriction enzymes  Foreign DNA can only be inserted in one direction Transformation  Transformation – means for introducing DNA (usually plasmids) into E. coli cells  Treat cells with 0.1 M C2Cl – renders them permeable to DNA or “competent”  Mix concentrated competent cells with ligation mixture from cloning experiment 6 Biochem 2B03 Jasmyn Lee  Heat shock at 42°C  Culture at 37°, 1 hour – plate on “selective medium”  Screening for recombinant plasmids o Replica plating o Blue/white screening o Colony PCR o E-lyse – in-well lysis procedure (not common)  Initial selection is on ampicillin: pBR322 and the chimera-containing cells both grow  Secondary test – plate a “replica” on tetracycline: pBR322 – but not chimera-containing cells grow  Colonies containing the plasmid you want (i.e. containing your cloned gene) grow on ampicillin but not on tetracycline 1. Cleavage of pBR322 with BamH1 2. Annealing and ligation of inserts generated by BamH1 cleavage of some foreign DNA 3. Creates a chimeric plasmid 4. The chimeric plasmid is then used to transform Ca2 treated heat-shocked E. coli cells and the bacterial sample is plated on a petri plate 5. Following incubation of the petri plate overnight at 37°C 6. Colonies of amp bacteria are evident 7. Replica plating of these bacteria on plates of tetracycline- con
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