Presentation on theme: "Recombinant DNA Technology"— Presentation transcript:
1 Recombinant DNA Technology ?Can you give me some examples of what chemicals you think youve used, or how you think chemistry may have impacted your life?
2 Why Do Genetic Engineering? 1. Produce desired proteins in vitro for therapeutic use.2. Have rice produce as much starch as a kernel of corn (in vivo production).3. Gene therapy
3 Steps in Genetic Engineering 1. Isolation of gene of interest2. Isolation of plasmid DNA3. Manipulation of DNA sequencea. Cutting- Restriction enzymesb. Splicing- DNA ligase4. Transformation of bacteria5. Selection of “correct” bacteria
5 Prokaryote Disadvantages 1. Can’t splice out introns2. Introns are needed for good expression3. Size of DNA that can be put into bacteria is limited4. Prokaryotes don’t glycosylate proteins
6 PlasmidsPlasmid- small, circular, extrachromosomal DNA which replicates independently of host chromosomal DNA
7 Isolation of Plasmid DNA ?Can you give me some examples of what chemicals you think youve used, or how you think chemistry may have impacted your life?
8 Plasmid map Ori antibiotic resistance gene(s) restriction sites Figure: Harpers Review of Biochemistry
9 Manipulation of DNA Sequence ?Can you give me some examples of what chemicals you think youve used, or how you think chemistry may have impacted your life?
10 Restriction enzymesRestriction enzyme- an enzyme which cuts specific DNA sequences, endonuclease“blunt end” vs. “sticky end”Cleavage is restricted to specific, 4-6 bp sequences (foreign bacteria); always palindromic sequenceMore than 800 are now known
11 Restriction Endonucleases Type I- multisubunit, endonuclease and methylase activities, cleave at random up to 1000 bp from recognition sequenceType II- cleave DNA within recognition sequence, require no ATP, most monomersType III- multisubunit, endonuclease and methylase about 25 bp from recognition sequence
12 Restriction Endonucleases EcoRI: E. coli strain R 1st enzyme foundGAATTC G AATTCCTTAAG CTTAA GHpaI GTTAAC GTT AACCAATTG CAA TTG
13 Generating a Plasmid map restriction sitessizes when insert included
14 Cloning Vectors 1. Plasmids- 5,000 to 400,000 bp useful for putting kb in2. Bacteriophages-virus that infects bacteriauseful for putting kb in3. Cosmids- artificially generateduseful for putting kb in4. YACs- yeast artificial chromosomesnew, 500 kb
15 Transformation of Bacteria ?Can you give me some examples of what chemicals you think youve used, or how you think chemistry may have impacted your life?
16 CaPO4 TransformationCells and DNA incubated together in CaCl2 at 0oC, then heat shock at 37oCHow this makes cells “competent” to take up DNA is not knownOnly a small percent of cells take up DNA- must select for them
17 Newer Methods of Transformation Lipofectin® and similar moleculesElectroporationMicroinjection
18 Last Time Restriction Enzymes Plasmid Maps Other Vectors Transformation
19 Selection of “Correct” Bacteria ?Can you give me some examples of what chemicals you think youve used, or how you think chemistry may have impacted your life?
20 Antibiotic Resistance Genes are a Part of Many (Constructed) Plasmids
21 Blue-White Screening Promega Corp; Madison, WI pGEM-3Z, e.g.AmprlacZpolycloning site in lacZ geneT7 promoter one side, SP6 other
22 DNA SequencingSanger’s firstRadiolabeled vs. fluorescent tag
23 Isolation of Gene of Interest ?Can you give me some examples of what chemicals you think youve used, or how you think chemistry may have impacted your life?
24 Isolation of Gene of Interest Use of Antibodies Ab ppt proteinProt being synth on mRNAGenerate cDNAreverse transcriptaseDNA polymeraseMust have protein in ~pure form
25 Isolation of Gene of Interest Genomic Library Screening Isolation of total genomeFragments and their sizesHow many fragments to get entire genome can be calculatedFragments put into a vectorVectors are hybridized with a probeDon’t need protein, but must know at least part of sequence
26 HybridizationBacterial colonies containing plasmid library are grown up“Paper” is used to pick up cells of each colonyPaper is incubated in radiolabeled probe and washedAutorad of paper Ids colonies containing gene of interestFigure: Stryer, Biochemistry
27 Isolation of Gene of Interest Polymerase Chain Reaction (PCR) Taq polymeraseEquipment- thermocyclerProcedure- Taq + template + primerDon’t need protein, but must know at least part of sequenceThe real power here is ability to amplify DNA