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Pan, Cerberus, and the Centaurs: Genetic Engineering Donna C. Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center Fun Things To Do With DNA Spool it onto a glass rod (Isolation) Put it in a necklace (Precipitation) Pull it apart (Denature) and put it back together (Anneal) Cut it up (Restriction Enzymes) and look at it (Gel electrophoresis) Map it (Southern Blots) Read it (Sequencing) Copy it millions of times (Cloning, Polymerase Chain Reaction) Move it from one place to another (Genetic Engineering) Diversity of Genetic Material Prokaryotic DNA Eukaryotic DNA Viral DNA or RNA Plasmid DNA Isolating Nucleic Acids for Molecular Analysis Cell Lysis Enzymatic or Chemical Extraction General DNA Isolation Procedures Lyse Cells • Detergent dissolves cell membrane and denatures protein. • EDTA chelates divalent cations required by nucleases. • Proteinase K degrades proteins. Organic Extraction Nonorganic Extraction Solid Phase Extraction • Uses phenol:chloroform:isoamyl alcohol • Denatures and removes proteins •Toxic Chemicals!! •Salt precipitation of protein • DNA precipitation •Apply sample •Wash •Elute DNA •Best method! (1 X 107 cells) X (6 pg DNA/cell) X 80% yield= 48 mg DNA!!! Looking at DNA: Nucleic Acid Analysis DNA (or RNA) is characterized using several different methods for assessing quantity, quality, and molecular size. UV spectrophotometry Agarose gel electrophoresis Colorimetric blotting How Much DNA Do You Have? Absorbance from UV Spectrophotometry DNA and RNA absorb maximally at a wavelength of 260 nm. Proteins absorb at 280 nm. Background scatter absorbs at 320 nm. Concentration of DNA = (A260 – A320) X dilution factor X 50 µg/mL Concentration of RNA = (A260-A320) X dilution factor X 40 µg/mL How Does Your DNA Look? Quality from Agarose Gel Electrophoresis Human Whole Blood DNA Lambda DNA marker Whole blood genomic DNA Lambda DNA cut with Hind III marker Pulling DNA Apart And Putting It Back Together: Denaturation and Annealing Reactions DNA Likes To Find Its Perfect Match: Denaturation and Annealing of DNA Hybridization Will Occur In Liquid Or On A Solid Surface Melting Temperature (Tm), Salt and G + C Content Basic Techniques for Analysis of Nucleic Acids Endonuclease digestion (DNAse, RNase, restriction enzymes) Electrophoresis (agarose and polyacrylamide gel electrophoresis) Enzymatic modification (polymerase, kinase, phosphatase, ligase) Cutting, Chewing, Tagging DNA: Nucleic Acid Modifying Enzymes Restriction endonucleases DNA polymerases (synthesize DNA) DNA ligases (join DNA strands) Kinases (phosphorylation of 5´ends of DNA) Phosphatases (dephosphorylate 5´ends of DNA) Ribonucleases (digest RNA molecule. Example: RNase A) Deoxyribonucleases (digest DNA molecules) Restriction Endonucleases (RE) Found only in microorganisms Exhibit novel DNA sequence specificities >2000 distinct restriction enzymes have been identified Recognize symmetrical dsDNA (palindromes) Utilized in the digestion of DNA molecules Nomenclature: EcoRI First letter of Genus + first 2 letters of species + order of enzyme discovery E co RI Restriction Enzymes Recognize Palindromes Palindrome reads the same in both directions BOB “Able was I ere I saw Elba.” (Napoleon Bonapart, following his exile from the European continent to the island of Elba) Sequences directly opposite one another on opposite strands of the ds DNA molecule MICROORGANISM ENZYME SEQUENCE NOTES HaeIII 5’GGCC3’ 3’CCGG5’ 1 Thermus aquaticus TaqI 5’TCGA3’ 3’AGCT5’ 2 Haemophilus haemolyticus HhaI 5’GCGC3’ 3’CGCG5’ 3 Desulfovibrio desulfuricans DdeI 5’CTNAG3’ 3’GANTC5’ 2,4 Moraxella bovis MboII 5’GAAGA(N)83’ 3’CTTCT(N)75’ 4,5 Escherichia coli EcoRV 5’GATATC3’ 3’CTATAG5’ 5’GAATTC3’ 3’CTTAAG5’ 1 2 PstI 5’CTGCAG3’ 3’GACGTC5’ 3 Microcoleus MstII 5’CCTNAGG3’ 3’GGANTCC5’ 2,4 Nocardia ototidis caviarum NotI 5’GCGGCCGC3’ 3’CGCCGGCG5’ 2,6 Haemophilus aegyptius EcoRI Providencia stuartii 1. 2. 3. Enzyme produces blunt ends.. Single strand is 5’ strand. Single strand is 3’ strand. 4. 5. 6. N= any purine or pyrimidine pair. Enzyme cuts 8 nts 3’ of recognition site. Cuts mammalian DNA very rarely. Looking At DNA: Electrophoresis Nucleic acids are separated based on size and charge. DNA molecules migrate in an electrical field Employs a sieve-like matrix (THINK JELLO!) and an electrical field. DNA is negatively charged and migrates towards the positively charged anode. Gel Electrophoresis Electrophoresis is the movement of molecules by an electric current. Nucleic acid moves from a negative to a positive pole. Nucleic acid has a net negative charge, they RUN TO RED Principles of Gel Electrophoresis The gel itself is composed of either agarose or polyacrylamide Agarose is a polysaccharide extracted from seaweed Polyacrylamide is a cross-linked polymer of acrylamide. Acrylamide is a potent neurotoxin and should be handled with care! “Submarine” Agarose Gel Electrophoresis Agarose Gel Apparatus Comparison Of Various Agarose Concentrations Electrophoresis Of Lambda DNA Digested Using Three Different RE 1 2 3 4 Lane 1 contains uncut lambda DNA. Lane 2 contains lambda DNA digested by PstI. Lane 3 contains lambda DNA digested by EcoRI. Lane 4 contains lambda DNA digested by HindIII. Restriction Enzyme Mapping Digest DNA with a restriction enzyme. Resolve the fragments by gel electrophoresis. The number of bands indicates the number of restriction sites. The size of the bands indicates the distance between restriction sites. Restriction Enzyme Mapping: Circular DNA Molecules BamH1 XhoI BamH1 XhoI BamH1 4.3 kb 3.7 kb 2.8 kb 4.0 kb 2.3 kb 1.9 kb 1.4 kb 1.3 kb 1.1 kb 2.8 kb 1.7 kb 1.7 kb XhoI 1.2 kb 1.2 kb 1.2 kb 1.1 kb 0.7 kb XhoI Mapping DNA: Southern Blots DNA immobilized on solid support Detect specific DNA fragments with a DNA probe using hybridization Ok, what the #&*^!!! is a probe? It’s a usually a clone or amplified DNA—we’ll get there in a minute. Southern Blot Hybridization: Overview Southern Blot Analysis of EHV-3 DNA Restriction Enzyme Map of EHV-3 DNA BamHI BclI BglII EcoRI Hind III CLONE: THE NOUN AND THE VERB To clone produce multiple identical copies of something A clone identical copy, derived from single progenitor may be DNA molecules, cells, or an organism Molecular Cloning Genetic engineering includes techniques that allow for the construction of novel DNA molecules by joining DNA sequences from different sources. Recombinant DNA Vector Clone Cloning Plasmid Replicator (ori) Selectable marker Cloning site Cloning into Plasmid Vectors Cut plasmid, target DNA with RE Treat plasmid DNA with alkaline phosphatase Mix plasmid and target DNAs to allow annealing Add DNA ligase Transfection And Transformation: Putting Genes Back Into Cells Calcium phosphate/chloride precipitation Aggregates of DNA precipitate and are endocytosed DEAE dextran Anion binding gel that aggregates DNA Biolistics DNA coated onto gold microprojectiles Electroporation High voltage shock that makes transient DNA permeable holes in cell membranes Transform bacterial cells Treat with CaCl2 Add media with antibiotic, incubate Streak on selective media plate High copy number plasmids give the best yield Efficiency Of Transformation 10% of cells treated take up DNA 1% of cells become stable transformants Most transfections are transient and must be forced to maintain the foreign DNA by selection pressure Antibiotic selection Color selection galactosidase gene (Lac Z) Green fluorescent protein (GFP) Selection Of Clones Containing Inserts Intact lac Z gene beta gal= blue colonies Interrupted lac Z gene No beta gal= white colonies Bacteriophage Vectors Most are derived from lambda phage Charon 16A vectors were named after the ferryman of Greek mythology who conveyed the spirits of the dead across the River Styx Yeast Vectors Accommodate large inserts Extremely stable Integrate into yeast chromosome Mini-chromosomes Artificial chromosome Eukaryotic system Post-translational modifications similar to mammalian systems Remember Eukaryotic Genes Contain Introns: Reverse Transcription of RNA to cDNA What about primers? Remember poly A tail? Primers are long string of Ts! What Are Cloned Genes Good For Anyway? Provide large quantities of DNA for analysis Mapping, sequencing studies Identification, disease diagnosis Provide source of specific gene product for commercial use Production of medically important molecules Provide source of specific genes for creation of transgenic animals IS THERE A BETTER WAY TO DO SOME OF THIS? PCR and Cary Mullis Cary Mullis and the Nobel Prize: The Basics Knew that you could expose template DNA by boiling ds DNA to produce ss DNA Knew that you could use primers to initiate DNA synthesis Knew that a cheap, commercial enzyme was available (Klenow fragment of E. coli DNA polymerase) Cary Mullis and PCR Wanted a way to generate large amounts of DNA from a single copy Initially used the “3 graduate student” method Denaturing (unwinding) DNA Annealing (hybridizing) primers Extending (copying) DNA THREE STEPS OF PCR Denaturation of target (template) Usually 95oC Annealing of primers Temperature of annealing is dependent on the G+C content May be high (no mismatch allowed) or low (allows some mismatch) “stringency” Extension (synthesis) of new strand Automation of PCR PCR requires repeated temperature changes. The thermal cycler changes temperatures in a block or chamber holding the samples. Thermostable (heat stable) polymerases are used to withstand the repeated high denaturation temperatures. Thermostable DNA Polymerase: Yellowstone National Park And Deep Sea Vents Thermostable Polymerases Polymerase Taq pol T ½, 95oC 40 min Extension Type of Rate (nt/sec) ends 75 3’A Source T. aquaticus Amplitaq (Stoffel fragment) Vent 80 min >50 3’A T. aquaticus 400 min >80 Deep Vent 1380 min ? Pfu >120 min 60 95% blunt 95% blunt Blunt Tth 20 min >33 3’A Thermococcus litoralis Pyrococcus GB-D Pyrococcus furiosus T. thermophilus Taq: Thermus aquaticus (most commonly used) PCR Cycle: Temperatures Denaturation temperature Annealing temperature Reduce double stranded molecules to single stranded molecules Controls specificity of hybridization Extension temperature Optimized for individual polymerases Combinations Of Cycle Temperatures TEMP 94-60-72 94-55-72 94-50-72 94-48-68 94-45-65 94-37-65 FOR COMMENTS Perfect, long primers Good or perfectly matched primers between 19-24 nt Adequate primers Higher temp can be used; maximum annealling temp Standard conditions Poorly matched primers Unknown match, likely poor Hail Mary Allows 4-5 mismatches/20 nt Allows 1-3 mismatches/20 nt Primers of questionable quality, long-shot PCR Uncontrolled results REAL TIME PCR Detects PCR products as they accumulate Detect ds DNA by two methods: Intercalator fluorescent markers (ethidium bromide, syber green dye): non specific Fluorogenic probes: specific Plot increase in fluorescence versus cycle number GEL ANALYSIS VS FLUORESCENCE DNA Detection: SYBR Green I Dye DENATURATION STEP: DNA + PRIMERS + DYE WEAK BACKGROUND FLUORESCENCE ANEALING STEP:DYE BINDS dsDNA, EMITS LIGHT EXTENSION STEP: MEASURE LIGHT EMMISSION Cycle Threshold (Ct) Cycle Threshold Threshold line (fluorescent units) Construction of Standard Curve Threshold fluorescence level Threshold cycles for each sample Real-Time PCR Labeled Probes Cleavage-based probes Molecular beacons TaqMan Assay Fluorescent reporter at 5’ end and a quencher at 3’ end Hairpin loop structure Fluorescent reporter at 5’ end and a quencher at 3’ end FRET probes Fluorescence resonance energy transfer probes What is DNA Sequencing? DNA sequencing is the ability to determine nucleotide sequences of DNA molecules. DNA Sequencing Methods Technology Chain termination Cycle sequencing Chemistry Maxam and Gilbert Sanger Platform Manual Automated Sanger (Dideoxy) DNA Sequencing Incorporation of 2´,3´-dideoxynucleotides by DNA polymerase Termination of elongation reaction Fragment size analysis (manual vs. automated) 5 CH OH OH 2 Gel Capillary O 4 C1 C H H 3C C 2,3-dideoxyribose H H 2 Dideoxy or Sanger DNA Sequencing A T G C A ATTA ATTAGA AT ATT ATTAGACGT ATTAG ATTAGACG ATTAGAC A T ATTAGACGT G C Sequencing Gels Cycle Sequencing Cycle sequencing is chain termination sequencing performed in a thermal cycler. Cycle sequencing requires a heat-stable DNA polymerase. Fluorescent Dyes Fluorescent dyes are multi-cyclic molecules that absorb and emit fluorescent light at specific wavelengths. Examples are fluorescein and rhodamine derivatives. For sequencing applications, these molecules can be covalently attached to nucleotides. Dye Terminator Sequencing A distinct dye or “color” is used for each of the four ddNTP. Since the terminating nucleotides can be distinguished by color, all four reactions can be performed in a single tube. A T G T AC GT The fragments are distinguished by size and “color.” Dye Terminator Sequencing The DNA ladder is resolved in one gel lane or in a capillary. G A T GA TC C G T C T G A Slab gel Capillary Dye Terminator Sequencing The DNA ladder is read on an electropherogram. Slab gel Capillary Electropherogram 5′ AGTCTG Automated Sequencing Dye primer or dye terminator sequencing on capillary instruments. Sequence analysis software provides analyzed sequence in text and electropherogram form. Peak patterns reflect mutations or sequence changes. T/T 5′ AGTCTG T/A 5′ AG(T/A)CTG A/A 5′ AGACTG APPLICATIONS OF GENETIC ENGINEERING in Produce Transgenics Medicine May be includes Plant Genetic screening Gene mapping Gene therapy Raise Legal and ethical questions Forensic medicine To affect Yield Disease resistant Herbicide resistance Drought, salt and cold tolerance Storage, appearance Raise or Animal to Increase yield For? Organ donors and Production therapeutic proteins