DNA Technologies Lecture 2 (PDF)

DNA TECHNOLOGIES @JordiPaps  Genetic engineering Set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries  Genetic engineering Research: loss of function experiments (gene knockout), gene gain of function, Green Fluoresce...

DNA TECHNOLOGIES @JordiPaps  Genetic engineering Set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries  Genetic engineering Research: loss of function experiments (gene knockout), gene gain of function, Green Fluorescent Protein (from jellyfish)  Genetic engineering Medicine: mass production of insulin inserted in bacteria  Genetic engineering Industry: making biofuels, cleaning up oil spills, carbon and other toxic waste  Genetic engineering Agriculture: pest resistant crops (less pesticides), Golden Rice (vitamin A), cows and goats have been engineered to express drugs or even silk!  Genetic engineering Cloning Biology research Biomedicine Gene function Production of Promoter analysis pharmaceuticals Cell signalling and Diagnosis environmental Gene therapy responses Development and evolution Protein purification Forensics Agriculture DNA fingerprinting Veterinary diagnosis Paternity testing Marker assisted selection Precision crop enhancement Cleanup and bioremediation Recommended Reading Campbell et al (2015) Biology A Global Approach 10th Edition Chapter 19 DNA Technology p448-475 * Take a look at p451 about next generation sequencing technologies! 1. PCR 2. GENE CLONING 3. ELECTROPHORESIS GELS Typical gene cloning strategy Isolate gene / Manipulate it Analyze its promoter from in vitro function organism Isolate gene / Manipulate it Analyze its promoter from in vitro function organism PCR Polymerase Chain Reaction We need multiple copies of a gene to be able to study it PCR is a simple technique to make many copies of a specific piece of DNA It can be used to isolate a gene from the genome of an organism PCR was invented by Kary Mullis in 1983 1993 Nobel Prize for Chemistry "Back in the 1960s and early '70s I took plenty of LSD. A lot of people were doing that in Berkeley back then. And I found it to be a mind-opening experience. It was certainly much more important than any courses I ever took." And in 1997, he told the BBC, "What if I had not taken LSD ever; would I have still invented PCR? I don't know. I PCR was invented by Kary Mullis in 1983 1993 Nobel Prize for Chemistry Mullis reported an encounter with a extraterrestrial fluorescent raccoon at his cabin in California. He denied being on psychedelic drugs at the time. He denied climate change and ozone depletion, that HIV causes AIDS, and believed in astrology Polymerase Chain Reaction Ingredients: Template DNA Primers flanking ends of DNA section that will be amplified and isolated Free nucleotides (A, G, C, T) to build DNA DNA polymerase (classically from Thermus aquaticus, “Taq polymerase”) Polymerase Chain Reaction DNA template to be amplified (e.g. DNA isolated from animal) Denaturalisation: Heat DNA to 95 °C for 1 minute to unzip the double strands Annealing: cool to ~60 °C to allow short ‘primers’ to bind to DNA Extension: Heat to 72 °C, the optimum temperature for a DNA polymerase (Campbell p455)  Polymerase Chain Reaction https://www.youtube.com/watch?v=MyLrs_h1  Universal Tree of Life (ssu rDNA) Two of the three domains populated exclusively by prokaryotes In the third domain (Eukarya) multicellular life is largely restricted to three recent branches (fungi, plants, animals) DNA barcoding PCR song https://youtu.be/mvvP90Cpdfc  GENE CLONING Isolate gene / Manipulate it Analyze its promoter from in vitro function organism Gene cloning overview  Escherichia coli is an important tool for gene cloning Plasmids are small circular pieces of DNA found in bacteria We can insert genes in plasmids, and then transform plasmids in bacteria Then, transformed bacteria divide making copies of the plasmid inside them Used before PCR, but still a main lab technique in genetics Plasmid = Cloning Vector We will insert our gene of interest in the plasmid using restriction enzymes We need to check that gene was inserted in the plasmid Then we will transform the plasmid (introduced it within a bacterial cell) We need to check that plasmis was introduced in the bacteria  Restriction Enzymes Restriction enzymes (or restriction endonucleases) recognize specific short sequences of DNA Then they cleave the DNA at or near that site Recognition sites are usually 4-8 bp long and palindromic – same on both strands  Palindromes Word which reads the same backward as forward  Restriction Enzymes In nature, these enzymes protect bacterial cells against invasion by viral DNA Bacterial DNA is protected by addition of methyl groups  Restriction Enzymes Each enzyme is named after its organism of origin – EcoRI is from Escherichia coli – HindIII is from Haemophilus influenzae BamHI Abbreviation Meaning Description B Bacillus Genus am amyloliquefaciens Species H H Strain I 1st Order of Identification Examples: EcoRI and HindIII EcoRI HindIII 5’ TGCGAATTCCGA 3’ 5’ TGTAAGCTTGCA 3’ 3’ ACGCTTAAGGCT 5’ 3’ ACATTCGAACGT 5’ 5’ TGCG AATTCCGA 3’ 5’ TGTA AGCTTGCA 3’ 3’ ACGCTTAA GGCT 5’ 3’ ACATTCGA ACGT 5’ Hundreds of commercially-available restriction enzymes for gene cloning All these enzymes recognize and cut specific but different DNA sequences Researchers select and order right enzyme for the job  Bacterial plasmid Restriction site 5′ 3′ G A AT T C DNA C T TA A G 3′ 5′ 1 Restriction enzyme digests (cuts) the sugar-phosphate backbones at each arrow. 3′ 5′ 5′ A AT T C 3′ G C T TA A G 5′ 3′ 3′ 5′ Sticky end  3′ 5′ 5′ A AT T C 3′ G C T TA A G 5′ 3′ 3′ 5′ Sticky end 5′ A AT T C 3′ 2a Base pairing of sticky G G C T TA A ends produces various 3′ 5′ combinations. Fragment from different DNA molecule cut by the same restriction enzyme 5′ 3′ 5′ 3′ 5′ 3′ G A AT T C G A AT T C C T TA A G C T TA A G 3′ 5′ 3′ 5′ 3′ 5′  5′ 3′ 5′ 3′ 5′ 3′ G A AT T C G A AT T C C T TA A G C T TA A G 3′ 5′ 3′ 5′ 3′ 5′ 2b DNA ligase seals the strands 5′ 3′ 3′ Recombinant DNA molecule 5′ Recombinant plasmid Features of typical gene cloning vector “Multiple cloning site” Origin of replication Transformation: introducing the plasmid in the bacteria Plus salts (e.g. CaCl2)  Two checks for gene cloning We need to check that plasmid was transformed in the bacteria Plasmid contains antibiotic resistance gene Bacteria are grown in a medium with antibiotic Only bacteria with the plasmid can grow, the bacteria with no plasmid die because of the antibiotic We need to check that our gene was inserted in the plasmid Plasmid cloning site is within a protein coding gene (lacZ) This gene produces pigmented colonies (e.g. blue) If no gene of interest is inserted, color gene does work, then colonies have colour (e,g, are blue). If our gene of interest is inserted, the colour gene is broken, then bacteria are white  Features of typical gene cloning vector Antibiotic resistance “Multiple gene with bacterial cloning promoter site” in e.g. b- colour lactamase gene Origin of replication Two checks for gene cloning 1+2 3 4  Cut and paste with restriction enzymes For example- using GFP to GFP study location of a protein in a cell A gene In the lab, the GFP encoding a gene is linked to the protein under gene encoding the investigation protein (isolated with (using restriction PCR) enzymes)  GEL ELECTROPHOR ESIS  Separation and visualization of DNA by gel electrophoresis Nucleic acids (DNA and RNA) are acid (they have a negative charge) Thus, these molecules are attracted to a positive charge  Agarose gels Agarose is a polysaccharide from red algae Liquid at high temperature, solid at room temperature Casting the gel Gel in the tank (with electrodes!!!) Loading the gel Separation and visualization of DNA by gel electrophoresis DNA for analysis is pipetted DNA migrates through the into small holes in the gel gel in response to an (called ‘wells’) electrical field The pores in the gel act as a sieve, A gel of an aequeous polymer so smaller DNA molecules travel (usually agarose) faster and further Agarose matrix  DNA in the gel is stained with a DNA intercalating dye Gel viewed from above under UV light Ethidium bromide is a large flat molecule It can bind to DNA It is fluorescent (visible under UV light) Larger DNA Smaller DNA  DNA Sequencing: PCR + electrophoresis Special dNTPs called terminators (they stop PCR chain extension)  Cutting (restriction enzymes) Checking Joining (gels and (ligation) sequencing) Screening Cloning (antibiotics) (in E. coli)