Analyzing and Engineering Genes - Biology - Lecture Slides, Slides of Biology

Download Analyzing and Engineering Genes - Biology - Lecture Slides and more Slides Biology in PDF only on Docsity! Chapter 19 ANALYZING AND ENGINEERING GENES Manipulation of genetic material docsity.com Summary Tools for genetic engineering Host/vector systems and DNA libraries Genetic engineering experiment Identifying genes in organisms Analyzing DNA Biotechnology and you docsity.com What is a Palindrome? Hint: What do all these have in common? Racecar Hannah Civic Kayak Level Rotator docsity.com Molecular Biologists’ Tools Restriction endonucleases cont’d 2 types: type I produces blunt end cuts, type II produces staggered cuts with “sticky ends”. Restriction sites have certain base sequences called palindromes, that are recognized by specific endonucleases. DNA Ligase – also borrowed from nature joins the ends of cut strands. docsity.com GAATTC CTTAAG GAATTC AATTC AATTC AATTC GAATTC G G G G G CTTAAG CTTAA CTTAA CTTAAG DNA ligase joins the strands. DNA from another source cut with the same restriction endonuclease is added. Restriction endonuclease cleaves the DNA. DNA duplex Sticky ends (complementary single-stranded DNA tails) Restriction sites Recombinant DNA molecule Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. docsity.com Host/Vector Systems Vectors - Plasmids Small circular pieces of DNA into which foreign DNA (10kb) can be inserted. “Engineered” plasmids usually contain a gene for antibiotic resistance and one for a metabolic enzyme. Plasmids move in and out of the host’s DNA (bacteria) and are replicated as the host reproduces. docsity.com Host/Vector Systems Vectors - Plasmids Why do we want to have a gene for antibiotic resistance? Hint: Artificial selection. docsity.com Host/Vector Systems Vectors - Phages Derived from virus DNA, can accept larger pieces of DNA (40 kb). After “infection” the DNA becomes part of the host’s DNA and is replicated. Other vectors are used to infect animal or yeast cells. docsity.com Genetic Engineering Experiment One of the 1st attempts was to produce a “safe” growth hormone to treat pituitary dwarfism. Procedure, fig 19.1: Isolate mRNA from pituitary cells and use reverse transcriptase to produce a piece of cDNA (c=complementary). Attach restriction sites to both ends of cDNA. docsity.com Genetic Engineering Experiment Procedure cont’d Cut cDNAs and plasmids with a restriction endonuclease and insert cDNAs into plasmids (DNA ligase). Insert plasmids into bacteria. Screen bacteria containing plasmids (& cDNA) by growing in the presence of antibiotic. Those that grow have antibiotic resistance carried by the plasmid and form the cDNA library. docsity.com CREATING A cDNA LIBRARY THAT CONTAINS THE HUMAN GROWTH HORMONE GENE Reverse {I transcriptase 5’ 37 SAATICD PDP Ud sears CTTAAG CTTAAG 3’ fl 5! TIC G AATTELOUIU Era, G AATTC DOCTTAA GPR Plasmid er Antibiotic-resistance gene Cy Figure 19-1 part 1 Biological Science, 2/e 1. Isolate mRNAs from cells in pituitary gland. 2. Use reverse transcriptase to synthesize a cDNA from each mRNA. 3. Attach a restriction endonuclease recognition site to ends of each cDNA. 4. Cut cDNAs and plasmids with restriction endonucleases; remaining sticky ends join by com- plementary base pairing. © 2005 Pearson Prentice Hall, Inc. docsity.com Genetic Engineering Experiment Screening of the library - Finding clones with cDNA insert cDNA is inserted within gene sequence for a metabolic enzyme. If bacteria DO NOT have a functional metabolic enzyme they have a cDNA insert. docsity.com Genetic Engineering Experiment Screening cont’d - Finding the “clones” with growth hormone cDNA: Transfer clones to filter paper. Prepare a DNA “probe” with growth hormone sequence and a radioactive marker (nucleotide). Treat filter paper with probe which will “stick” or hybridize to growth hormone cDNA. Lay photographic film over filter paper. Clones with radioactive probe will cause spots to appear on film. docsity.com FINDING THE GROWTH HORMONE GENE INA cDNA LIBRARY - 9 — <0 a ©& eae ss wy Figure 19-4 part 1 Biological Science, 2/e 1. Grow E. coli cells containing plasmids on many plates. Each colony contains a different cDNA. 2. Lay a filter on each plate, then remove. Some cells from each colony stick to filters. 3. Treat filters with chemical to make DNAs single stranded. © 2005 Pearson Prentice Hall, Inc. docsity.com 1. Electrophoresis is performed, using radioactively labeled markers as a size guide in the first lane. 3. Pattern on gel is copied faithfully, or “blotted,” onto the nitrocellulose. 4. Blotted nitrocellulose is incubated with radioactively labeled nucleic acids, and then rinsed. 5. Photographic film is laid over the paper and is exposed only in areas that contain radioactivity (autoradiography). Nitrocellulose is examined for radioactive bands, indicating hybridization of the original nucleic acids with the radioactively labeled ones. 2. The gel is covered with a sheet of nitrocellulose and placed in a tray of buffer on top of a sponge. Alkaline chemicals in the buffer denature the DNA into single strands. The buffer wicks its way up through the gel and nitrocellulose into a stack of paper towels placed on top of the nitrocellulose. Test nucleic acids Radioactively labeled markers with specific sizes Electrophoretic gel Nitrocellulose paper now contains nucleic acid "print" Sealed container Size markers Hybridized nucleic acids Film Radioactively labeled nucleic acids Gel Buffer Sponge Stack of paper towels Nitrocellulose paper GelE le c tro p h o re s is Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. docsity.com Identifying Genes in Organisms PCR – Polymerase Chain Reaction Use: produce large amounts of DNA from a small sample. Basis: DNA replication in a test tube. Procedure: Isolate DNA or make cDNA from RNA Add a solution with DNA polymerase, primers and deoxynucleotides to produce copies of DNA. docsity.com POLYMERASE CHAIN REACTION 3’ a5’ > | | I 4 IE &- part1 Biological Science, 2/e 1. Start with a solution containing template DNA, synthesized primers, and an abundant supply of the four dNTPs. 2. Denaturation Heating leads to denaturation of the double-stranded DNA. © 2005 Pearson Prentice Hall, Inc. docsity.com Analyzing DNA DNA sequencing Use: identify gene base sequence from normal organisms and mutations that cause disease. Sequencing Procedure: Isolate specific gene (DNA) Prepare 4 PCR solutions with labeled probes and deoxynucleotides (dNTPs) + 1 dideoxynucleotide (ddNTPs, have no OH group on 5’ end). docsity.com ddNTPs terminate DNA synthesis. e e P P e oy 5'CH2 Base 5'CH2 Base 3’ 3 OH H~<s~— No OH Normal dNTP ddNTP (extends DNA strand) (terminates synthesis) e Biological Science, 2/e © 2005 Pearson Prentice Hall, Inc. 3 docsity.com Analyzing DNA Sequencing Procedure cont’d When a ddNTP is incorporated in new DNA strands, DNA synthesis stops. Separate DNA fragments on a polyacrylamide gel. Transfer bands to filter paper and expose to photographic film. Band patterns indicate base sequence of gene. docsity.com Analyzing DNA RFLP = restriction fragment length polymorphism. Uses: forensic analysis, identifying patients with genetic alterations that may cause disease. Principle: DNA fragments of identical genes from different individuals will not be exactly the same size (point mutations, multiple copies, etc.). docsity.com Restriction endonucleases produce DNA fragments of various lengths (1 kb = 1000 bases). Section of chromosome 2.5 kb 15 kb LP LY 84kb a MA? } Sites where restriction endonuclease cuts DNA * Polymorphic sites: some { individuals have this site, others don’t Figure 19-10a Biological Science, 2/e © 2005 Pearson Prentice Hall, Inc. ® docsity.com Analyzing DNA RFLP Procedure: Cut DNA with several restriction endonucleases and separate pieces using gel electrophoresis; compare band patterns. Band pattern (distribution of fragments by size) acts as a DNA fingerprint. docsity.com oe " 20 A < = vil AC BC BC s > a > ao > an > 2@; > a > an > > A @ 2 > > ao nO Figure 19-8 Biological Science, 2/e ew ev O oO Oo C AB AB AB AB BC AB AB BC/BB BC AC A BO Ol AA BC}; CD BC BC Ge RB Og BC BC a SY cc Almost all people who inherit the C pattern (——, see Figure 19.10) also inherit Huntington's disease (indicated in purple) © 2005 Pearson Prentice Hall, Inc. @ docsity.com Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. _ Victim Rapist’s semen Suspect’s blood ‘ : ‘ Victim es Rapist’s semen Suspect’s blood docsity.com WIIFM – How does biotechnology improve our lives? Medical Applications Agriculture Genetic Testing of Humans Bio-Warfare and Terrorism Bio-Ethics docsity.com Biotechnology and you Agricultural Disease resistance to crops and animals Herbicide and insect resistance to plants Delay rotting of fruits and vegetables Introduce N-fixation ability to plants Some plants naturally can do this docsity.com Biotechnology and you Agricultural Disease resistance to crops and animals Herbicide and insect resistance to plants Delay rotting of fruits and vegetables Introduce N-fixation ability to plants Genetic engineering of “super” plants and animals. Risks????? docsity.com Biotechnology and you Agricultural Disease resistance to crops and animals Herbicide and insect resistance to plants Delay rotting of fruits and vegetables Introduce N-fixation ability to plants Genetic engineering of “super” plants and animals. Triploid Carp for aquatic vegetation control. docsity.com Now, the “dark side” of Biotechnology Biowarfare and terrorism – What could “bad guys” do to us? Movie: Outbreak - Movie: Andromeda Strain - Reality may be worse “How far we have come – how far we have to go”. Many of yesterday’s great victories for Biology & Medicine – represent future challenges docsity.com Biotechnology Can be used to either preserve life, or destroy it Many of the scourges overcome in the last 200 years now represent threats to the world…as weapons. docsity.com Biological Weapons History 1940 – Japanese used Bubonic plague against China. (Flea bombs) 1950 – 53 – US weaponized Brucellosis (disease of Cattle) potentially for use in Korea Later US and others weaponized Anthax. Iraq admitted (1991) to producing 19,000 Liters of botulinum toxin (3x the amount needed to kill entire world population. (How do you say “weapons of mass destruction”) docsity.com 15,000 lethal doses of Botulinum Toxin docsity.com Castor Beans – Useful for castor oil, but a source of Ricin docsity.com In case you need something to worry about: Infectious agents: What makes a “good” infectious agent? High infectivity Not rapidly fatal Delivery – Aerosol Or: Epidemics Infection not immediately obvious Highly transmissible No established immunity or vaccine docsity.com Ebola Virus Zaire and Sudan Africa 50% Fatality rate Spread by contact with infected animal host No vaccine One variety may be spread by aerosol docsity.com Foot & Mouth Disease Highly contagious virus Sometimes fatal Mainly affects cattle, sheep, goats, pigs, - rarely affects humans Devastating to agriculture 2001 – outbreak in Britain, again in 2007 docsity.com Anthrax Bacillus Anthracis Commonly a livestock disease Spores may persist a long time in soil Used in the US in 2001 by a terrorist who has not been identified. Soviet accident 1979 – 66 deaths docsity.com Smallpox Eradicated from the world, except for one lab in US and one in Russia North Korea suspected of possession in weaponized form. Viral disease – formerly 20-60% fatal, 80% in children. 300-500 million dead in 20th century December 1979 - eradicated Airborne transmission docsity.com docsity.com Potential Biological Agents CDC’s List of Highest Concern: Anthrax (Brucilus anthracis) Botulism (Botulinum toxin) Plague (Yersinia pestis) Small Pox (Variola major) Tularemia (Francisella tutarensis) Viral hemorrhagic fever (Fibroviruses) Severe Acute Respiratory Syndrome (SARS) docsity.com Some Bioethics Questions Listing (Alphabetical Order) Human enhancement (example height) Human embryonic stem cell research Life support Organ donation – allocations MANY others We do live in a “Brave new World” docsity.com Biotechnology Great potential for good – or harm Appreciate the potential Unleash the power for good. docsity.com