Genetic Engineering & Recombinant DNA

Transcription

1 Genetic Engineering & Recombinant DNA Chapter 10 Copyright The McGraw-Hill Companies, Inc) Permission required for reproduction or display. Applications of Genetic Engineering Basic science vs. Applied science Artificial manipulation of genetic information Characterize organisms Indentify organisms Modify organisms More useful organisms Produce useful products 1

2 10.1 Basic Elements and Applications of Genetic Engineering Six applications and topics in genetic engineering Tools and techniques Methods in recombinant DNA technology Biochemical products of recombinant DNA technology Genetically modified organisms Genetic treatments Genome analysis 10.2 Tools and Techniques of Genetic Engineering DNA is the raw material Enzymes such as restriction endonucleases and ligases are used to manipulate the DNA 2

3 Enzymes: Restriction Endonucleases Enzymes that cut DNA Each has a known sequence of 4 to 10 pairs as its target Can recognize and clip at palindromes Madam, I m Adam GAATTC CTTAAG (1) (2) Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA Organism 1 DNA Organism 2 TGCA ACGT Site of cut C T A G G A T C C T A G G A T C Sticky ends ACGT TGCA Restriction endonuclease makes staggered cut at palindrome. (c) Action of restriction endonucleases. (1) A restriction endonuclease recognizes and cleaves DNA at the site of a specific palindromic sequence. Cleavage can produce staggered tails called sticky ends that accept complementary tails for gene splicing. (2) The sticky ends can be used to join DNA from different organisms by cutting it with the same restriction enzyme, ensuring that all fragments have complementry ends. TGCA ACGT ACGT TGCA Some Common Restriction Enzymes BamHI - Bacillus amyloliquefaciens G GATCC CCTAG G EcoRI - Escherichia coli G AATTC CTTAA G EcoRV - Escherichia coli GAT ATC CTA TAG 3

4 Enzymes: Ligase and Reverse Transcriptase Ligase: Enzyme necessary to seal sticky ends together Reverse transcriptase: enzyme that is used when converting RNA into DNA Copied DNA is referred to as complementary DNA or cdna cdna 4

5 Analysis of DNA Gel Electrophoresis Electric gradient made DNA passes through agarose gel Small fragments move fastest Readable pattern of DNA fragments Nucleic Acid Hybridization and Probes Two different nucleic acids can hybridize by uniting at their complementary regions Gene probes: specially formulated oligonucleotide tracers Short stretch of DNA of a known sequence Will base-pair with a stretch of DNA with a complementary sequence if one exists in the test sample Can detect specific nucleotide sequences in unknown samples Probes carry reporter molecules (such as radioactive or luminescent labels) so they can be visualized 5

6 Probes for Diagnosis Isolate DNA from sample Denature DNA and combine with probe for hybridization Some diagnostic probes Salmonella Campylobacter Shigella Chlamydia Methods Used to Size, Synthesize, and Sequence DNA Relative sizes of nucleic acids usually denoted by the number of base pairs (bp) they contain DNA Sequencing: Determining the Exact Genetic Code Most detailed information comes from the actual order and types of bases- DNA sequencing Most common technique: Sanger DNA sequence technique 6

7 Sanger DNA Sequencing Polymerase Chain Reaction: A Molecular Xerox Machine for DNA Some techniques to analyze DNA and RNA are limited by the small amounts of test nucleic acid available Polymerase chain reaction (PCR) rapidly increases the amount of DNA in a sample So sensitive- could detect cancer from a single cell Can replicate a target DNA from a few copies to billions in a few hours 7

8 Three Basic Steps that Cycle Denaturation Heat to 94 C to separate in to two strands Cool to between 50 C and 65 C Priming Primers added in a concentration that favors binding to the complementary strand of test DNA Prepares the two strands for synthesis Extension 72 C DNA polymerase and nucleotides are added Polymerases extend the molecule The amplified DNA can then be analyzed Polymerase Chain Reaction 8

9 10.3 Methods in Recombinant DNA Technology Primary intent - deliberately remove genetic material from one organism and combine it with that of a different organism Form genetic clones Gene is selected Excise gene Isolate gene Insert gene into a vector Vector inserts DNA into a cloning host Recombinant DNA strategies Obtain target gene Insert into vector Introduce into hosts Expression of desired gene product Many applications 9

10 Technical Aspects of Recombinant DNA and Gene Cloning Strategies for obtaining genes in an isolated state DNA removed from cells, separated into fragments, inserted into a vector, and cloned; then undergo Southern blotting and probed Gene can be synthesized from isolated mrna transcripts Gene can be amplified using PCR Once isolated, genes can be maintained in a cloning host and vector (genomic library) Characteristics of Cloning Vectors Capable of carrying a significant piece of the donor DNA Readily accepted by the cloning host Must have a promoter in front of the cloned gene Vectors (such as plasmids and bacteriophages) should have three important attributes: An origin of replication somewhere on the vector Must accept DNA of the desired size Contain a gene that confers drug resistance to their cloning host 10

11 Cloning Vectors Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. puc19 (2,686bp) MCS EcoRI SacI KpnI SmaI BamHI XbaI SalI PstI SphI HindIII ori Construction of a Recombinant Insertion into a cloning host Gene expression Location of recombinant clones 11

12 Selecting Clones Use selectable marker Selective media E.g. antibiotics Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Bacteria lacking plasmid (1) Culture of cloning host after incubation with recombinant plasmid Bacteria with recombinant plasmid (2) Differential media Bacteria carrying plasmid Ampicillinresistance gene E.g. X-gal Indicates inserts Regular nonselective medium with two types of colonies Selective medium with ampicillin Pure culture of bacteria containing cloned gene 10.5 Genetically Modified Organisms Transgenic or genetically modified organisms (GMOs) Recombinant organisms produced through the introduction of foreign genes Can be patented 12

13 Recombinant Microbes: Modified Bacteria and Viruses Genetically altered strain of Pseudomonas syringae Can prevent ice crystals from forming Frostban to stop frost damage in crops Strain of Pseudomonas fluorescens Engineered with a gene from Bacillus thuringiensis Codes for an insecticide Drug therapy Bioremediation Transgenic Animals: Engineering Embryos Several hundred strains have been introduced Can express human genes in organs and organ systems Most effective way is to use viruses 13

14 10.6 Genetic Treatments: Introducing DNA into the Body Gene Therapy For certain diseases, the phenotype is due to the lack of a protein Correct or repair a faulty gene permanently so it can make the protein Two strategies ex vivo in vivo Ex vivo Gene Therapy 14

15 in vivo Gene Therapy Skips the intermediate step of incubating excised patient tissue Instead the naked DNA or a virus vector is directly introduced into the patient s tissues Gene Silencing Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Disease due to aberrant production of a protein sirna attaches to proteins to form RNA silencing complexes sirna Turn off the expression of the gene RNA-induced silencing complex (RISC) Anneal to mrna Complex is destroyed sirna unwinding Binding to target mrna mrna cleavage by RNA silencing complexes RNA silencing complexes Activated RNA silencing complexes 15

16 10.7 Genome Analysis: Maps and Profiles Genome Mapping and Screening Locus: the exact position of a particular gene on a chromosome Alleles: sites that vary from one individual to another; the types and numbers are important to genetic engineers Mapping: the process of determining location of loci and other qualities of genomic DNA Linkage maps Physical maps Sequence maps DNA Profiles: A Unique Picture of a Genome 16