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Microbiology: A Systems Approach, 2nd ed. Chapter 10: Genetic Engineering- A Revolution in Molecular Biology 10.1 Basic Elements and Applications of Genetic Engineering • Basic science: when no product or application is directly derived from it • Applied science: useful products and applications that owe their invention to the basic research that preceded them • 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: The Raw Material – Heat-denatured DNA • DNA strands separate if heated to just below boiling • Exposes nucleotides • Can be slowly cooled and strands will renature Restriction Endonucleases • Enzymes that can clip strands of DNA crosswise at selected positions • Hundreds have been discovered in bacteria • Each has a known sequence of 4 to 10 pairs as its target • Can recognize and clip at palindromes Figure 10.1 • Can be used to cut DNA in to smaller pieces for further study or to remove and insert sequences • Can make a blunt cut or a “sticky end” • The pieces of DNA produced are called restriction fragments • Differences in the cutting pattern of specific restriction endonucleases give rise to restriction fragments of differing lengthsrestriction fragment length polymorphisms (RFLPs) Ligase and Reverse Transcriptase • Ligase: Enzyme necessary to seal sticky ends together • Reverse transcriptase: enzyme that is used when converting RNA into DNA Figure 10.2 Analysis of DNA • Gel electrophoresis: produces a readable pattern of DNA fragments Figure 10.3 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 • Southern blot- a type of hybridization technique Figure 10.4 Probes Used for Diagnosis Figure 10.5 Fluorescent in situ Hybridizaton (FISH) • Probes applied to intact cells • Observed microscopically for the presence and location of specific genetic marker sequences • Effective way to locate genes on chromosomes 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 Figure 10.6 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 Figure 10.7 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 (amplicons) for synthesis • Extension – 72°C – DNA polymerase and nucleotides are added – Polymerases extend the molecule • The amplified DNA can then be analyzed 10.3 Methods in Recombinant DNA Technology • Primary intent of recombinant DNA technology- 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 Figure 10.8 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 Figure 10.9 Characteristics of Cloning Hosts Construction of a Recombinant, Insertion into a Cloning Host, and Genetic Expression Figure 10.10 Figure 10.11 Synthetic Biology: Engineering New Genetic Capabilities • Scientists are attempting to create microbes that produce hydrogen as fuel • Can use recombinant techniques mentioned previously 10.4 Biochemical Products of Recombinant DNA Technology 10.5 Genetically Modified Organisms • Transgenic or genetically modified organisms (GMOs): recombinant organisms produced through the introduction of foreign genes • These organisms can be patented 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 Plants: Improving Crops and Foods • Agrobacterium can transfect host cells • This idea can be used to engineer plants Figure 10.12 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 Figure 10.13 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 Figure 10.14 in vivo • 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 DNA Technology as Genetic Medicine • Some diseases result from the inappropriate expression of a protein • Prevent transcription or translation of a gene Antisense DNA and RNA: Targeting Messenger RNA • Antisense RNA: bases complementary to the sense strand of mRNA in the area surrounding the initiation site – When it binds to the mRNA, the dsRNA is inaccessible to the ribosome – Translation cannot occur • Single-stranded dNA usually used as the antisense agent (easier to manufacture) • For some genes, once the antisense strand bound to the mRNA, the hybrid RNA was not able to leave the nucleus • Antisense DNA: when delivered into the cytoplasm and nucleus, it binds to specific sites on any mRNAs that are the targets of therapy Figure 10.15 10.7 Genome Analysis: Maps, Fingerprints, and Family Trees • Possession of a particular sequence of DNA may indicate an increased risk of a genetic disease • Genome Mapping and Screening: An Atlas of the Genome – 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: show the relative proximity and order of genes on a chromosome • Physical maps: more detailed arrays that also give the numerical size of sections in base pairs • Sequence maps: produced by DNA sequencers – Genomics and bioinformatics: managing mapping data DNA Fingerprinting: A Unique Picture of a Genome • DNA fingerprinting: tool of forensic science • Uses methods such as restriction endonucleases, PCR, electrophoresis, hybridization probes, and Southern blot technique Figure 10.16 Microarray Analysis • Allows biologists to view the expression of genes in any given cell Figure 10.17