Foundations in Microbiology

Chapter10

PowerPoint to accompany

Fifth Edition

Talaro

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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Genetic Engineering: A Revolution in Molecular Biology

Chapter 10

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Genetic engineering

• direct, deliberate modification of an organism’s genome

• bioengineering

• Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production

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I. Tools & Techniques of genetic engineering

• enzymes for dicing, splicing, & reversing nucleic acids

• analysis of DNA

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Enzymes for dicing, splicing, & reversing nucleic acids

1. restriction endonucleases – recognize specific sequences of DNA & break phosphodiester bonds

2. ligase – rejoins phosphate-sugar bonds cut by endonucleases

3. reverse transcriptase – makes a DNA copy of RNA - cDNA

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Analysis of DNA

• gel electrophoresis- separates DNA fragments based on size

• nucleic acid hybridization & probes – probes base pair with complementary sequences; used to detect specific sequences

• DNA Sequencing – reading the sequence of nucleotides in a stretch of DNA

• Polymerase Chain Reaction – way to amplify DNA

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Gel electrophoresis

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Southern blot hydridization

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In situ hybridization

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Sanger DNA sequence technique

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Polymerase chain reaction (PCR)

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II. Methods in Recombinant DNA Technology

• concerned with transferring DNA from one organism to another

1. Cloning vectors & hosts2. Construction of a recombinant plasmid

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Characteristics of cloning vectors

• must be capable of carrying a significant piece of donor DNA

• must be readily accepted by the cloning host

• plasmids – small, well characterized, easy to manipulate & can be transferred into appropriate host cells through transformation

• bacteriophages – have the natural ability to inject their DNA into bacterial hosts through transduction

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Vector considerations

• origin of replication• size of donated DNA vector will accept• gene which confers drug resistance to their

cloning host

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pBR322

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Characteristics of cloning hosts

1. rapid overturn, fast growth rate2. can be grown in large quantities using ordinary culture

methods3. nonpathogenic4. genome that is well delineated 5. capable of accepting plasmid or bacteriophage vectors6. maintains foreign genes through multiple generations7. will secrete a high yield of proteins from expressed

foreign genes

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III. Biochemical Products of Recombinant DNA Technology

• enables large scale manufacturing of life-saving hormones, enzymes, vaccines– insulin for diabetes– human growth hormone for dwarfism– erythropoietin for anemia– Factor VIII for hemophilia– HBV vaccine

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IV. Genetically Modified Organisms (GMO)

• Recombinant microbes – Pseudomonas syringae – prevents ice crystals– Bacillus thuringienisis –encodes an insecticide

• Transgenic plants– Rice that makes beta-carotene– Tobacco resistant to herbicides– Peas resistant to weevils

• Transgenic animals– Mouse models for CF, Alzheimer’s, sickle cell anemia– Sheep or goats that make medicine in their milk semen

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Bioengineering of plants

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Transgenic mice

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V. Genetic Treatments

• Gene therapy• Antisense DNA• Triplex DNA

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Gene therapy

• correct faulty gene in human suffering from disease– ex vivo – normal gene is is added to tissues taken from

the body, then transfected cells are reintroduced into the body

– in vivo – naked DNA or viral vector is directly introduced into patient’s tissue

• Most trials target cancer, single gene defects & infections

• Most gene deliveries are carried out by viral vectors

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Gene therapy

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Antisense DNA: targeting mRNA

• Antisense – a nucleic acid strand with a base sequence that is complementary to the translatable strand

• Antisense DNA gets into the nucleus and binds to mRNA, blocking the expression of an unwanted protein– cancers– Alzheimer’s disease– autoimmune diseases

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Triplex DNA

• A triple helix formed when a third strand of DNA inserts into the major groove, making it inaccessible to normal transcription

• oligonucleotides have been synthesized to form triplex DNA – oncogenes – viruses– receptor for IL-2

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Antisense DNA & triplex DNA

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VI. Genome Analysis

• Gene Mapping• DNA Fingerprinting • Microarray analysis

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Gene Mapping

• determining the location of specific genes on the chromosomes

• Human Genome Project – to determine the nucleotide sequence of the >30,000 genes in the genome & the importance of these sequences & how they relate to human disease

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Map of chromosome 16

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DNA Fingerprinting

• Every individual has a unique sequence of DNA

• Used to:– identify hereditary relationships– study inheritance of patterns of diseases– study human evolution– identify criminals or victims of disaster

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DNA fingerprints

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Pedigree analysis

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Microarray analysis

• Method of determining which genes are actively transcribed in a cell under various conditions– health vs disease– growth vs differentiation

• could improve accuracy of diagnosis and specificity of treatment

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Microarray