Foundations in MicrobiologyChapter10PowerPoint to accompanyFifth EditionTalaroCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Genetic Engineering: A Revolution in Molecular BiologyChapter 10

Genetic engineeringdirect, deliberate modification of an organisms genomebioengineering

Biotechnology use of an organisms biochemical and metabolic pathways for industrial production

I. Tools & Techniques of genetic engineeringenzymes for dicing, splicing, & reversing nucleic acidsanalysis of DNA

Enzymes for dicing, splicing, & reversing nucleic acidsrestriction endonucleases recognize specific sequences of DNA & break phosphodiester bondsligase rejoins phosphate-sugar bonds cut by endonucleasesreverse transcriptase makes a DNA copy of RNA - cDNA

Analysis of DNAgel electrophoresis- separates DNA fragments based on sizenucleic acid hybridization & probes probes base pair with complementary sequences; used to detect specific sequencesDNA Sequencing reading the sequence of nucleotides in a stretch of DNAPolymerase Chain Reaction way to amplify DNA

Gel electrophoresis

Southern blot hydridization

In situ hybridization

Sanger DNA sequence technique

Polymerase chain reaction (PCR)

II. Methods in Recombinant DNA Technologyconcerned with transferring DNA from one organism to another

Cloning vectors & hostsConstruction of a recombinant plasmid

Characteristics of cloning vectorsmust be capable of carrying a significant piece of donor DNAmust 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

Vector considerationsorigin of replicationsize of donated DNA vector will acceptgene which confers drug resistance to their cloning host

pBR322

Characteristics of cloning hostsrapid overturn, fast growth ratecan be grown in large quantities using ordinary culture methodsnonpathogenicgenome that is well delineated capable of accepting plasmid or bacteriophage vectorsmaintains foreign genes through multiple generationswill secrete a high yield of proteins from expressed foreign genes

III. Biochemical Products of Recombinant DNA Technologyenables large scale manufacturing of life-saving hormones, enzymes, vaccinesinsulin for diabeteshuman growth hormone for dwarfismerythropoietin for anemiaFactor VIII for hemophiliaHBV vaccine

IV. Genetically Modified Organisms (GMO)Recombinant microbes Pseudomonas syringae prevents ice crystalsBacillus thuringienisis encodes an insecticideTransgenic plantsRice that makes beta-caroteneTobacco resistant to herbicidesPeas resistant to weevilsTransgenic animalsMouse models for CF, Alzheimers, sickle cell anemiaSheep or goats that make medicine in their milk semen

Bioengineering of plants

Transgenic mice

V. Genetic TreatmentsGene therapyAntisense DNATriplex DNA

Gene therapycorrect faulty gene in human suffering from diseaseex vivo normal gene is is added to tissues taken from the body, then transfected cells are reintroduced into the bodyin vivo naked DNA or viral vector is directly introduced into patients tissueMost trials target cancer, single gene defects & infectionsMost gene deliveries are carried out by viral vectors

Gene therapy

Antisense DNA: targeting mRNAAntisense a nucleic acid strand with a base sequence that is complementary to the translatable strandAntisense DNA gets into the nucleus and binds to mRNA, blocking the expression of an unwanted proteincancersAlzheimers diseaseautoimmune diseases

Triplex DNAA triple helix formed when a third strand of DNA inserts into the major groove, making it inaccessible to normal transcriptionoligonucleotides have been synthesized to form triplex DNA oncogenes virusesreceptor for IL-2

Antisense DNA & triplex DNA

VI. Genome AnalysisGene MappingDNA Fingerprinting Microarray analysis

Gene Mappingdetermining the location of specific genes on the chromosomesHuman 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

Map of chromosome 16

DNA Fingerprinting Every individual has a unique sequence of DNAUsed to:identify hereditary relationshipsstudy inheritance of patterns of diseasesstudy human evolutionidentify criminals or victims of disaster

DNA fingerprints

Pedigree analysis

Microarray analysisMethod of determining which genes are actively transcribed in a cell under various conditionshealth vs diseasegrowth vs differentiationcould improve accuracy of diagnosis and specificity of treatment

Microarray