KSU BSCI 30050 - Chapter 19 Biotechnology

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Chapter 19Biotechnology- Use or alteration of cells or biological molecules for specific application- Transgenic organisms are possible because the genetic code is universalPCR- Polymerase Chain Reaction- Increases the amount of DNA sequences in tube- Rapidly replicates the sequence millions of times- Four Components are needed: ** NEED stable temperature1. Knowing parts of a target DNA sequence2. 2 types of lab: made single stranded short pieces of DNA called primers3. Large supply of 4 types of DNA nucleotide building blocks4. Tag, a polymerase produced by a microbe that inhibits hot springs (does not fall apart when DNA is heated)\Recombinant DNA- Recombinant DNA is a molecule that combines DNA from two sources (also known as gene cloning)- Creates a new combination of genetic material – Human gene for insulin was placed in bacteria to make large quantities for diabetics - Genetically modified organisms are possible because of the universal nature of the genetic codeCreating Recombinant DNA Molecules• Cut DNA from donor and recipient with same restriction enzyme• Cut DNA fragment is combined with a vectoro Plasmid Vectors: up to 15o Bacteriophage: up to 90o Bacterial Artificial Chromosome: 100-500o Yeast Artificial Chromosome: 250-2,000• Vector DNA cut with same restriction enzyme • Donor DNA and vector DNA combined• The complementary ends of the DNAs (sticky ends) bind and ligase enzyme reattaches the sugar-phosphate backbone of the DNAIsolating Genes of Interest• Genomic library o Collections of recombinant DNA that contain pieces of the genomeo To find specific fragments within the library, use DNA probe or hemology within and between the species• DNA probe– Radioactively (or fluorescently) labeled gene fragments• cDNA library– Genomic library of protein encoding genes produced by extracting mRNA and using reverse transcriptase to make DNA– Reverse transcriptase: works backwards, proteins  RNA  cDNA– cDNA: complimentary DNA, product of reverse transcriptase, making ½ DNA to match a sequence in microarraySelecting for cells with vectors• Vectors are commonly engineered to carry antibiotic resistance genes • Host bacteria without a plasmid die in the presence of the antibiotic• Bacteria harboring the vector survive• Growing cells on media with antibiotics ensures that all growing cells must carry the vectorTransgenic Organisms• When recombinant DNA is applied to multicellular organisms, individuals must be bred to yield homozygous individuals• Plants may be produced by asexual reproduction (cuttings) so easier to make than transgenic animals• Different vectors and gene transfer techniques can be used• Bt gene: From bacterium Bacillus thuringiensis (bt)o Specifies a protein that destroys the stomach lining of certain insect larvaGenetically Modified Crops• Benefits: o Resistant to pests and diseaseso Delayed fruit ripening, extending the shelf lifeo Elimination of allergenso Production of pharmeceuticals and edible vaccineso Production of biodegradable plastics• Risks o “Escape” of the transgene beyond the fieldo Economical and political repercussions of displaced traditional productso Harm to farmers as other nations boycott GM productso Reduction of biodiversityBioremediation• Transgenic organisms can provide process as well as products• Ability to detoxify pollutants• Examples:o Hg-contaminated soilso GFP gene reveal locations of land minesMonitoring Gene Function• Gene Expression Profilingo Indicates genes transcribed• DNA Variation Screeningo Detects mutations in Single Gene Polymorphisms (SNPs)• Microarray Comparative Genomic Hybridizationo Deletions and amplifications of DNA sequences between cells or specieso Shows which genes are activated in different types of cellso Way to figure out how specific groups of cells functionSilencing DNA• In some situations, silencing gene expression may be useful:– Blocking transcription of oncogenes, so cancer cells wont form• Three techniques can be used to control gene expression:o RNA interference Short, double-stranded RNAs sent into cells separate into single strands One of these strands binds its complement in mRNA, preventing it from being translatedo Antisense sequences Antisense-induced exon skipping silences mutations that cause exons to be cut out of maturing mRNAo Knockouts from gene targeting  Gene targeting is a technique that uses homologous recombination to replace a normal DNA sequence with one that cannot be transcribed or translated This silences gene expression by creating a “knockout” gene Moreover, observing what happens (or not) can reveal the gene’s normal function Chapter 20Genetic Counseling• Reasons to Seek:o Family History of abnormal chromosomes, cancer or multifactorial disordero Elevated risk of a single gene disordero Sought out for infant difficultiesGenetic Tests• Population Carrier Screen: identifies heterozygotes—people with one copy of a mutant gene• Prenatal Test: detects mutant allele in a fetus for a condition present in a family• Prenatal Screen: tests embryos or fetuses from a population for increased risk in a condition, not based on familyhistory• Newborn Screen: population wide testing for several treatable inborn errors of metabolism• Diagnostic Test: confirms diagnosis based on symptoms• Direct to consumer: concerns are cardiovascular health, interpretation of results may not be 100% accurate or 100% penetrance, osteoporosisEnzyme Replacement Therapy• Replaces defective enzymes like insulin for diabetes• Type 1 Gaucher disease: lysosome storage diseaseso Breaking down moleculeso Liver and spleen break down o Buildup of stuff enzymes should break down o To combat this disease: replace enzyme, substrate reduction therapy, pharmacological chaperone therapy (make enzyme functional)Gene Therapy• Altering genes hopefully can provide a longer-lasting treatment– Treatments have been focused on inherited disorders with a known disease mechanism– Uses bacteriophage vectors, Germline vs. somatic vectors• Germline gene therapy – gamete or zygote alteration– is heritable– not done in humans• Somatic gene therapy – specific cells– not heritable• Ex vivo gene therapy alters cells outside of body • In situ gene therapy occurs in a localized area• In vivo gene therapy vector is introduced directly into the body; most

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