Chapter 19 Biotechnology Use or alteration of cells or biological molecules for specific application Transgenic organisms are possible because the genetic code is universal PCR Polymerase Chain Reaction Increases the amount of DNA sequences in tube Rapidly replicates the sequence millions of times Four Components are needed NEED stable temperature 1 Knowing parts of a target DNA sequence 2 2 types of lab made single stranded short pieces of DNA called primers 3 Large supply of 4 types of DNA nucleotide building blocks 4 Tag a polymerase produced by a microbe that inhibits hot springs does not fall apart when DNA is heated Recombinant DNA Recombinant DNA Creates a new combination of genetic material is a molecule that combines DNA from two sources also known as gene cloning 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 code Creating Recombinant DNA Molecules Cut DNA from donor and recipient with same restriction enzyme Cut DNA fragment is combined with a vector o Plasmid Vectors up to 15 o Bacteriophage up to 90 o Bacterial Artificial Chromosome 100 500 o 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 DNA Isolating Genes of Interest Genomic library species DNA probe cDNA library o Collections of recombinant DNA that contain pieces of the genome o To find specific fragments within the library use DNA probe or hemology within and between the Radioactively or fluorescently labeled gene fragments Genomic library of protein encoding genes produced by extracting mRNA and using reverse Reverse transcriptase cDNA transcriptase to make DNA microarray works backwards proteins RNA cDNA complimentary DNA product of reverse transcriptase making DNA to match a sequence in Selecting 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 vector Transgenic 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 larva Genetically Modified Crops Benefits o Resistant to pests and diseases o Delayed fruit ripening extending the shelf life o Elimination of allergens o Production of pharmeceuticals and edible vaccines o Production of biodegradable plastics Risks o Escape of the transgene beyond the field o Economical and political repercussions of displaced traditional products o Harm to farmers as other nations boycott GM products o Reduction of biodiversity Transgenic organisms can provide process as well as products Bioremediation Ability to detoxify pollutants Examples o Hg contaminated soils o GFP gene reveal locations of land mines Monitoring Gene Function Gene Expression Profiling o Indicates genes transcribed DNA Variation Screening o Detects mutations in Single Gene Polymorphisms SNPs Microarray Comparative Genomic Hybridization o Deletions and amplifications of DNA sequences between cells or species o Shows which genes are activated in different types of cells o Way to figure out how specific groups of cells function Silencing 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 translated Antisense induced exon skipping silences mutations that cause exons to be cut out of maturing o Antisense sequences mRNA o 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 20 Genetic Counseling Reasons to Seek Genetic Tests o Family History of abnormal chromosomes cancer or multifactorial disorder o Elevated risk of a single gene disorder o Sought out for infant difficulties 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 family history 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 osteoporosis Enzyme Replacement Therapy Replaces defective enzymes like insulin for diabetes Type 1 Gaucher disease lysosome storage diseases o Breaking down molecules o 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 In situ In vivo gene therapy alters cells outside of body gene therapy occurs in a localized area gene therapy vector is introduced directly into the body most invasive Gene Therapy Target Tissues Skin skin grafts can secrete therapeutic proteins Endothelium can secrete needed proteins directly into bloodstream Muscle accessible comprises body mass and has a good blood supply Liver many functions and can regenerate Lungs are easily accessed with