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Chapter 14: Genetic Engineering -Modification of the DNA of an organism to produce new genes with new characteristics Biotechnology • Use of organisms to benefit humanity Recombinant DNA technology • DNA from different organisms is spliced together • Allows scientists to make many copies of any DNA segment (clone) • Can introduce foreign DNA into cells of microorganisms Recombinant DNA technology • Restriction enzymes – cut DNA – Bacteria produce for defense against viruses • Vector – transports DNA into a cell – Ex: bacteriophage – Plasmid – separate, smaller circular DNA that maybe be present and able to replicate inside bacteria • Transformation – uptake of foreign DNA by cells – How plasmids can get into bacteria Bacterial Conjugation and Recombination Recombinant DNA technology • Palindromic sequences – reads the same as complement, in opposite direction – AAGCTT – TTCGAA – Many restriction enzymes cut these sequences • Restriction enzymes cut on a stagger sticky ends (can pair with complementary singlestranded end of other DNA cut with same enzyme) • DNA Ligase – links 2 fragments recombinant DNA Fig. 20-3-3 Restriction site DNA 1 5 3 3 5 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination 3 DNA ligase seals strands. Recombinant DNA molecule Restriction Enzymes Steps of Creating a Recombinant DNA Plasmid (Basic) • 1. Plasmids and desired DNA cut by same restriction enzyme • 2. Mix 2 types of DNA so sticky ends pair • 3. DNA ligase forms bonds between fragments Fig. 20-2 Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial chromosome Plasmid Recombinant DNA (plasmid) Gene of interest DNA of chromosome 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Basic Protein harvested 4 Basic research and various applications research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth Cloning a Gene Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restriction site ampR gene Sticky ends Bacterial plasmid Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones Cloning DNA • Genome – total DNA per cell • Genomic library – collection of DNA fragments more or less representative of all DNA in genome • Genetic Probe – single stranded DNA or RNA that is radioactively labeled and can attach to target sequence by base pairing rules • A probe can be synthesized that is complementary to the gene of interest • For example, if the desired gene is 5 … G G C T A A C T T A G C … 3 – Then we would synthesize this probe 3 C C G A T T G A A T C G 5 DNA Probe Using a DNA probe Polymerase Chain Reaction (PCR) • Can amplify a small sample of DNA quickly • DNA replication in vitro • 2 strands separated by heating so special heat-resistant DNA polymerase called Taq polymerase used (thermophile) • MAJOR BONUS: Only specific sequences can be replicated • Study: crime scenes, archaeological remains PCR Gel Electrophoresis • Separates fragments like DNA, RNA or polypeptides (they carry charge and can migrate in an electrical field • RNA and DNA (-) --- so they move to (+) pole • Smaller fragments go further • Compare sample to standard • Usually “blot” - transfer DNA from gel to nitrocellulose filter for further analysis • DNA Fingerprinting Gel Electrophoresis Fig. 20-9a TECHNIQUE Power source Mixture of DNA molecules of different sizes Anode – Cathode + Gel 1 Power source – + Longer molecules 2 Shorter molecules Fig. 20-9b RESULTS DNA Fingerprint Transgenic Organisms • Plants and animals in which foreign genes have been incorporated • Animals – Inject DNA into nucleus of egg or stem cell – Eggs implanted in uterus; stem cells injected into blastocysts + then implanted into foster mother • Plants – Disease resistance – Pesticide resistance Transgenics Fig. 20-20 Embryonic stem cells Early human embryo at blastocyst stage (mammalian equivalent of blastula) Adult stem cells From bone marrow in this example Cells generating all embryonic cell types Cells generating some cell types Cultured stem cells Different culture conditions Different types of differentiated cells Liver cells Nerve cells Blood cells Fig. 20-18 TECHNIQUE Mammary cell donor Egg cell donor 2 1 Egg cell from ovary Cultured mammary cells 3 Cells fused 3 4 Grown in Nucleus removed Nucleus from mammary cell culture Early embryo 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development RESULTS Lamb (“Dolly”) genetically identical to mammary cell donor Fig. 20-19 Cloning Video GE Plants Application of GE • Human proteins – Insulin – Hormones - HGH • Human treatments for disease – Multiple sclerosis, certain cancers, heart attacks, forms of anemia • Vaccines Fig. 20-23 Fig. 20-24 (a) This photo shows Earl Washington just before his release in 2001, after 17 years in prison. Source of sample STR marker 1 STR marker 2 STR marker 3 Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 Kenneth Tinsley 17, 19 13, 16 12, 12 (b) These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. Forensics