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Biotechnology Engineering 2 Ch.12 Ms. Haut Biotechnology: Training & Careers Career Spotlight: Bioengineer DNA technology has many useful applications – The Human Genome Project – The production of vaccines, cancer drugs, and pesticides – Engineered bacteria that can clean up toxic wastes Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings •DNA and Crime Scene Investigations – Many violent crimes go unsolved • For lack of enough evidence – If biological fluids are left at a crime scene • DNA can be isolated from them – DNA fingerprinting is a set of laboratory procedures • • That determines with near certainty whether two samples of DNA are from the same individual That has provided a powerful tool for crime scene investigators Investigator at one of the crime scenes (above), Narborough, England (left) BACTERIAL PLASMIDS AND GENE CLONING •Plasmids are used to customize bacteria: An overview – Gene cloning is one application of DNA technology • Methods for studying and manipulating genetic material The Bacterial Chromosome • One double-stranded, circular molecule of DNA • Located in nucleoid region, so transcription and translation can occur simultaneously • Many also contain extrachromosomal DNA in plasmids Binary Fission Plasmids • Short, circular DNA molecules outside the chromosome • Carry genes that are beneficial but not essential • Replicate independently of chromosome en.wikipedia.org/?title=Plasmid R Plasmids • Contain genes that confer antibiotic resistance • Medical consequences: resistant strains of pathogens due to overuse of antibiotics http://www.slic2.wsu.edu:82/hurlbert/micro101/images/AntibioticSelection.gif Bacteria as Tools • Bacterial Transformation— – Uptake of DNA from the fluid surrounding the cell – Causes genetic recombination Transformation • Biotech companies use this technique to artificially introduce foreign genes into bacterial genomes (human insulin, human growth hormone) – Researchers can insert desired genes into plasmids, creating recombinant DNA • Figure 12.1 And insert those plasmids into bacteria (transformation) E. coli – If the recombinant bacteria multiply into a clone • The foreign genes are also copied 1 Isolate DNA Human cell from two sources 2 Cut both Plasmid DNAs with the same restriction enzyme DNA Gene V Sticky ends 3 Mix the DNAs; they join by base-pairing 4 Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid Gene V 5 Put plasmid into bacterium by transformation 6 Clone the bacterium Bacterial clone carrying many copies of the human gene Restriction Enzymes •Used to “cut and paste” DNA •The tools used to make recombinant DNA are – Restriction enzymes, which cut DNA at specific sequences – DNA ligase, which “pastes” DNA fragments together Genes can be cloned in recombinant plasmids: A closer look – Bacteria take the recombinant plasmids from their surroundings – And reproduce, thereby cloning the plasmids and the genes they carry Cloned genes can be stored in genomic libraries •Genomic libraries, sets of DNA fragments containing all of an organism’s genes – Can be constructed and stored in cloned bacterial plasmids or phages Genome cut up with restriction enzyme Recombinant plasmid Recombinant phage DNA or Bacterial clone Plasmid library Figure 12.4 Phage clone Phage library Nucleic acid probes Radioactive probe (DNA) •A short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA ATCCGA Mix with singlestranded DNA from various bacterial (or phage) clones Single-stranded G DNA TC T TA A T C GC G C T G A AT T T C C G A – Can tag a desired gene in a library Master plate A A T G G C G C TA G G A C TA Base pairing indicates the gene of interest Master plate Probe DNA Radioactive single-stranded DNA Solution containing probe Colonies containing gene of interest Gene of interest Single-stranded DNA from cell Filter A Film Filter lifted and flipped over Hybridization on filter A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest. CONNECTION •Recombinant cells and organisms can mass-produce gene products – Applications of gene cloning include • The mass production of gene products for medical and other uses Table 12.6 Genetically modified organisms are transforming agriculture • New genetic varieties of animals and plants are being produced – A plant with a new trait can be created using the Ti plasmid • “Golden rice” has been genetically modified to contain beta-carotene – This rice could help prevent vitamin A deficiency Figure 12.18B Drought resistant corn Flavr Savr Tomato (CalGene) •Transgenic organisms – Are those that have had genes from other organisms inserted into their genomes – Different organisms, including bacteria, yeast, and mammals • Can be used for this purpose These sheep carry a gene for a human blood protein that is a potential treatment for cystic fibrosis Figure 12.6 CONNECTION •DNA technology is changing the pharmaceutical industry – DNA technology • Is widely used to produce medicines and to diagnose diseases DNA technology is changing the pharmaceutical industry and medicine • Hormones, cancer-fighting drugs, and new vaccines are being produced using DNA technology – This lab equipment is used to produce a vaccine against hepatitis B Figure 12.17 •Therapeutic hormones – In 1982, humulin, human insulin produced by bacteria • Became the first recombinant drug approved by the Food and Drug Administration Figure 12.7A •Diagnosis and Treatment of Disease – DNA technology • Is being used increasingly in disease diagnosis •Vaccines – DNA technology • Is also helping medical researchers develop vaccines Figure 12.7B