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Biotechnology and Recombinant DNA Bio 205 Chandler Community College Biotechnology • Biotechnology – The use of microorganisms, cells, or cell components to make a product • Foods • Vaccines • Antibiotics • Vitamins • Mining – extract minerals from ore Recombinant DNA • Recombinant DNA (rDNA) – Also called genetic engineering – Inserting genes into cells to make chemicals that the organisms do not naturally make • Usually commercial or medicinal value – Insulin – Vaccines – Amplify DNA – Enzymes – Hormones – Insect resistant plants rDNA Procedures • Insulin produced in bacterial cells rDNA Procedures • Human Growth Hormone (hGH) • Some humans lack hGH • Use to get hGH from the dead • Now bacteria are used to make the hormone Tools of Biotechnology • Natural Selection – normal process • Humans use Artificial Selection to select desirable breeds of animals or strains of plants Tools of Biotechnology • Mutation – Responsible for diversity of life – New bacteria strains can be created by exposing them to mutagens • Chemicals or UV radiation – Produced over 1000 new strains of penicillin producing organisms • Site Directed Mutagenesis – Used to make a specific change in a gene Site Directed Mutagenesis Restriction Enzymes • Restriction Enzymes – Special class of DNA-cutting enzymes that exist in many bacteria – Used naturally by some bacteria to kill invading viruses • Cuts, or digests, only one particular sequence of nucleotide bases in DNA • Cuts the same way each time Restriction Enzymes Vectors • A Plasmid or virus used in genetic engineering to insert genes into a cell – A mosquito is a vector also – inserting parasites into a human body • Vehicle for the replication of a desired DNA sequence • Shuttle Vector can be used to move cloned DNA sequences among organisms • Viral DNA can be used as a vector – Retroviruses, adenoviruses, herpesviruses Polymerase Chain Reaction • Polymerase Chain Reaction (PCR) • Process by which small pieces of DNA can be quickly amplified for analysis – Only good for small pieces of DNA – Within 30 minutes the sample can be amplified 1 billion times • Used for diagnostic tests to detect the presence of infectious particles PCR PCR Inserting Foreign DNA into Cells • rDNA must first be manipulated outside the cell before it can be inserted into a new cell • Several ways to introduce DNA into new cells – Transformation – Electroporation – Protoplast fusion – Microinjection Transformation • Procedure where cells can take up plasmids (DNA) from the surrounding environment • The cell receiving the new DNA must be Competent • Made able to take up DNA • Chemicals used to prepare the cell – Calcium chloride – Heat also used Electroporation • Uses an electrical current to form microscopic pores in the membrane of the cells receiving the DNA • Procedure works on most cells • Some must be made into protoplasts first – Removing the cell wall – The plasma membrane is more susceptible for receiving DNA Protoplast Fusion • Protoplast in solution will fuse at slow but significant rate • Adding polyethylene glycol will increase the frequency of fusion • New “hybrid” cell is formed by the fusion of the two cells • Used more in plant and algae cells Microinjection • Uses glass micropipette with very small diameter • DNA injected through animal cell membrane into cell Obtaining DNA • Biologists can get the tools for rDNA by using two main sources for genes: • Gene Libraries • Synthetic DNA Gene Libraries • DNA is extracted from a cell • Broken into smaller pieces of the cell’s entire genome • Pieces are then spliced into a plasmid or a virus to make a collection of clones • The collection of clones (one clone for each fragment) containing different fragments of DNA from a single organism • Each organism and it’s DNA fragments has a “book” of genetic information Synthetic DNA • Some genes can be made in the cell (in-vitro) • Synthesis machines • Used to enter a desired sequence of nucleotides • Like a word processor • Order is determined by a the use of stored nucleotides and reagents Selecting a Clone • Must select the particular cell that contains the specific gene of interest – Can be difficult (number of cells) • Blue-White Screening procedure • Colony Hybridization – DNA probes – Radioactive marker or – Fluorescent marker – Identifies the marker – Photography Making a Gene Product • Organisms used most effectively – Eschericia coli – Saccromyces cerevisiae (Baker’sYeast) – Mammal cells – Plant cells Applications of rDNA • Therapeutic (pharmaceuticals) – Insulin production – Somatostatin production – Subunit vaccines • Contain protein portion of a pathogen – DNA vaccines – Gene therapy • Replace a defective gene with a better one – Gene silencing • Defense in microbes against viruses Human Genome Project • 13-year effort to sequence the entire human genome • Used rDNA technology • 3 billion nucleotide pairs • 20,000 to 25,000 genes • Only 2% of genome produces functional products • 98% is junk DNA Scientific Applications • rDNA makes a large amount of copies of DNA • Many techniques to analyze the DNA once it is made Scientific Applications • DNA sequencing – Determination of the exact sequence of nucleotides • Random Shotgun sequencing – Small pieces of a genome are sequenced and then assembled using a computer • Bioinformatics – The science of understanding the function of genes through computer assisted analysis Scientific Applications • Proteomatics – Science of determining all of the proteins expressed in a cell • Southern Blotting – Hybridization technique that determines the sequence of a gene – Electrophoresis – Used for genetic screening for certain diseases Scientific Applications • Forensic Microbiology – Identification of a particular DNA from many others – DNA Fingerprinting • Determines paternity • Blood analysis • Etc. • Nanotechnology – Design and manufacture of extremely small electronic circuits using molecular level of matter – Robots and computers Agricultural Applications • Developing new strains of plants • Resistance • Crossing plant species • Cloning of plants • Plant breeding • Ti plasmid used to introduce genes into plants • Produces stronger plants Ti Plasmid Ethics and rDNA