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Recombinant DNA and Genetic Engineering Chapter 16 Definitions Recombinant DNA technology: techniques & tools used to analyze genes Genetic Engineering: uses the above technology to isolate & modify genes in organisms or even to insert new genes Genetic Engineering Genes are isolated, modified, and inserted into an organism Made possible by recombinant technology Cut DNA up and recombine pieces Amplify modified pieces Restriction Enzymes Molecular scissors that cut DNA at a specific nucleotide sequence Over 200 different restriction enzymes are known, each isolated from bacteria and able to cut DNA in a unique manner Restriction enzymes Recombinant DNA Technology “Cutting and Pasting” Enzymes: Restriction enzymes = “cut” Ligase = “paste” DNA (Gene) cloning Want to study or isolate a particular gene Need to get many copies (amplification) of the gene so it can be studied adequately Most organisms only have one or two copies of any gene per cell, so we need a way to amplify copies of that gene Do that via cloning into a vector This allows scientists to make additional copies of the gene using bacteria Using Plasmids Plasmid is small circle of bacterial DNA Foreign DNA can be inserted into plasmid Forms recombinant plasmids Plasmid Can is a cloning vector deliver DNA into another cell Using Plasmids Polymerase Chain Reaction A faster method of amplifying or making more copies of a gene is PCR PCR requires short pieces of single-stranded DNA which match up to a regions at the beginning & end of the gene to be amplified, called primers Primers are required as a starting point for the DNA polymerase, the same enzyme used in DNA replication DNA polymerase then makes copy after copy of the gene. In a couple of hours more copies can be made by PCR than are made using bacteria & cloning vectors Polymerase Chain Reaction Sequence to be copied is heated Primers are added and bind to ends of single strands DNA polymerase uses free nucleotides to create complementary strands Doubles number of copies of DNA at each cycle Polymerase Chain Reaction Double-stranded DNA to copy DNA heated to 90°– 94°C Primers added to base-pair with ends Mixture cooled; base-pairing of primers and ends of DNA strands Stepped Art Figure 16.6 Page 256 DNA polymerases assemble new DNA strands Polymerase Chain Reaction Mixture heated again; makes all DNA fragments unwind Mixture cooled; basepairing between primers and ends of single DNA strands Stepped Art Figure 16.6 Page 256 DNA polymerase action again doubles number of identical DNA fragments Gel Electrophoresis DNA is placed at one end of a gel A current is applied to the gel DNA molecules are negatively charged and move toward positive end of gel Smaller molecules move faster than larger ones Gel Electrophoresis What are these techniques used for? Forensic: identifying criminals & victims Identifying disease genes in animals & humans Gene Therapy: inserting of new working copies of genes into humans Animal knockouts: turning off of a specific gene in order to discover its function DNA Fingerprinting Engineered Proteins Bacteria can be used to grow medically valuable proteins Insulin, interferon, blood-clotting factors Vaccines Engineered Plants Cotton plants that display resistance to herbicide Aspen plants that produce less lignin and more cellulose Tobacco plants that produce human proteins Mustard plant cells that produce biodegradable plastic Cloning Dolly 1997 - A sheep cloned from an adult cell Nucleus from mammary gland cell was inserted into enucleated egg Embryo implanted into surrogate mother Sheep is genetic replica of animal from which mammary cell was taken The Human Genome Initiative Goal - Map the entire human genome Initially thought by many to be a waste of resources Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes Sequencing was mostly completed ahead of schedule in early 2001 Ethical Issues Who decides what should be “corrected” through genetic engineering? Should animals be modified to provide organs for human transplants? Should humans be cloned?