Chapter 9 - Recombinant DNA
I.
Introduction to Biotechnology
A. Biotechnology is the use of microorganisms, cells, or cell components to make a product.
B. Recombinant DNA (rDNA) technology is the technology used to insert foreign DNA into an organism (commonly referred to as genetic engineering)
C. Recombinant DNA technology was developed in the 1970s and 80s to express and amplify selected DNA in novel hosts.
D. An overview of recombinant DNA procedures.
1. Genes of interest are isolated from the environment.
2. These genes are inserted into a genetic transport system, called vector, most commonly this is a plasmid when dealing with prokaryote rDNA technology.
3. The modified vector (with inserted gene) is inserted into a carrier organism (commonly a bacterium), which is allow to multiply.
4. Multiplied bacteria carrying the modified vector are called clones, since they derive from a single progenitor cell.
E. Common uses for the clones
1. Harvesting large numbers of the gene of interest for further manipulations.
2. The cloned gene converts the phenotype of the modified host (i.e. BT toxin in modified plants).
3. The protein is produced and harvested for other uses (i.e. insulin for the treatment of diabetes).
II. Tools of
Biotechnology
A. Selection artificial selection is the selection of desirable traits in plants and animals. Microbiologists use the increased number of generations in bacteria to rapidly select for desired traits.
B. Mutation where as random mutations can result in unpredictable changes, microbiologist can make directed predicted mutations with the intent of creating desired reactions.
C. Restriction Enzymes a special class of DNA cutting enzymes that recognize specific DNA sequences.
D. Vectors DNA vehicles to carry inserted genes having a number of specific traits.
1. ori origin of replication to allow replication of the vector
2. Selection Sequence some phenotype encoded by the vector that can be selected for to determine the clones containing the vector (a common example is antibiotic resistance).
3. Cloning Site a small sequence stretch filled with specific restriction enzyme sites.
E. Polymerase Chain Reaction a process by with a specific DNA polymerase is used to amplify a specific sequence of DNA in a test tube.
III.
Techniques of Genetic Modification
A. Inserting Foreign DNA into Cells
1. Transformation as in the last chapter it can occur naturally in some species, but mainly it is accomplished through the use of chemicals to push DNA into cells.
2. Electroporation uses an electric current to force DNA into cells.
3. Protoplast fusion stripping away the cell wall of a cell and pushing DNA through the cell membrane with osmotic pressure.
B. Obtaining DNA
1. Genetic Libraries random fragments of DNA from a specific organism.
2. Synthetic DNA gene sequences can be formulated in the lab and used.
IV.
Applications
A. Genetic Screening the testing for the presence of specific sequences associated with a disease condition (i.e the breast cancer gene)
B. Forensic Microbiology tracking DNA fingerprints to determine the source of an infection.