Biotechnology and Recombinant DNA Chandler Community College

Biotechnology Biotechnology Foods Vaccines Antibiotics Vitamins The use of microorganisms, cells, or cell components to make a product Foods Vaccines Antibiotics Vitamins Mining – extract minerals from ore

Recombinant DNA Usually commercial or medicinal value Insulin Vaccines Amplify DNA Enzymes Hormones Insect resistant plants Recombinant DNA (rDNA) Also called genetic engineering Inserting genes into cells to make chemicals that the organisms do not naturally make

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