Produce Useful Product Study Guide

BIOTECHNOLOGY -use of microbiological and biochemical techniques to produce useful products and solve practical problems 1. IDENTIFICATION TECHNOLOGIES 2. DNA CLONING TYPES OF IDENTIFICATION TECHNOLOGIES DNA SEQUENCING Determining the sequence of bases in a whole genome Human Genome Project DNA PROBES short, labeled, single strand of bases used to detect complementary sequence of DNA Colony BlotÝng, FISH, DNA microarrays DNA FINGERPRINTING Isolation and visualization of DNA sequences to help identify an unknown RFLPs and PCRs 1. DNA SEQUENCING -Human Genome Project  3 billion base pairs in humans  Approximately 25,000 genes  Lots of noncoding DNA (introns), found remnants of viruses -Other organisms  E.Coli- 4,500 genes  Mice -25,000 genes  Plants (Rice) – 55,000 genes Why do plants have more genes? -Needs more diversity b/c needs more methods of reproduction and survival 2. DNA PROBES  The probe is complementary some unique part of the organism  If probe attaches to unknown, probe will only attach to that particular sequence  Because it is labeled, there is evidence that the attachment took place (glow) Colony BlotÝng - we can use probes to look for organisms that were cultured (grown on a plate) -by doing this process, we don’t destroy all the unknowns because we don’t add probe directly to original plate Fluorescence in situ hybridization (FISH) – for non-cultured organisms Ex: adding probe directly into a pond water sample, cells would die, but we get wet mount instead and put under fluorescent microscope. DNA Microarrays (DNA Chips) -able to detect many genes at one time -have multiple test ran at the same time -Little holes = reaction area and probe looking for something 3. DNA FINGERPRINTING RFLPs Restriction Fragment Length Polymorphism CutÝng using restriction enzymes (DNA fragments all @ different lengths) Result: Fragments of DNA separated by size PCR Polymerase Chain Reaction Amplification the sequence of interest (producing more) Results: 1 thick band = amplified DNA sequence Restriction fragment length polymorphisms (RFLPs) o These restriction enzymes cut DNA at specific. 4-6 base pair sequences o some restriction enzymes cut symmetrical, and some make stager cuts o works by finding specific sequence on DNA strand that it recognizes cuts it o If cuts are made at different places for DNA strands, we know the strands have different DNA from each other We use gel electrophoresis to visualize Samples added to wells We compare the unknown fragment to known DNA fragments Electricity ON (DNA IS NEGATIVELY CHARGED, goes to positive side) Long DNA fragments are slow, short fragments are fast Fragments will separate by size, and migrate in a straight line after an hour of electricity, the fragments have completely separated and are called bands. we achieve a profile of bands and their sizes (each organism has a unique profile of bands) we can match the size of bands with the size of known DNA fragments Polymerase Chain Reaction (PCR) -amplifying a DNA sequence of interest, then running the amplified DNA on a gel to visualize 1) Starts with finding particular DNA sequence that you want to produce more of 2) Put DNA sequence through the thermal cycler 3) Add specific primers for specific gene, and bases If gene is present, you get multiple copies of it If the gene is not present, nothing happens (negative) 4) Run the amplified DNA on a gel like gel electrophoresis RESULT: one amplified gene that is the same size  stacks up on each other DNA CLONING -procedure in which a gene of interest is extracted and inserted into another cell, where it can be expressed and replicated -involves recombinant DNA (rDNA) and genetic engineering Recombinant DNA: DNA formed from joining DNA, from two sources (rDNA) Genetic Engineering: altering the organism by changing its DNA (the process) Procedure (gene from bacteria to another bacteria) 1. Obtain source with gene of interest 2. We need an origin of replication, usually a plasmid 3. We insert gene into the plasmid, which is then given to the cell 4. Product can either be a protein or multiple copies of the gene itself Examples of Protein products: insulin, human growth hormone (product of the gene) Example of gene products: frost resistance gene for plants 1. Get DNA from source. Must lyse the source to get the gene. (pop the cell) 2. Cut the DNA to get the gene using restriction enzyme, cutÝng it up into fragments -We want stagger cuts, so we can hybridize the cut DNA with something else - stager cuts leave sticky ends 3. Goal is to hybridize the fragments into the plasmid, we want it to be part of the plasmid 4. Cut open plasmid with same restriction enzymes 5. Fragment can now enter the plasmid 6. Plasmid with the gene of interest into the mass producer (host cell) -host cells: E. Coli, Bacillus Sub. -by artificial transformation (plasmids) -heat shock (plasmids) -Electroporation (plasmids) -artificial transduction for phages 7. Culture bacteria 8. Collect product If product is protein made by gene  lyse the cells (lysing) Or collect the secreted protein (B. Sub) Eukaryote to Prokaryote (Human to bacteria) -bacteria can’t handle the INTRON (non-coding DNA)  WE MUST HAVE cDNA Which is made from mRNA (introns already removed) Made from reverse transcriptase  cDNA can be put into plasmids, then given to bacteria Alternative host cells other than bacteria: -plant cells -animal cells -yeast DNA into host cells by: -gene gun (plants only) -micro injection (for animals) -artificial transformation -viral/bacterial- mediated gene delivery  introduce something to a pant Give gene to plant virus Have plant virus infect the plant Protein Products Vaccine Products Commercial PRODUCTS: EUKAROTIC TO PROKARYOTIC Insulin Factor 8 (Hemophilia) Hepatitis B. HPV Bovine Growth Hormone Chymosin (curdle cheese) Transgenic organism: foreign DNA in EVERY CELL aka GMO Concerns about Transgenic Organisms  introduces allergens/toxins/carcinogens  changes ecology by: -killing good insects, not just bad -create plant monocultures  we lose genetic diversity, b/c we only get one type of plant - escape and outcompete wild counterparts salmon getÝng into the ocean GMO Plants: pest resistance, herbicide resistance, golden rice, canola oil Edible vaccines: similar to the shot, but now in food 1. Find gene from pathogen that makes protein 2. Introduce it to a plant cell when it’s a single cell 3. Let it grow into a plant 4. Becomes transgenic plant 5. Eating the plant, we get the protein the pathogen makes 6. Body sees it as foreign  creating an immune-response GMO Animals: cancer mouse: oncomouse fluorescent mouse: brainbow fluorescent animals: marker for some other gene you are testing spidergoat Food production: AquaAdvantage fish Makes salmon grow faster Pharming: using animal products to make pharmaceuticals (milk or egg) -ATyrn Gene Therapy: -only changing dysfunctional cells (THE BAD ONES ONLY) -Replace dysfunctional gene w/normal one  Severe Combined Immunodeficiency (SCID)  caused by mutated ADA gene  Leber’s Congenital Amaurosis (LCA- A retinal disease) -Alter functioning of cell genetics “tweaking DNA”  Melanoma and blood cancers  HIV “immunotherapy”  using your own body to kill the cancer HIV Gene Therapy  Shortening the CCR5 gene receptor gene  Shorter gene = no receptor  Longer gene= receptor - we can cut the gene to get a shorter gene (mimicking the mutated one)