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Biotechnology • Often referred to as genetic engineering, it’s basically how scientist and researchers manipulate genes. • Recall, DNA is the genetic material of all living organisms that carry genetic code that may code for proteins. • Genes from one kind of organism can be transcribed and translated (protein synthesis!) when put into another kind of organism. • For example, human and other genes are routinely put into bacteria in order to synthesize products for medical treatment and commercial use. Human insulin, human growth hormone, and vaccines are produced by bacteria.
First, What is……………… • Recombinant DNA DNA from different sources sliced then spliced together. • Called transgenic • Plasmid Vector Circular, double-stranded DNA used to transfer genes into a host bacterial • Viral Vector Harmless virus typically used in gene therapy • Bacterial Transformation The host bacterium, such as E. coli, takes up the plasmid, which includes the foreign gene. Bacteria will replicate the foreign DNA along with their own DNA producing wanted protein such as human insulin • Restriction Enzymes proteins that digest DNA at specific regions for preparation of bacterial transformation
Recombinant DNA– Insertion of a Functional gene from one organism into Bacterial Plasmid Transformation*This process is pretty involved but here’s the basic idea
*Recombinant DNA Technology – Bacterial Transformation and Cloning of a target gene*Insulin is created this way
First Step – “cut” DNA OF PLASMID VECTOR AND HUMAN (IN THE CASE OF INSULIN GENE) WITH THE SAME ENZYME CTTAAG Backwards ** These enzymes, CALLED RESTRICTION ENZYMES, recognize a very specific, single palindrome nucleotide sequence. ** Palindromes are sequences that are the same forwards as they are backwards…like the word racecar EcoRI CTTAAG Forwards
Restriction Enzyme EcoRI Step 1: A Restriction Enzyme Cleave DNA of Vector and Foreign DNA • Restriction enzyme (green guy called EcoRI) recognizes nucleotide sequence specific to that enzyme • When it recognizes the palindrome, it cuts the DNA ATTCGCGGAATTCATTACGCATCG TAAGCGCCTTAAGTAATGCGTAGC ATTCGCGG TAAGCGCCTTAA AATTCATTACGCATCG GTAATGCGTAGC ** “Sticky” ends are created (the black letters) that stick out. This allows a gene, or other segment of DNA, to be inserted by base-pairing **The black letters represent the palindrome recognition site
Ligase Step 2. Inserting the Gene Into Bacterial Plasmid Vector • The human insulin gene is inserted at the “Sticky” ends of the plasmid at the palindrome through base-pairing • Another enzyme “glues” (ligates) them together GAATTGCTAACTTACGCATGGCAATTC CTTAACGATTGAATGCGTACCGTTAAG ATTCGCGG TAAGCGCCTTAA AATTCATTACGCATCG GTAATGCGTAGC **The yellow letters represent the gene for human insulin (of course it is much larger than this, you get the idea!). The white letters are just the restriction site. **The black letters represent the binding site of the plasmid
Step 3. Host Cell (E. coli here) Takes Up The Plasmid Vector *NOTICE…Plasmid vector also carries a gene that makes them resistant to ampicillin, an antibiotic that kills bacteria!! This is important in the next step, cloning and the production of insulin!!
Step 4. Bacteria Growth ?? **Bacteria are spread out on petri dishes that contain a medium (food) and ampicillin. (or other antibiotic) They grow, creating clones of themselves. **No growth..bacteria were killed by ampicillin thus contained NO vector and NO insulin gene **Growth!! Bacteria were ampicillin resistant thus contained the vector that carried the insulin gene!!
Step 5. Collecting Cloned Product **Those colonies that contain plasmid vector are allowed to grow, cloning themselves. As they do so, the gene for insulin is expressed (protein synthesis!!!) thus the protein is produced. Eventually, the crude protein can be extracted, purified and you’ve got insulin! **This is done on a much wider scale than we’ve learned here. Pharmaceutical companies produce enough insulin for millions of Americans who have insulin-dependent diabetes.
In Summary…… ** “Slice” a gene from a donor and “Splice” it into the vector…bacteria in this illustration, but viruses can be used too!!
PCR – Performed before gel electrophoresis • Artificial “cloning” of DNA strands in a machine called a thermocycler • Heat separates the DNA strands from each other • Nucleotides (A,G,T,C) are added in reaction tubes • Put into a PCR machine called a thermal cycler • Added nucleotides find their compliments, and millions of strands of DNA are cloned
What is it? • Simply put, gel electrophoresis is a technique used to separate molecules such a DNA, RNA and proteins according to size (number of bases) • Requires an electric current as these molecules are negatively charged • We’ll be looking at separating DNA into strands of varying sizes to determine the sequence of a DNA fragment as well as determining DNA fingerprints
First, DNA is Extracted • The same DNA is found in the nucleus of nearly all of your cells (mature red blood cells have no nucleus). • Mitochondrion have a totally different set of genes than what is in the nucleus. You inherit them from your mother! Many studies use this DNA to determine how closely related different species of organisms are! • Nuclear DNA can be extracted from blood, cheek cells, hair follicles, tooth pulp or mostly any other tissue • There are quite a few protocols that use a variety of chemicals and buffers to isolate ONLY DNA and get rid of everything else
Restriction Enzymes • Long strands of DNA, restriction enzymes • Each enzyme recognizes different sites to cut • This results in many fragments of different sizes (number of bases long)
Prepare for gel electrophoresis After the gel cools and becomes jelly-like, the comb is removed and is placed in the chamber filled with buffer Power supply A compound called agarose is melted in a buffer, poured into a mold and a tooth comb inserted to create wells Gel Chamber with buffer
“running a gel” A loading dye (seen in blue) is mixed with DNA sample and loaded into a well Electrical supply is turned on…DNA fragments run through the gel from the negative electrodes toward the positive DNA Ladder (or size standard) is also loaded. This lets allows you to determine how long each fragment is (in kilobases) The gel is stained and examined. The smallest (shortest) fragments travel the farthest down the gel
Reading an Electrophoresis Gel….DNA Sequence • Determine the sequence of bases in a gene, or DNA fragment…read from top to bottom **This particular sequence is: ATGCTTCGGCAAGACTCAAAAAATA **Notice it’s one stranded!
Sequences That are Non-CodingCan Be Used to Produce a DNA Fingerprint • ID of individuals • ID of parents • Crime scene applications **Restriction enzymes cut DNA , fragments that contain repeats such as AGGTAGGTAGGT, over and over but the number of repeats differ among individuals. We can run these on a gel (CSI stuff) and see their DNA fingerprint!
Example of Fingerprint (Remember, these are not genes) **Suspect 3’s Fingerprint is a perfect match of the blood stain!!!
Gel Electrophoresis uses an electric current. DNA fragments that have been cut by enzymes separate by size. The pattern gives you a “fingerprint” - Largest Fragments ATTCGCGG TAAGCGCCTTAA AATTCATTACGCATCG GTAATGCGTAGC Smallest Fragments +
Paternity A person should have ½ of their DNA fragments that match their mother, and ½ that match their father. Mom Child AF1 AF3 AF2 The child received these fragments from its mother… - …So Alleged Father #2 is the father of the child. ..which means that they must have received these from the father… …so, which alleged father has the same size fragments as the child at these positions??? +
Lab Animals • Mouse chromosomes are similar to humans’ chromosomes and used in many studies
Lab Organisms • The roundworm Caenorhabditis elegans is another organism with well-understood genetics that is used for transgenic studies.
Lab Orgsanisms • A third animal commonly used for transgenic studies is the fruit fly, Drosophila melanagaster
Genetically Engineered (slice and splice) Transgenic Organisms Contain Recombinant DNA • Plants and animals that contain FUNCTIONAL foreign DNA from another organism are known as transgenic organisms • Such as E. coli bacteria plasmid that contains the gene for human insulin.
Genetically Modified Foods • Crops have been developed that are better tasting, stay fresh longer, and are protected from disease and insect infestations. • Soybeans that contain a component that helps lower cholesterol • Alfalfa that’s resistant to herbicides
Pros and Cons Pros: *More nutritious food *Tastier food *Disease- and drought-resistant plants that require fewer environmental *resources (water, fertilizer, etc.) *Decreased use of pesticides *Increased supply of food with reduced cost and longer shelf life *Faster growing plants and animals *Food with more desirable traits, such as potatoes that absorb less fat when fried *Medicinal foods that could be used as vaccines or other medications Cons: *Modified plants or animals may have genetic changes that are unexpected and harmful. *Modified organisms may interbreed with natural organisms and out- compete them, leading to extinction of the original organism or to other unpredictable environmental effects. *Plants may be less resistant to some pests and more susceptible to others.
In Medicine • Transgenic bacterial used to produce insulin, hormones, antibodies, enzymes, vaccines…It’s crazy!
Other Genetically Modified Organisms (GMOs) I Gots me a big ole rooster…. names Jethro
Transgenic Animals • Transgenic cattle were created to produce milk containing proteins that aid in the treatment of emphysema. • Transgenic Horses were once created to produce human insulin for diabetics; Now we us E. Coli
Mice that Glow ** insertion of a gene taken from coral and inserted it into the mouse genome
Sheep Milk That Contains a Clotting Protein that is Isolated for People with Hemophilia
Other Transgenics • Transgenic cattle were created to produce milk containing proteins that aid in the treatment of emphysema. • Transgenic Horses were once created to produce human insulin for diabetics; Now we us E. Coli
Hybrid Animals – Genetically Modified but not Transgenic Lion and Tiger hybrid
Recall Bacterial Transformation • Transformed bacteria that contain a functional gene replicate creating millions of clones thus a lot of product (whatever the gene codes for)
Cloning of Entire Organisms • A clone is basically a copy of an organism that contains the same DNA…but, since the clone is carried by a surrogate organism, it won’t be exactly the same… • Dolly the sheep is the most famous cloned animal…although it took numerous tries! • What are the ethical aspects of cloning??