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DNA Technology and Genomics Human Genome Project Started in 1990 Goal was to map the human genome by determining the sequence of nucleotides in human DNA. o Information available on the Internet for people to access. Completed around 2003 Benefits: o Embryonic development o Evolution o Disease diagnosis and treatment o Prevent heart disease, allergies, diabetes, schizophrenia, alcoholism, Alzheimer’s and cancer Humans have roughly 25,000 genes – fewer than expected o A human gene might code for more than one polypeptide utilizing alternative splicing. 97% is noncoding DNA o Gene control sequences – promoters and enhancers o Introns o Noncoding regions between genes o Telomeres – repetitive ends on DNA Download the human genome Human Genome Project: After the human genome was sequenced, scientists started on scientifically valuable organisms o Over 180 eukaryotic species Yeast * Worm * Mustard plant * Fruit fly * White lab mice * Mosquito Dog Rat Chicken Frog Neanderthal o Around 4000 bacteria (Archaea and Eubacteria) Eukaryotic Genomes Largest – rare Japanese flower – 149 billion base pairs (50X the size of the human genome) o Human – 3 billion base pairs Smallest – mammalian parasite – 2.25 million base pairs Comparisons of genome sequences from different species allows us to evaluate evolutionary relationships o More similar genome = more closely related The success of genomics has focused attention on proteins Full set of proteins – proteomes o Proteomics – Studying the whole set of proteins and their interactions. Restriction Enzymes – Biological scissors An enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Gel Electrophoresis and DNA Sorting Gel electrophoresis is a technique that uses a gel (jellylike substance) to sort DNA/RNA/Proteins based on their size or electrical charge. Steps: DNA samples are exposed to the same restriction enzymes DNA samples are loaded into different wells o Each sample contains DNA fragments of different lengths An electrical current is applied (negative at the top, positive at the bottom) DNA has a strong negative charge so it moves towards the positive end (bottom) Smaller fragments move through the spaces in the gel faster than longer fragments Gel electrophoresis allows numerous samples to be compared based on their number of fragments and fragment length Must pick variable region (introns) Since your DNA is unique to you (except identical twins*), your number of fragments and their lengths will be unique as well – DNA fingerprint This technique can be used to compare a suspect’s DNA to samples collected at a crime scene This technique can be used to determine if a person is carrying a harmful allele After gel is produced, use DNA probe complementary to the gene in question to determine if the gene is present o One or more fragments will be detected http://www.youtube.com/watch?v=UYAGhRi30oM https://www.youtube.com/watch?v=mN5IvS96wNk Gene Probes How do scientists find the right DNA fragment to insert into the plasmid or make a fragment more unique? Scientists can use their understanding of complementary base pairing if a gene sequence or partial sequence is known. Use radioactive isotopes to produce a radioactive complementary strand to the gene called a nucleic acid probe o Gene with TAGGCT o Probe has ATCCGA Use heat or chemicals to separate the DNA then mix with the probe Stem Cells A zygote divides by “Mitosis” to produce a multicellular embryo made of stem cells – undifferentiated cells. The cells in the first few rounds of division are Totipotent – they can differentiate into any type of cell. As the embryo develops further, the cells begin to specialize and lose their ability to differentiate into any type of cells. These cells are Pluripotent. Also known as embryonic stem cells. These cells can become any body cell, but not all cells of the original embryo. Adult stem cells produce new cells for tissues that need continual renewal. These cells are Multipotent. They can produce a limited number of cell types. Skin Blood cells Animal Cloning Cloning History: 1979 – First genetically identical mice produced by embryo splitting. Clones produced by nuclear transfer of embryo into an emptied nucleus (cows, sheep and chickens) 1996 – First mammal clone from a body cell taken from an adult animal (sheep – Dolly) C 1998 – Cloned eight calves from a single cow Somatic cell cloned animals: Sheep Cattle Cat Deer Mule Ox Rabbit Rat Embryo splitting: Rhesus monkey Dog Horse A B C Basic steps: 1. A diploid nucleus is removed from sheep A. 2. The nucleus is removed from an egg from sheep B. 3. The nucleus from sheep A is transferred to the empty egg from sheep B. 4. Electricity is used to stimulate mitosis. 5. The multicellular embryo is transferred into sheep C for gestation. 6. Sheep C gives birth to the lamb. To which sheep is the lamb identical (a clone)? Would you pay $100,000 to clone a pet? Chimera Injecting cells from one species into the embryo of another creates mixtures called chimeras. From left to right: an ordinary mouse, a mouse that’s partly rat, a rat that’s partly mouse, a white rat. Chimeras are defined as organisms composed of cells or genes obtained from two or more different organisms or species. Recombinant DNA Technology Recombinant DNA Technology – Laboratory techniques for combining genes from different sources (same species or different) into a single DNA molecule. Used to alter the genetics of organisms to make them more useful to humans o Use bacteria as chemical factories to produce human proteins. o Make plants more drought resistant o Increase the growth rate of plants/animals o Make animals glow in the dark Heavily dependent on bacteria. Why? o Plasmid – small, circular double stranded DNA molecules Carry few genes Replicate on their own Great for gene cloning – multiple copies of gene-carrying piece of DNA Are passed from bacterial cell to cell Great for recombinant DNA – DNA in which genes from two different sources are combined into the same DNA molecule How do we get the “gene of interest” out of the original chromosome/DNA and into the bacterial plasmid? Use Restriction Enzymes Restriction Enzymes and “Sticky Ends” Restriction enzymes are the cutting tools used to remove a gene from one molecule of DNA and to open the new DNA molecule for “pasting”. Original use was to protect bacteria from foreign DNA that enters their cell. Restriction enzymes recognize specific DNA sequences and cut the DNA at points within the sequence. o Need cuts that leave jagged, “sticky ends” Between which nucleotides was the molecule cut? A and C How can a new gene be inserted into the broken DNA molecule? Cut it out with the same restriction enzyme. It will have complementary “sticky ends” allowing base pairing. What enzyme do you think is used to covalently bond the sugars/phosphates to complete the DNA backbone? DNA ligase The final result is a recombinant DNA molecule Restriction Enzymes http://www.dnalc.org/resources/animations/restriction.html https://www.youtube.com/watch?v=lWXryzgRces Bacteria with recombinant DNA plasmids can be used as biological factories to clone useful genes for humans. Ex. Insulin – taken for diabetes In the past, people used insulin produced by other animals. Similar to human insulin o Rejection/allergic reactions possible Now we inject human genes into bacteria and have them make human insulin. Identical to insulin produce by humans o No rejection/reaction What is used to cut the plasmid? Restriction Enzymes How many times is the plasmid cut? What is used to cut the DNA? 1 The same restriction enzyme How many times is the human DNA cut (minimum)? 2 What would you do? 1. You are a tomato farmer whose crops are threatened by a persistent species of beetle. Each year, you spend large sums of money for pesticides to protect your crops. A biotechnology company introduces a new strain of tomato plant that produces a natural pesticide, making it resistant to the beetle. By switching to this new strain, you could avoid both the beetle and the chemical pesticides traditionally needed to fight it. 2. As a family physician, you often treat children who suffer from infectious diseases that could easily be prevented through vaccination. But the parents of many of your patients cannot afford the cost of vaccinations. You hear of a new approach that would reduce the cost to a fraction of its current price: genetically modified fruits and vegetables that contain various vaccines. By simply eating a banana, a child could be protected against disease— without getting a shot! 3. You are the leader of a developing nation. Hunger is a problem among your citizens: the salty coastal wetlands of your country can't support the growth of needed crops, and your slow economy can't support importing enough food for everyone. A biotechnology company has genetically modified a rice plant that can thrive in salt water, providing your nation with the opportunity to feed its citizens while bolstering its economy. Genetically Modified Organisms (GMO’s) have acquired one or more genes from artificial means rather than by traditional breeding methods. A term usually associated with commercially available foods. Grow faster Produce greater fruit Improved nutritional value Drought resistance Chemical/herbicide resistance Cold weather tolerance If the newly acquired gene is from a different species the GMO is called a transgenic organism. Concerns are being raised about long-term consequences to human health and the environment, as well as access to resources. Allergens Organ damage Resistance transferred to pest species Harm nontarget species Affordable seeds SciShow Bill Nye Are GMOs Good or Bad? Organisms that have been genetically engineered: In 2007, South Korean scientists altered a cat’s DNA to make it glow in the dark and then took that DNA and cloned other cats from it, creating a set of fluffy, fluorescent felines. Scientists have recently taken the gene that programs poison in scorpion tails and combined it with cabbage. Why would they want to create venomous cabbage? To limit pesticide use while still preventing caterpillars from damaging cabbage crops. These genetically modified cabbages produce scorpion poison that kills caterpillars when they bite leaves — but the toxin is modified so it isn’t harmful to humans. Strong, flexible spider silk is one of the most valuable materials in nature, and it could be used to make an array of products — from artificial ligaments to parachute cords — if we could just produce it on a commercial scale. In 2000, Nexia Biotechnologies announced it had the answer: a goat that produced spiders’ web protein in its milk. Researchers inserted a spiders’ dragline silk gene into the goats’ DNA in such a way that the goats would make the silk protein only in their milk. This “silk milk” could then be used to manufacture a web-like material called Biosteel The Enviropig, or “Frankenswine,” as critics call it, is a pig that’s been genetically altered to better digest and process phosphorus. Pig manure is high in phytate, a form of phosphorus, so when farmers use the manure as fertilizer, the chemical enters the watershed and causes algae blooms that deplete oxygen in the water and kill marine life. So scientists added an E. Coli bacteria and mouse DNA to a pig embryo. This modification decreases a pig’s phosphorous output by as much as 70 percent — making the pig more environmentally friendly. Starlight Avatar