Biology: Applied Genetics and Biotechnology notes
(revised 11/2015)
Selective Breeding—increasing “good” alleles in an organism’s population by breeding individuals with desired traits.
Inbreeding—mating between closely related individuals. This practice has been used in animal husbandry (show dogs, race horses, farm animals, etc.) to insure that many desired traits show consistently in individual organisms. However, inbreeding also increases the chances of harmful recessive traits occurring. Biology mythbuster: inbreeding does NOT directly lead to mental retardation; many people think this, but research does not support it.
Hybrids—organisms created by crossing parents that were purebred for different traits.
Hybrid plants and animals are often bigger, stronger and more productive than purebred organisms. This is called “hybrid vigor”.
Genetic Engineering
Genetic engineering—a method of cutting a desired piece of DNA from one organism and inserting it into another.
Recombinant DNA—the DNA that is made by connecting (recombining) fragments of DNA from different sources.
Transgenic organisms—organisms that have functional pieces of DNA from other organisms in them.
Restriction enzymes—bacterial proteins that cut DNA at specific points in the nucleotide sequence. Hundreds of restriction enzymes exist.
Gel electrophoresis—fragments of DNA are separated by passing them through a gel that has electric current running through it. Negatively charges pieces of DNA move from wells at one end towards the positive electrode at the other. Small fragments move farthest, large fragments move least. This is similar to paper chromatography (used in the chlorophyll pigment lab).
Vectors—anything used to carry DNA into a new organism.
Mechanical
Micropipette—tiny needle-like pipette injects DNA into a cell.
DNA bullet—tiny DNA coated bullet is shot into a cell with an air gun.
Biological
Viruses—viruses that have desired pieces of DNA in them can transfer the foreign DNA into a new organism.
Plasmid—a circular piece of bacterial DNA can carry the foreign DNA into a new organism.
Gene cloning
When a piece of DNA has been introduced into a new species, that foreign DNA will be reproduced every time the new cell divides. Transferring human DNA to bacterial DNA can result in millions of copies made in a short time. Currently this only works for short pieces of human DNA.
Reproductive cloning
Cloning one gene is much simpler than cloning an animal’s entire genome. Ian Wilmut created Dolly the cloned sheep (1996) by taking an udder cell (any 2n somatic cell should work) from an adult sheep and fusing it with an egg cell that had its nucleus removed (called “enucleated”.) The enucleated egg cell has no DNA. The DNA from Dolly’s udder cell became the egg cell’s DNA. The fused egg was implanted back into a surrogate mother (intentionally not the cell donor sheep to prove the clone was not like the surrogate), and Dolly developed normally from there. Cows (@UConn), pigs, mice, monkeys and a horse (the only true clone made yet since cell donor was also the surrogate mother) have been cloned. Reproductive cloning success (<0.5% births per attempt; lots of birth defects) has not improved in 13 years since Dolly and has almost been abandoned completely except...Big news: Scientists have the entire genome of the extinct woolly mammoth (gotten from a frozen mammoth carcass, 2008). They found out the Asian elephant is its closest living relative and are trying to clone a mammoth with an Asian elephant as surrogate mother, no success yet. Stay tuned!
DNA sequencing
Cloning billions of sections of DNA allows the sequence of DNA to be determined. This technique allows genome sequencing (see Human Genome Project)
Uses for recombinant bacteria (gene clones grown in bacteria)
Industry
· Used to clean up oil spills. The bacteria “eat” the oil and break it down into harmless substances.
· Being developed to extract minerals from ore (bacteria “eat” the ore, leave the minerals.)
Medicine
· Produce insulin to treat diabetes, and human growth hormone used to treat dwarfism.
· Used to produce phenylalanine that is used to make aspartame (Nutra-sweet®).
Agriculture
· Bacteria prevent frost damage in strawberries, and produce nitrogen fertilizer for plants.
Uses for transgenic organisms (also called GMO’s or genetically modified organisms)
Plants
· Have been engineered to resist herbicides, resist pests, and to increase the protein or vitamin content of the plant (e.g. golden rice has vitamin A added to it.) The majority of corn, cotton and soybeans grown in the U.S. and world are GMO.
Animals
· Animals have been created with human diseases, so the cure for those diseases might be found without excessive human testing. Mice given human Huntington’s disease and Alzheimer’s have led to breakthroughs in treatments.
· Pigs (cow milk and chickens soon) with omega-3 fatty acids (good for the heart). The natural source of omega-3 fatty acids is some oily fishes like tuna and salmon, but they are overharvested and often have high mercury levels in them.
· Glo-fish—the gene for gfp (green fluorescent protein) from a jellyfish was added to create a novelty pet. Not as well-known, (but way more important) this discovery led to a Nobel Prize in medicine (Shimomura, Chalfie, Tsien 2008) since the gene is tacked on to other GM attempts, allowing visual proof that cells in a sample or organism got the new gene being studied.
The Human Genome
Genome = the complete set of genes for an organism.
The human genome contains approximately 21,000-23,000 protein coding genes, made up of about 3 billion base pairs. (ATACGACCTG, etc., 3 billion times!) All bases have been sequenced (finished 2001) but exactly what each gene is or does isn’t yet known. Up until 2001, it was thought that the human genome might contain around 100,000 genes because that is how many different proteins are in humans (this was known as the "one gene-one protein hypothesis from 1941.) Scientists now know that many genes can make more than one kind of protein (the same sequence is edited in different ways). 98.5% of the 3 billion pairs are “junk” (do NOT code for any proteins). Many scientists originally thought the “junk” is old viruses that have infected our genome over the billion years it has evolved, but are ignored by the cell when making proteins. MicroRNA (miRNA) and other gene control factors are now known to be coded for by the “junk”. The current scientific term for “junk” is non-coding DNA, because it does not code directly for proteins.
How did they do it?
In order to sequence genes, thousands of copies (clones) of the gene are needed. A technique called PCR (polymerase chain reaction) allowed machines to clone DNA. The process involves heating and cooling DNA fragments (to unwind them) and using DNA polymerase enzyme to induce natural replication. The problem is that DNA polymerase from eukaryotes can’t be heated and cooled without breaking down. PCR works because an American scientist Dr. Kary Mullis (Nobel Prize 1993) had the simple, brilliant idea to use DNA polymerase enzyme from bacteria that survive in hot springs in Yellowstone NP. The copies are then cut with restriction enzymes. Each clone is cut so that it is one base shorter than the other. That way, the last base on each piece is known. The pieces are then sorted by “tagging” the last base in the piece with a different colored dye (i.e. yellow for T and red for A, etc.) and the order is determined.,
Why bother? The human DNA sequence is used for a variety of applications scientists thought impossible just 30 years ago:
Current (or very near future) uses:
· Diagnosing genetic disorders accurately before birth. Create a DNA chip listing a person's entire DNA sequence. Good genes or bad genes will all be known without having to wait for the problem to occur.
· Used for making normal genes for gene therapy. Gene therapy involves inserting normal genes (by using a vector) into a human cell hoping they will take over for "bad" genes. Recent breakthroughs: cure red-green colorblindness in primates, restore vision to a boy blind with Leber’s Congenital amaurosis, halt progression of Alzheimer’s disease and restore some nerve function by introducing NGF (nerve growth factor) to patient's cells.
· Gene editing. More direct than gene therapy, bad genes are removed from a cell' DNA and replaced with good genes. No vector is needed and no "hoping" the vector successfully takes over enough cells with "good" genes. A technique called TALENS (Transcription activator-like effector nucleases) was used in Nov. 2015 to treat a baby with leukemia. T-cells (part of the immune system) were edited three ways: to attack the leukemia, to resist chemotherapy drugs, and to not trigger the patient's immune system to attack the foreign T-cells back. CRISPR (clustered regularly interspaced short palindromic repeats) technique has successfully modified genes in cells in human embryos (in China), and in another test it was used to modify T-cells more cheaply and effectively than the TALENs technique. A human test for treating LCA blindness w/ CRISPR T-cells is proposed for 2017. CRISPR is being used to delete genes in pig organs that make proteins that trigger immune responses in the recipient of an organ donation. The hope is to use pigs as organ donors for humans.
· Giving cell cultures a genetic disease so the cells, not a whole test organism, can be studied for a disease cure. Fewer lab rats, monkeys, etc. that need to be harmed in search for a cure. Only promising treatments will go on to further testing.
· DNA “fingerprinting”. Because every person’s DNA is unique, it is the best way to identify suspects from crime scene evidence. DNA from a crime scene can be matched to a suspect’s DNA with almost 100% certainty (more than 1.8 million “markers” or potential match points are known), far better than traditional fingerprints which only uses 7 matching factors.
Future (in your lifetime) uses (currently these are still in research, but likely available in 10-30 years):
Ø Cure many forms of cancer, AIDS, CF, MD, sickle cell and other diseases that involve specific changes to normal genes. Gene therapy works for some treatments already; more will be here soon. A more promising breakthrough for more complicated diseases is gene “silencing” with a microRNA piece. Bad genes can be permanently stopped without actually fixing them.
Ø By knowing what genes cause certain side effects with medicines, pharmacists can provide individualized medicines that will work as close to 100% efficiently as possible for each person’s specific genetic make-up.
Stem cells – are undifferentiated, which means they do not have the specialized genes turned on to make them be a particular kind of
cell (such as a liver cell, blood cell, bone cell, etc.)
· Embryonic stem cells-come from 3-5 day old embryos donated to science. Benefit: they can become any kind of cell (fancy word for that is “pluripotent”) and reproduce forever. Drawback: the embryo dies when the cells are collected, very controversial for human embryos.
· Adult Stem cells-found in a few tissue types in adults, such as heart, blood and bone. Benefit: can be collected without harm. Drawback: not pluripotent, limited cell reproduction lifespan.
· Induced pluripotent stem cells (iPSCs) – discovered by Shinya Yamanaka in 2007 (won 2012 Nobel Prize for it, fastest award ever). Yamanaka discovered how to reverse differentiation of any adult cell back to a pluripotent stem cell.
Uses-many human diseases that are degenerative (happen as cells get “old” and no longer do their job well) are likely to be treatable with stem cells. Good examples are Alzheimer’s, Parkinson’s, ALS (Lou Gehrig’s disease), heart disease and diabetes. Hockey Hall of Famer, former Whaler and former Glastonbury resident Gordie Howe received stem cell treatment (in Mexico) in November 2014 after a stroke that left him unable to walk or talk. He can now do both. NY Mets pitcher Bartolo Colon had successful stem cell treatment on his shoulder that healed damage that had reduce is fastball by 20 MPH.