Keri+Maeschen

= DNA Cloning/Recombinant DNA/Genetic Engineering =

Gene Cloning with Bacterial Plasmids
__Describe__: Gene cloning is the process where genes are made into many copies. They are used to sequence the gene, produce its encoded protein, or in other types of research. Bacterial plasmids are DNA strands which are typically circular and double stranded that include an origin of replication, a marker gene, and a cloning site. Plasmids are used to transfer genetic material because they carry the genetic information. __Analyze__: First, the plasmids are isolated. Next, the plasmid and the DNA of the subject are cut out and the fragments join together. After this, the fragment is then inserted into the bacterial cell and it multiplies by mitosis. This is made up of DNA, plasmid, and the bacterial cell. __Apply__: This process is used for making copies of genes. Some examples include putting in a new gene into plants, such as corn, for pest resistance. Another example is using a cloned gene to alter bacteria to clean up toxic waste from things like an oil spill. __Synthesize__: I think it would be neat if somehow scientists could find a way to alter the genes to make humans constantly healthy. In other words, if there could be a way to make it so the cells could constantly be healthy and never be sick again. An example would be cancer, because sometimes cancer can come back, but if there could be a way to make the human body unable to get it again, there would be more healthy people. __Argue__: I am for this type of cloning, because it can protect and help living things. I live on a farm, so it is important that all of our crops are healthy in order to harvest a large amount of commodity. If new genes are in crops to protect from pests, it is helpful because of two reasons. One is that bugs will stay away from the plant and the other reason is that no pesticides will needed to be used and that is significant because the chemicals in pestisides are not very good for the environment.



For additional information about gene cloning with bacterial plasmids, click here.

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** Nucleic Acid Hybridization **
__Describe__: A strand of DNA is denatured and is added onto a strand of RNA or a probe. If you could see it, you would notice that at first the DNA is a strand but then a piece is taken off so there are two separate strands because they were split, although there is still a covalent bond. The nucleotides match up to an RNA strand and become a hybrid of DNA/RNA. __Analyze__: Hybridization take place when a nucleic acid probe is used. This probe finds a DNA sequence from a mix of DNA molecules. Each probe is bonded to a completmentary sequence by a hydrogen bond and is labeled with a radioactive isotope or flourescent tag so it is able to be visualized. __Apply__: This process is used to identify similar or identical genes in DNA from different types of sources. This technique can also detect pathogens. Things like chlamydia and gonorhoeae can be tested with this process. __Synthesize__: When I see this method, I think of a metal detector. I think of this because it is attracted to the metal and makes a sound or somehow notifies you, while the nucleic acid probe is attracted to the sequence. __Argue__: I am for this, because it can be used as a test to check for gene sequences in bacterial DNA and can help to diagnose many types of infections. The probes can detect problems to help find out what could be wrong with a patient.



Click here for more information about nucleic acid hybridization!

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__Describe__: This is the set of plasmid-containing clones that have copies of a segment from the genome. It is the collection of bacteria that have been made to hold DNA of a different type of organism. There are three different types; one is a plasmid library which appears to be like books in a library which are actually the clones. Each of the clones contains a copy of foriegn genomes. Another type is the phage library, which show genome segments. The last type shows bacterial artificial chromosome clones. These clones (known as BAC) are stored in a plastic plate with wells, kind of like a deep beehive. Each type of clone is inside one of the cavities of the plate. __Analyze__: In order to do this, DNA must be broken down and then organized. After this they are put into cloning vectors (plasmids) which can store different amounts and types. A genomic library can be made using phages. The fragments of foreign DNA is spliced and then made into a smaller version of a phage genome. A restriction enzyme and DNA ligase are used to do this and after infection, many new phage particles are produced. __Apply__: Genomic libraries are very important to scientists. They can use it to find DNA that they specifically need with probes and can also store the vectors by freezing them for another time when they are needed. With the help of a genomic library, scientists can learn more about the genomic function and structure of different types of organisms and can locate mutations. Another helpful thing is that they can be used to clone DNA when the vectors are replicated by the host. __Synthesize__: When i hear about a genomic library, I think of a cupboard. I think of this because the bacteria holds the DNA until it is taken out, like a cupboard holds things like food until it is needed and someone removes it. __Argue__: I am for this because it is very helpful to researchers. With this technology, researchers can clone and store DNA. They can also perform experiments on the DNA to see the differnet actions of seperate genes along the strand. They can also compare and contrast healthy and unhealthy organisms by viewing the different genetic coding.
 * Genomic Library **

To learn more about genomic libraries, click here.

Sources: [|www.wisegeek.com/what-is-a-genomic-library.htm], Campbell Reece AP Biology Book Picture from: []

__Describe:__ If you were to see a polymerase chain reaction, you would first see a DNA segment. Other parts that hel this process include //taq// polymerase, nucleotides, and primers. //Taq// polymerase is an enzyme that comes from hot springs bacteria and with this attribute, it is able to withstand a high amount of heat that derives from a PCR reaction. The primers are typically complementary in sequence to the DNA nucleotide and the free nucleotides also bind. Another characteristic is that it takes heat to do this reaction and it takes several hours to complete it with many repeating cycles. After 25 cycles, there are approximently 33,554,432 copies of the DNA. __Analyze__: First, it must be heated up to 95 degrees Celcius which denatures the DNA and breaks hydrogen bonds. Next, the temperature goes down to 60 degrees so the primers can bond to the complementary sequences on the DNA strand. Once again, the temperature is raised, but only to 72 degrees and the //taq// binds to the 3 prime end of the new strand. It then moves down the strand and adds new nucleotides to the ends. __Apply__: This can be useful for scientists who are analyzing DNA but need more copies than there actually are in a sample. It can also be helpful in DNA fingerprinting, virus detection, and diagnosing genetic disorders. __Synthesize__: When i think of the polymerase chain reaction, I think of a copy machine. A copy machine can make numerous copies of the same thing. The PCR is the same way because it copies DNA fragments by having the //taq// bind to the strand and binds the nucleotides to it to create copied DNA. __Argue__: I am for this because DNA is copied and is useful for scientists. It seems like this process would be quicker and more efficent than using the genomic libraries because once the DNA is replicated a few times, each strand continues to replicate making it double each time. The DNA is useful to the scientists because there are many copies that can be used.
 * Polymerase Chain Reaction **

Click here to watch a video about the Polymerase Chain Reaction! Sources: [], [], Campbell Reece AP Biology Book  Picture from: []

= Studying Expression and Function of Gene = Gel Electrophoresis __Describe__: If you were to visualize the process of gel electrophoresis, you would notice gel is used. Gel is used because it is kind of like a sponge and the gel pores are sized perfectly to enable the movement of molecules. An agarose gel can be used, which is made out of purified seaweed and the gel is put inside a tank filled with solutions so the process may take place. Other objects used in gel electrophoresis include a pipette, gel comb, colored DNA, an ultraviolet light, and a photograph. __ Analyze __: Gel Electrophoresis is the process of separating molecules, such as DNA, by their size and this is done by using gel in an electrical field. The phosphates in DNA have a negetive charge which then gives the entire molecule a negetive charge. Gel, usually aragose, is used to separate the DNA fragments and holes are made to hold the fragments before the process begins by using a gel comb. Many different sizes of DNA, which are colored so they can be easily identified, are put into the cavities. Since the DNA is negetively charged, they are attracted to the positive charge which is on the other side. The smaller DNA molecules move faster than the larger ones because it is easier for the smaller molecules to move along the holes in the matrix rather than the large ones and then molecular weight markers are used to decide the sizes of each DNA fragment. Sometimes a photograph is taken to visualize the results. __Apply__: This process can be used for restriction fragment analysis to create information about DNA sequences that can be very helpful. Restriction fragment analysis enables DNA fragments to be made by restriction enzyme digestion (or cutting) of the DNA molecules which are hen sorted by gel electrophoresis. When this hapens, the DNA molecules of viruses and plasmids can be seen by the restriction fragment patterns and it provides a way to make pure samples of each fragment. Another way restriction fragment analysis is useful is to compare and contrast DNA molecules by recognizing sequences in nucleotides or changes in base pairs. __Synthesize__: When I think of Gel Electrophoresis, I think of children and adults running through an obstacle course. The small children are able to move much quicker through the different obstacles and holes rather than the adults who are larger and cannot move as fast. This means that the small children typically make it to the end first, like the smaller fragments of DNA. __Argue__: I think it is important to know about Gel Electrophoresis. This process helps scientists recognize the different sizes of DNA molecules. THe process of restriction fragment analysis can tell the difference between the alleles of sickle cells and the normal ones.



For more information on gel electphoresis, click here.

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Southern Blotting
__Describe__: The southern blot technique is similiar to how gel electrophoresis works and looks. This process includes the aragose gel where DNA restriction fragments are placed and this part is the same as the electrophoresis process. When you next see the southern blot technique, you will notice that the DNA is transferred to a nylon filter which is a special kind of blotting paper. A probe is then added, which is radioactively labeled as nucleic acid. An x-ray film is also used to cover the nylon in order to find location. __Analyze__: Southern blotting is used to detect specific DNA fragments and to identify the size of the restriction fragment. First, the DNA must be isolated and digested with a restriction enzyme and after this they are added to the holes in the agarose gel, just like the beginning of the gel electrophoresis process. They then undergo the that process and the larger molecules stay back while the small ones move forward toward the positive pole. Next, the DNA is moved from the gel to a blotting paper such as a nylon filter and a nucleic acid probe labeled radioactively is added to the substance, which binds to the DNA. An X-ray film is placed over top to see which probes are visible and which cannot be seen. __Apply__: This technique was specifically designed to locate a DNA sequence in a mixture, but can be used in many processes including gene discovery, evolution study, and in forensics. It is also used to make sure the genetic sequence has been incorporated into the host organism's genome and is used to determine the molecular weight of restriction fragments. __Synthesize__: When I think of southern blotting, I think of a GPS. A Global Positioning System is able to find specific things and places in the world. This is similar to southern blotting because this process is used to locate DNA sequences. __Argue__: This form of genetic engineering is very important. Scientists can use southern blotting to find out more about Chronic Myelogenous Luekemia. Two genomic probes and three restriction endonucleases were used to determine results.



Click here to learn more about southern blotting!

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Mircoarrays
__Describe__: If you saw an example of the microarray process, you would see that it first starts with a tissue sample. DNA, mRNA, test tubes, a DNA chip, and microarray are all used to help this process. __Analyze__: This method allows thousands of genes in a single RNA sample to be measured. First, cells are grown to be activated or repressed for survival. Next, the cells are spun and are in pellets and the liquid is removed from the mixture. The mRNA is then extracted and RNA floats in the extraction buffer and is put in a new tube. Next, color DNA is created and mRNA is degraded. The color DNA is then mixed. A DNA pellet is then used and in each spot there is a sequence that will base pair with the colored DNA and all of this is incubated. After this, the DNA binds to specific spots in the pellet because they have the same base pair. Next, a microarray is used to detect the DNA by lasers. Each type of DNA shows up in the color of laser; the green dyed DNA shows up under the green laser and the red dyed DNA shows up under the red laser. __Apply__:This is important because it helps find new types of treatments for diseases. It helps with developing drugs, immunotherapeutics, and gene therapy. __Synthesize__: When i see this method, I think of cat urine under a black light because nder the black light, the cat urine glows. This is the same as microarrays because only specific types of DNA are seen under the lasers. __Argue__: I think this is a good method because people who need treatment for diseases are able to get it when needed.



For more information on microarrays, click here!

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= Cloning Organisms =

Plant Cloning
__Describe__: If you were to see plant cloning, you would first see fragments of the plant that is going to be cloned, such as a carrot. Next, you would see single cells, an embryo, and then finally a plantlet that will turn into the plant, such as a carrot. They are first planted in a test tube but later can be put into soil. The clone will physically and genetically be exactly like its parent plant. __Analyze__: First, fragments are cut out of the plant adn are cultured in a nutrient medium. This causes single cells to break away in the liquid and they begin to divide. Next, an embryo is formed from the cell and after it grows, a plantlet forms. The plantlet is first on a cultured agar medium but then put into soil to further the growing process. When it is fully grown, it should look exactly like the parent plant. __Apply__: This type of cloning is used in agriculture because certain plants with valuable characteristics are cloned. Plants that can resist a pathogen, for example, are cloned because this can protect the daughter plants and make production more successful. Some businesses who grow plants only use cloning as a technique to grow plants becaue all of the daughter plants will be the same as the parent. __Synthesize__: When I think of plant cloning, I think of mitosis because during mitosis many copies of the same thing are made. This is the same as plant cloning because the clones are exactly the same as the parent. __Argue__: I am for this because it is very important to agriculture. Without this, there wouldn't be as many food plants that are the best they can be because they would be grown normally with some being perfect and some with mutations.



Click here to learn more about plant cloning!

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Animal Cloning
__Describe__: If you were to see animal cloning, you would see cells from the mother and put into a culture medium. An ovary from another female, but a different one is used with a removed nucleus. A third female is used to implant both the nucleus and the ovary and a lamb is born after it grows. This means that during this process, three animals of the same species, but with different traits, are used. __Analyze__: First, mammary cells are taken from animal number one, such as a sheep. The cells are cultered and semistarved (this arrests the cell cycle) and causes dedeifferentiation. During this time, sheep number 2 donates an egg cell and the nucleus is removed. Next, the mammary cells and the egg cell are fused together so the nucleus of the mammary cell is inside the egg. The cell is cultured and creates an embryo and implanted in sheep number three to take on the growth of the embryo. It develops and a lamb is born that looks exactly like sheep number one, which was the mammary cell donor. __Apply__: Animals can be cloned to create animals exactly like its parent because it may look perfect. Cloning still has problems though because they do not have the same personality or act the same as the parent and usually the clone has problems such as obesity or premature death. __Synthesize__: This process reminds me of human twins. Human twins are very similar, but they are not exactly alike, like clones. __Argue__: I am against this because there are many problems with cloning animals. The clones have many defects and only a very small percent actually develop normally. Studies have show that mice who have ben cloned are very prone to obesity, pneumonia, liver failure, and premature death.



Click here for more information on animal cloning!

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= Practical Application of DNA Technology = = ﻿Restriction Fragment Length Polymorphism (RFLPs) = __Describe__: The main substance used in this process is DNA. In the DNA, the restriction enzymes recognize specific nucleotides and the DNA strand is cut, creating new lengths. The process of gel electrophoresis is also used. __Analyze__: Restriction enzymes first recognize sequences in the DNA nucleotides and are cut. The DNA rarely has the same sequences in the restriction sites, which is how they got their name polymorphic. After the cutting of the fragments, there are many different sizes which are separated by gel electrophoresis and creates a pattern __Apply__: RFLPs are used as genetic markers and can be useful in a couple different ways. One is in forensic science by using the "DNA fingerprints" during investigations. In this process they take the two different DNA samples, cut them, and see the similarities after the gel electrophoresis process and they also help to identify people who have a risk of obtaining a genetic disorder. __Synthesize__: When I think of this, I think of chopping carrots in a salad because the DNA is chopped into different sizes and is put in the gel electrophoresis to determine sizes. __Argue__: I am for this because this helps forensic scientists compare different types of DNA. When they identify the DNA, they can determine who is the victim or suspect.

To watch a video on RFLPs, click [|here]!

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Gene Therapy
__Describe__: About 1 in 10 people suffer from a genetic disease and gene therapy is a way to treat these people. Gene therapy is introducing genes into an individual for therapeutic needs to treat disorders from a defective gene. If you were to see the process of gene therapy, you would notice that a person is injected with normal alleles in places such as their bone marrow. The alleles injected must first be created using viral DNA, cells, and a normal allele. __Analyze__: There are four different ways to correct these genes. One- A normal gene is inserted into the genome to replace the faulty gene. Two- A normal gene through a homologous recombination is swapped with the faulty gene. Three- Reverse mutation is used to repair the nonfunctional gene. Four- The gene's regulation is altered. In most cases, though, a normal gene is inserted to replace the faulty one. A vector is used to carry the gene to the cell and one of the most common vectors is a virus because it can be genetically altered to carry DNA that is normal. There are typically four types of viruses used including retroviruses, adenoviruses, adeno-associated viruses, and the herpes simplex virus. During the process, cells are used to be infected with the viral vector, next the retrovirus infects the cells removed from the patient, then the viral DNA with the normal allele is put into the chromosome, and lastly it is injected into the patient. __Apply__: Gene therapy can be used to help treat many genetic diseases. It can be used to replace cells such as bone marrow cells if the patient has cancer. It can also treat viral infections, other cancers, and inherited disorders. This technique is only used to treat diseases and is still a risky and sometimes dangerous process. __Synthesize__: When I think of gene therapy, I think of a knee replacement on a person. During that process, a new knee is put into the patient so they can walk more efficently and live healthier. This is similar to gene therapy because a normal allele is inserted into a chromosome, which is then injected into the patient. __Argue__: I definately think this is a positive and important process. Even though there are still tests being done to improve it and make it safter, I still think this is helpful. I think it's helpful because this means that a normal allele is inserted into the gene and the cells are inserted into the patient. This process is much quicker and easier than surgery or taking medication.



Click here to learn about gene therapy and cancer!

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Transgenic Animals (Genetically Modified Organisms ~ GMO's)
__Describe__: A transgenic animal carries a foreign gene that was inserted into its genome. Many different types of animals have been tested including mice, pigs, sheep, rabbits, rats, and cows to become transgenic. __Analyze__: In order for this process to take place, a gene must be transmitted into an animal. There are three ways to do this including DNA microinjection (the most common), retrovirus-mediated gene transfer, and embryonic stem cell-mediated gene transfer. During DNA microinjection, a gene is taken from a different subject and the gene is transferred into the pronucleus of a reproductive cell and is grown on a culture medium. Lastly, it is at the embryonic phase and transferred to a female. When retroviruses are used, they are vectors so they can transfer the genes into a host cell. This animal is inbred until the transgenic offspring are born. In the embryonic stem cell-mediated gene transfer, stem cells are taken from an embryo, a gene is inserted into the cells, and are incorporated into the hosts embryo. __Apply__: There are two common reasons why they are produced. First, they are produced to help economically because some types of cattle were produced transgenically to carry a specific protein to produce milk. This type of milk with human proteins is possibly a treatment for emphysema. Another reason is because they are used to study diseases. Animals can be genetically engineered to carry diseases, including cancer, so scientists can study them to find new information to cure it for humans. This process can also be helpful in breeding farm animals to increase production. __Synthesize__: This process reminds me of a person who dyes their hair. They are the same person but with different traits. This is like a cow who is transgenic because it is the same cow but it may produce more milk. __Argue__: I am for this because it helps scientists study diseases and it helps farmers produce high quality milk or meats. Since scientists can study diseases on animals, they are able to help find cures for diseases in humans. There is a downfall to this process though, because animals then have to suffer through the disease so scientists can test and study them.



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Transgenic Plants (Genetically Modified Organisms ~ GMO's)
__Describe__: Transgenic plants are types of plants that contain a gene that has been artifically inserted into the plant through pollination. The transgene may come from the same type of plant or a completely different species. If you were to see a transgenic plant, you would see the plant and the genes that are implanted in them with a microscope. __Analyze__: There are a few ways gene transfer can take place. In electroporation, the DNA sequence is removed from a donor from restriction enzymes. Next, it is subject to the PCR because it amplifies the DNA. Then wall denaturing enzymes move the plant cell wall and the cells become protoplasts or plant cells stripped of the cell wall but still in the cell's membrane. A high electrical charge is sent through a protoplast solution which causes the membrane to form pores and through these pores the donor gene's DNA is injected. The cell wall forms back and the cells are placed in a culture to reproduce. Lastly, they are transferred to a regular environment to grow. __Apply__: There are many reasons why plants today are transgenic. They can resist insects, diseases, and herbicides which helps the environment because farmers do not need to use harmful chemicals to prevent their plants from getting sick or eaten. This technology can also improve nutritional value to foods. Rice, for example, can be transformed to produce beta-carotene, which is a precurser to vitamin A. When there is more vitamin A, the people who are deficient to this vitamin are able to eat rice in areas that need it including Africa. Biopharmaceuticals can also be made using transgenic plants because the genes for proteins used in medicine can be inserted into plants. __Synthesize__: I think this process can change by putting an animal cell into the plant cell. I'm not quite sure if that is possible or if it would work, but maybe it could cure diseases in plants or help the plant in some way. __Argue__: I think transgenic plants are very useful. This is because medication, healthy foods, and the production of foods is much more efficient and beneficial to people. With this process, farmers earn more profit because they do not need chemicals to guard their plants and there is much more growth in the plants to produce higher yeilds.



Click here for a video on transgenic plants!
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Genetic profiles (DNA forensic testing)
__Describe__: Genetic profiling is a process where labs determine specific characteristics of DNA by collecting and analyzing it to show the unique sizes of alleles. This is also known as a genetic fingerprint. __Analyze__: First, enzymes are used to cute DNA. Next, the DNA goes throught the process of gel electrophoresis (DNA is loaded into a gel with an electrical current. The size is determined by how quickly the DNA goes to the positive end. The smaller the DNA, the further it moves.). After this, probes are used to select pieces of DNA to view in the lab. These are kept in labs to view and use for identification of suspects or victims. __Apply__: They are used in forensics to identify people so investigators can settle personal estates. This process is not only used in forensics, but is also used to for future paternity DNA test and for those who have high-risk professions (firefighters, police officers, ect.). Another helpful thing genetic profiles can do is determine a person's chance of developing a diseases or characteristic that has been inherited. __Synthesize__: This reminds me of using an actual fingerprint to determine identity. Just like the fingerprints, DNA from one person cannot be the same size and sequence as DNA from another. Both methods are used to identify people. __Argue__: I am for this because it is useful for forensic scientists. They use this process to find the DNA and see if the DNA matches a suspect or the victim. Without this, criminals would not be able to be punished.

For more information on genetic profiles, click here!

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