Kendall+Lindeman

__ Part 1~ DNA Cloning/Recombinant DNA/Genetic Engineering __ **__ Gene cloning with bacterial plasmids __** Replication of DNA, but with the use of recombinant DNA instead. It is done //in vitro.// Foreign DNA is inserted
 * __ Description __**


 * __ Analyze __**
 * 1) Isolate plasmid DNA from bacterial cells.
 * 2) Cut both DNA samples with the same estriction enzyme, preferably one that makes a single cut within the lacZ gene and many cuts within the DNA.
 * 3) Mix the cut plasmids and DNA fragments. Some of them will join by base pairing. Add DNA ligase to seal them together. The products are recombinant plasmids and many nonrecombinant plasmids.
 * 4) Mix DNA with bacterial cells that have a mutation in their own lacZ gene. Some cells take up a recombinant plasmid or other DNA molecule by transformation.
 * 5) Plate the bacteria on agar containing ampicillin and X-gal, a molecule resembling lactose. Incubate until colonies grow.[[image:riedell-dna-tech-project-2012/gene_cloning_with_b_plasmids_rig.jpg width="157" height="238" align="left"]]


 * __ Apply __**
 * Gene cloning is the production of multiple copies of a single gene to produce a protein product
 * Gene cloning can be used to treat stunted growth
 * Used to create more proteins that dissolve clots in heart attack therapy

__** Synthesize **__ It reminds me of when they cloned the sheep Dolly. Of course they both have to deal with cloning, so I bet it reminds a lot of people about that. It reminds me that it uses recombinant DNA instead because they used a sheep instead a human.

__** Argue - with **__ I like this because it is important when it comes to a couple health issues such as stunted growth and heart attack therapy. It also plays a part in the making of pest resistance crops.

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**__ Nucleic Acid Hybridization __** Nucleic acid hybridization is a technique in which single stranded nucleic acids, including DNA or RNA are allowed to interact so that complexes called hybrids are formed by molecules with similar complementary sequences. Can be done using any combination. DNA-DNA, RNA-DNA, RNA-RNA It is also a screening of a library for clones carrying a certain gene of interest
 * __ Description __**

__** Analyze **__ The main function of this process is to detect a genes detect a genes DNA by observing the ability to base pair with a complementary sequence. Nucleic acid probes are needed. They are complementary molecules that are short, and single stranded nucleic acids that can either be DNA or RNA. You only need to kow part of a sequence of the gene of interest in order to synthesize a probe that is complementary to it. Example: 5' ...GGCTAACTTAGC...3' (desired gene) 3' ...CCGATTGAATCG...5' (probe) (example found on page 402)

__** Apply **__ This technique is used to detect and isolate specific sequences, measure homology, or to define other characteristics of one or both strands. Example: Test a samle of human DNA for mutations in the gene suspected of causing an inherited disease

__** Synthesize **__ This reminds me of when the try to match pople fingerprints on CSI. It basically has the same concept where they are trying to trace someone (or a certain gene), and they are trying to match them with a fingerprint (DNA/RNA sequence with a probe).

__** Argue - with **__ This is an important topic because of the fact that it is essential when it comes to things such as tracing diseases down a family's history. Not only does it help traces diseases, but it helps us find a gene of interst in general.



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**__Genomic Library__**

**__Description__**
 * The complete set of plasmid-containing cell clones, each carryng cpies of a particular segment from the initial genome.
 * Each plasmid clone in the library is like a book containing specific information.

__** Analyze **__
 * These libraries are constructed using clones of bacteria and yeast that contain vectors into which fragments of partially digested DNA have been inserted
 * Certain bacteriophages have been used as cloning vectors for making libraries.
 * Phages are used over vectors normally because a standard plasmid can carry a DNA insert no larger than 12,000 base pairs, and a phage can carry 25,000.
 * If the library is made of phages, it is stored as a collection of phage clones.
 * BAC are anoher type of vector used in library contstruction. They are just large plasmids that are trimmed down so they contain the genes that are necessary and carry iserts of 100,000 to 300,000 base pairs.
 * cDNA is another type of library. They start with mRNA that is extracted from cells. They use the enzyme reverse transcriptase to make single stranded DNA transcripts of the mRNA molecules.


 * __ Apply __**
 * Using genomic libraries, researchers can explore the genome of an organism to learn more about genomic structure and function. They can map the genome as well, identifying the location of specific genes.

__** Synthesize **__ Like it has been referred to many times, it is basically a library. It is organized, and it helps find specific things a lot faster than usual.

The concept of a genomic library is a good idea. It makes the lives of researchers easier with the organization. It also helps them learn more about organisms genome structure and function.
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 * __ Polymerase Chain Reaction __**
 * __ Link for help: __**
 * __ Description __**
 * When the source of DNA is scanty or impure, the PCR is quicker and more selective.
 * Any specific target segment within one or many DNA molecules can be quickly amplified in a test tube.
 * It can make billions of copies of a target segment of DNA in a few hours which is significantly faster than screening a DNA library.
 * Being used increasingly to make enough of a specific DNA fragment to insert it directly into a vector.

1. The mixture is heated to denature 2. It is then cooled to allow hydrogen bonding 3. A heat stable DNA polymerase extends the primers in the 5' to 3' direction
 * __ Analyze __**
 * The specific parts are what ae amplified during the PCR
 * A three step cycle brings abou the chain reaction that produces an exponentaill growing population of identical DNA molecules.
 * Automation plays a key role in PCR, because it is what allows it to make billions of copies
 * A heat stable polymerase that was discovered which is the key to automating the PCR


 * __ Apply __**
 * Quicker and more selective then DNA cloning
 * It is starting to be used more and more because in order to make enough of a specific DNA fragment to insert directly into a vector, they can skip all the steps of making and screening it to a library.

__** Synthesize **__ This could also be used on things like crime scene investigations.

This can be very important when it comes to things like genetic counseling, and like I stated above, crime scene investigations.
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**__ Part 2 ~ Studying Expression and Function of a Gene __** __** Gel electrophoresis **__ __** Description **__
 * Used to study DNA molecules
 * Uses a gel made of a polymer, such as a polysaccharide
 * Used to seperate nucleic acids or proteins that differ in size, electical charge, or other physical properties

Here is a vitual lab to help explain!
 * __ Analyze __**
 * The gel acts as a molecular sieve to seperate nucleic acids or proteins on the basis of size, electrical charge, and other physical properties.
 * Gel electrophoress seperates macromolecules on the basis of their rate of movement through a polymeric gel in an electric field.
 * A mixture of DNA molecules is placed in a seperate well near one end of a thin slab of gel. The gel is set into a small plastic support and immersed in n aqueous solution in a tray with electrodes at each end. Once the current is turned on, the (-) charged DNA molecules move toward the positive electrode, with the shorter molecules moving faster than the longer ones. Once done, a DNA binding dye is added which reveals the seperate bands to which it binds.

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Gel electrophoresis is used for many things including:
 * __ Apply __**
 * To get DNA fingerprints from forensic purposes
 * To get DNA fingerpints for paternity testing
 * To check a PCR reaction

Once again, this kind of thing could be used in a crime scene investigation. It is used to get fingerprints and such, so it is ideal for forensics purposes.
 * __ Synthesize __**

This is an important process. You see people using it if you've ever watched any sort of CSI or Law and Order. It is important for forensics teams.
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 * __ Southern blotting __**


 * __ Description __**
 * Combines gel electrophoresis and nucleic acid hybridization
 * Allows us to detect just those bands that include parts of the B-globin gene
 * Principle is the same as in nucleic acid hybridization for screening bacterial clones


 * __ Analyze __**


 * The probe is usually a radioactive single-straned DNA molecule that is complementary to the gene of interest.
 * Each DNA sample is mixed wih the same restriction enzyme. The restriction fragments are then seperated by electrophoresis, forming a characteristic pattern of bands. Capillary action pulls the alkaline solution upward through the gel, transferring the DNA to a nitrocellulose membrane, producing the blot. The DNA is denatured in the process. The single strands of DNA stuck to the nitrocellulose are positioned in bands corresponding to those on the gel. The nitrocellulose blot is exposed to a solution containing a radioactively labeled probe. A sheet of photographic film is laid over the blot. The radioactivity in the bound probe exposes the film to form an image corresponding to those bands containing DNA that base-paired with the probe.

Here is a video to help you out! []

**__ Apply __**
 * Southern blot allows investigators to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples
 * Also used in gene discovery and mapping, evolution and development studies, and diagnostics and forensics.

**__ Synthesize __** In gene mapping and discovery, with genetically modified organisms, it is used as a definitive test to ensure that a particular section of DNA of known genetic sequence has been succesfully incorporated into the genome of the host organism.

__** Argue - with **__ This is important when it comes to gene discovery. Without it, it wouldn't be as easy to identify carriers of mutant alleles associated with genetic diseases is only one of the ways it is used.

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 * __ Microarrays __**
 * __ Description __**
 * Makes genome-wide expression studies possible
 * Consists of a large number of singlestranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array or grid.
 * Known also as a DNA chip.
 * These fragments represent all the genes of an organism

__** Analyze **__

Researchers can test thousands of genes simultaneously to determine which ones are expressed in a particular tissue, under different environmental conditions, in various disease states, or at different developmental stages. The four steps of Microarray include:
 * 1) Isolating the mRNA
 * 2) Make cDNA by revers transcription, using fluorescently labeled nucleotides
 * 3) Apply the cDNA mixtur to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organisms genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.
 * 4) Rinse the excess cDNA and scan the microarray for fluorescence. The fluorescent spots represent gene expresse in the tissue sample.

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They can test thousands of genes simultaneously. They can look for coordinated gene expression as well. Using microarrays, researchers have performed DNA microarrays on more than 90% of the genes of the nematode Caenorhabditis elegans, during every stage of its life cycle.
 * __ Apply __**

__** Synthesize **__ It reminds me of patches of grass where some are green and some are brown. The green grass sticks out, which would be the gene that is expressed where as the brown grass nobody really cares about so much.

This, of course, is very important for a couple reasons. Testing thousands of genes simultaneously is very important to a researcher and looking for coordinated gene expression is important as well.
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 * __ Part 3 ~ Cloning Organisms __**
 * __ Plant Cloning __**
 * __ Describe __**
 * Succesful cloning of whole plants from single differentiated cells was accomplished during the 1950's by F.D. Steward and his students at Cornell University. They used carrot plants as their palnt of choice.

Click this link for additional help []
 * __ Analyze __**
 * Differentiated cells taken from the root and incubated in culture medium could grow into normal adult plants that were genetically identical to the parent plant
 * This showed that differntiated cells do not neccessarily involve irreversible changes in the DNA
 * There was a cross section of the carrot root, turned that into 2 mg fragments, they were cultured in a nutrient medium where the stirring caused single cells to shear off into the liquid, the single cells began to divide, embryonic plant developed from a cultured single cell, and the plantlet was cultured on agar medium and later planted in soil.
 * Some differentiated somatic cells in a plant are totipotent (Mature cells that can dedifferentiate and then give rise to all the specialized cell types of the organism)


 * __ Apply __**
 * Plant cloning is used extensively in agriculture
 * For plants like orchids, cloning is the only commercially practical means of reproducing plants
 * Used to reproduce plants with valuable characteristics

__** Synthesize **__ This reminds me exactly of basically what it is, for example, using part of a plant that you grew and re-planting it to grow another of the same plant.

I find plant cloning to be very beneficial to humans. Reproducing a plant that can resist certain pathogens, or possibly has a higher nutritional value for us and things like that are very important.
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**__ Animal Cloning __**


 * __ Describe __**
 * Differentiated cells from animals do not divide in culture, much less develop into the multiple cell types of a new organism
 * Researchers needed a different approach to the question of whether differentiated animal cells could possibly be totipotent

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 * __ Analyze __**
 * They took the approach where they would remove the nucleus of an unfertilized or fertilized egg and replace it with the nucleus of a differentiated cell. This procedure is known as nuclear transplantation
 * If the nucleus from the differentiated donor cell retains its full genetic capability, then it should be able to direct development of the recipient cell into all the tissues and organs of an organism
 * These experiments were conducted on frogs by Robert Briggs and Thomas King in the 1950's and by John Gurdon in the 1970's. They transplanted a nucleus from an embryonic or tadpole cell into a nucleus lacking egg of the same species.
 * The transplanted nucleus was often able to support normal development of the egg into a tadpole. He found that the potential of a transplanted nucleus to direct normal development was inversely related to the age of the donor.
 * The older the donor nucleus, the lower percentage of normally developing tadpoles

Animal cloning could be essential to human life as it develops. There may be a shortage of food in the near future, which could bring the world to a downfall. The cloning of animals could be a necessity if it does come down to it. Of course the first thing I think of is Dolly the sheep. This just gives me a more in depth process of what had happened.
 * __ Apply __**
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__** Argue - neutral **__ I definitely have mixed feelings about this topic. I find it kind of wrong to do when it is not neccessarily needed. I can understand why they would do it if there was such a thing as a shortage of meat/food.

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__** Restriction Fragment Length Polymorphism **__
 * __ Part 4 ~ Practical Application of DNA technology __**
 * __ Describe __**
 * Alterations in the change of length of the restriction fragments formed by diestion with that enzyme
 * Some SNP's alter the sequence recognized by a restriction enzyme as is the case for the single-nucleotide difference between the alleles for normal and sickle-cell B-Globin.

__** Analyze **__
 * The SNP alter the sequence recognized by a restriction enzyme
 * Can occur in either coding or noncoding regions
 * Serve as useful genetic marker

Here is a link to help!

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It is used for the screening of sickle cell gene. It is also used as an analysis for epidemilogical studies of tuberculosis in developing countries.
 * __ Apply __**

**__Synthesize__** I think of RFLP as a GPS. Marking the positions of things makes it easier to find them, which is what a GPS can do.

__** Argue - with **__ Of course I agree with this. I feel like it is important to be able to detect certain diseases at an early stage.

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 * __ Gene Therapy __**
 * __ Describe __**
 * Introduces genes into an afflited individual for therapeutic purposes
 * Holds potential for treating disorders traceable to a single defective gene

__** Analyze **__
 * In theory, a normal allele of the defective gene could be inserted into the somatic cells of the tissue affected by the disorder
 * For gene therapy of samatic cells to be permanent, the cells that receive the normal allele must be ones that multiply throughout the patient's life
 * Bone marrow cells are prime candidates due to the fact that they include stem cells that give riseto all the cells of the blood and immune system
 * A possible procedure for gene therapy of a patient whose bone marrow cells do not produce a vital enzyme because of a single defective gene:
 * 1) Inserting the RNA version of the normal allele into the retrovirus
 * 2) Let the retrovirus infect he bone marrow cells that have been remove from the patient and cultured
 * 3) Viral DNA carrying the normal allele inserts into the chromosome
 * 4) Then inject the engineered cells into the patient
 * In France, ten patients were treated for SCID (Severe combined immunodeficiency). Nine of the patients showed improvement after two years. Three of the patients developed leukemia and one had died. Researchers decided that in two of the cases, the retroviral vector used to carry the normal allele into bone marrow cells had inserted near a gene involved in the proliferation and development of blood cells, which possibly could have led to leukemia.
 * How can the activity of a transferred gene be controlled so that cells make appropriate amounts of the gene product at the right time and in the right place? How can we be sure that the insertion of the therapeutic gene does not harm some other necessary cell function?

__** Apply **__ Gene therapy is used in trying to treat genetic diseases so that they do not end up getting passed from generation to generation.

__** Synthesize **__ As weird as it may sound, it kind of reminds me of medieval times when a doctor would try to cure a patient not knowing exactly what he is fixing. The results can turn out very random and turn for the worst, and sometimes it can come out in the best scenario.

__** Argue- Against **__ I do feel like this could come into play sometime in this world, but as for right now and people not knowing exactly what is happening, they definitely need more research before they start doing this. There is no point going out and trying to do such things when some people die and some people live.



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 * __ Transgenic Animals __**
 * __Describe__ **
 * Scientists can introduce a gene from an animal of one genotype into the genome of another individual, often of a different species; known as a transgenic animal

__** Analyze **__
 * To establish a transgenic animal, they first need to remove eggs from the female of the recipient species and fertilize them in vitro. Meanwhile, they have the cloned desired gene from the donor organism. They will then inject the cloned DNA directly into the nuclei of the fertilized eggs.
 * Some of the cells integrate the foreign DNA, the transgene, into their genoes and are able to express the foreign gene. The engineered embryos are then surgically implanted in the surrogate mother.
 * If an embryo develops succeesfully, the result will be a transgenic animal that contains a gene from the third parent, which could possibly be of a different species
 * If the introduced gene encodes for a desired protein, these animals can act as pharmaceutical factories.

__** Apply **__ Using transgenic animals can come in handy. Using these animals to produce things like proteins that we really need can be beneficial to humans in several ways. Researchers have engineered transgenic chickens that express large amounts of the transgene's product in eggs.

I consider this to be a lot like plant cloning in a way. I feel like it is similar to plant cloning because of the fact that we do it because we may need more food/by-products from the organism. We are basically taking advantage of certain characteristics of certain organisms to harvest their products for our well-being.
 * __ Synthesize __**

As beneficial as this is for us, I feel like it is necessary. It helps us out in many different ways. I can understand why people would think that it might be wrong and such, but I don't find this to be near as bad as things like animal cloning and such.
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 * __ Transgenic Plants __**
 * __ Describe __**
 * Plants are very necessary to take advantage of
 * A lot of the drugs and such that we use now-a-days are from plants
 * Researchers are also creating plants that make human proteins for medial use and viral proteins for use as vaccines

**__Analyze__** Here is a sight to help understand how it works!
 * Basically, a transgenic plant is one that obtained a gene or more that was artificially inserted instead of the plant acquiring it through pollination
 * Plant crops are combined so that they can make the most productive crop possible. They try to combine all of the best qualities from each of the plants and combine them so that they can for a resistant, strong, nutritious plant.
 * Transgenic plants contain almost all of the qualities of transgenic animals and follow almost the same exact steps in achieveing a transgenic plant

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Transenic plants are very important to humans in so many ways. They help make some of the crops that we eat produce more, make them more resistant, and give them nutritional value that they would not be able to produce on their own. They also provide all kinds of pharmaceuticals that we take in all of he time. Without transgenic plants, I think life would be a lot different then what it is now.
 * __ Apply __**

Once again, I will refer to this as being a lot like farming. Being used to it in my area, crop owners want to produce the best yield possible, and can only do so with the best plant. Those best plants are the transgenic ones that are bug resistant, stronger, need less water, and have better nutritional value. Crop owners look at those things when they buy their seed because they would like the best result.
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I think transgenic plants are very important to us as I have stated several times above. They are very important to the well being of humans when it comes to health. They even play a big role in our economy.
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 * __ Genetic Profiles (Forensic Testing) __**
 * __ Describe __**
 * A genetic profile is your unique set of genetic markers
 * Everyone's genetic profile is unique to them
 * This is the preferred name over "DNA fingerprinting" because of the fact that they want to emphasize the heritable aspect of these markers rather han simply the fact that they produce a pattern on a gel that is visually recognizable


 * __ Analyze __**
 * FBI started using DNA technology in forensics in 1988, using RFLP analysis by Southern Blotting to detect similiarities and differences in DNA samples
 * Today, forensic scientists use an even more sensitive method that takes advantage of variations in length of the genetic markers which is called short tandem repeats (STR) ~ they are tandemly repeated units of 2 to 5 base sequence in specific regions of the genome
 * The number of repeats present in these regions is highly variable from person to person (polymorphic)
 * PCR is used to amplify particular STRs, using sets of primers that are labeled with different colored fluorescent tags; the length of the region, and thus the number of repeats, can then be determined by electrophoresis
 * Quicker than RFLP analysis because southern blotting is not required
 * Samples containing as litle as 20 cells can be sufficient enough for PCR amplification

**__ Apply __** This technique is used a lot in forensic testing. It is used to determine certain individuals who have done certain things. The Innocence Project uses STR analysis to try to overturn wrongful convictions. **__ Synthesize __** This of course brings me back to CSI. Of course, with all of the forensics and stuff going on, it is what I'd think of. They do this kind of stuff all of the time to determine who did what and tracing fingerprints and blood samples.

__** Argue **__ Of course I agree with this. This is a major step in solving a vast majority of crime scene investigations. This plays a big part in identifying who has done what.



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