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UB BIO 200 - chapter 20

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Zoey RentzDecember 19, 2012Biology 110Chapter 20 BiotechnologyStem CellsA stem cell is an unspecialized cell. These types of cells are able to produce more stem cells, but more importantly they have the potential to become specific specialized cells when under the proper conditions. In humans stem cells can be isolated during an early embryonic stage, giving these cells the name embryonic stem (ES) cells. This stage is referred to as the blastocyst stage. Stem cells that come from this blastocyst stage are able to, under specific conditions, become differentiated cells. For Example, stem cells from the blastocyst stage are able to become: Liver cells, Nerve cells, or Blood cells. Due to their ability to become a wide variety of new cells these stem cells are called pluripotent. The adult human also has stem cells. However they are slightly more restrictive. There are fewer stem cells in an adult human, and the few stem cells that are presentare unable to function as those in the blastocyst stage do. An example of an adult stem cell is located in the bone marrow. The stem cells located there are able to become different types of blood cells. The only way to obtain these stem cells is from human embryos, which is the core aspect of the stem cell research debate. Luckily, in 2007 it was announced that researches have the ability to reprogram fully differentiated cells, resulting in them functioning as ES cells. Researchers used retroviruses (an RNA virus that uses an enzyme to produce DNA from its own RNA, and incorporating it into the hosts DNA) to introduce four copies of the stem cell genes. The retroviruses incorporated the stem cells genes into the host cell and inevitably creating a stem cell. These engineered cells are calls induced pluripotent stem (iPS) cells. iPS cells and ES cells are similar but there are some differences that are not fully understood.The Polymerase Chain Reaction (PCR)PCR is a quick and selective process in cloning DNA. This process can be used if the DNA that is being cloned is impure or there is a small amount of it. Some examples would be: body fluids at a crime scene, DNA of a 40,000-year-old woolly mammoth, or genetic testing on a single embryonic cell. The PCR process is compared to photocopying a few pages from a book instead of checking that book out from the library. The PCR process is a three-step cycle involving several steps. The process prompts the DNA molecule population to grow. The first cycle is three steps. Beginning with the denaturing of the DNA strands using heat. This step breaks the hydrogen bonds between the nucleobases, essentially unzipping the DNA. Next, the DNA cools and hydrogen bonds begin to form between DNA primers (strand of nucleic acid that is the starting point for DNA)and their counterparts on the strands of unzipped DNA. The primers attach to opposite ends of each strand, which becomes important in the final step. Finally, a DNA polymerase, that is able to withstand high levels of heat, extends the primers (backbone) in the 5 prime to 3 prime direction. It fills in the gaps, by building new nucleotides and lining them up with the primers. The first cycle ends with two molecules being produced. The first cycle is able to happen due to extremophiles, specifically the bacteria Thermus aquaticus, which live in hot springs. Due to their natural environment this type of bacteria have a heat stable polymerase. Since this type of polymerase is able to withstand high levels of heat, when the DNA is denatured by heat, the polymerase is able to stay intact. Without this the polymerase would denature along with the DNA in the initial stage. At the beginning of cycle two there are two molecules, which go through the same steps in cycle one and produce 4 molecules. The third cycle goes through the same steps as the first two cycles and the result is 8 molecules. Most importantly about the third cycle is that two of the molecules match the initial DNA sequence exactly. As these cycles continue more and more molecules match the initial DNA. Only small amounts of DNA are needed to start the PCR process. The DNA needed tostart the cycle doesn’t have to be perfect either, so long as a few of the molecules contain the complete target sequence. This is because the primers (nucleic acid starting point) hydrogen bond to sequences at opposite ends of the initial double helix of DNA that was denatured (target sequence).Gel ElectrophoresisA laboratory technique used to analyze DNA is gel electrophoresis. A polymer gel is used to separate nucleic acids or proteins based on their physical properties (size and electrical charge). The gel has wells dug into it on the negatively charged side, the side opposite of the wells is given a positive charge. Since DNA carries a negativecharge due to its phosphate group, this causes them to move towards a positive charge in an electrical field. The gel makes it harder for heavier longer molecules to move, but will separate the linear DNA into bands. Each band contains all of the DNA molecules that are the same length. Restriction Fragment Analysis can rapidly provide information on DNA sequences. The DNA is taken and broken into specific pieces using an enzyme. These now cut up pieces are placed into the gel and separated using electrophoresis. The starting molecule and the enzyme used to cut it yield a specific type of band pattern. Just looking at the pattern can identify some molecules. The DNA is undamaged during the gel electrophoresis process and can be recovered after the bands have separated.When large DNA molecules are used the bands just appear to be one long smear, dueto the amount of fragments contained in the molecule).Restriction Fragment Length Polymorphism (RFLP) RFLP is a restriction sequence on an allele, where an enzyme is supposed to cut DNAinto smaller segments. RFLP help to show particular variations in DNA sequences among a population. If an allele contains a RFLP it will produce a different length of fragments than an allele without it. Resulting in their band patterns being different.Sickle cell disease is a mutation of one nucleotide within a RFLP in the human-globin gene. In a normal -globin allele the DdeI restriction enzyme recognizes twocoding sites. It cuts the -globin at two sites, making it three pieces. In the sickle cell mutant -globin allele the DedI restriction enzyme only recognizes one coding site.


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UB BIO 200 - chapter 20

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