DNA Technology

DNA Extraction

A buccal swab collects skin cells (stratified squamous epithelial cells) from the inside of a cheek. Scissors are used to cut off the end of the swab and place it into an eppendorf tube. Micro pipette is used to add lysis solution, which is a combination of detergent, which breaks apart the cell and nuclear membranes, and proteinase k, which cuts away histones. This tube with lysis is added to a warm water bath, facilitating the action of the lysis. We then use a micro pipette to add concentrated salt solution. This solution causes proteins and other cellular debris to clump together. Then, the tube is added to a micro centrifuge, along with a "balancing" water test tube. The fast spinning in the centrifuge causes the clumped debris to fall to the bottom of the tube, and a micro pipette can then be used to remove the top level of liquid and place it in a different tube. Isopropyl alcohol is added to this solution, which separates out clumped DNA into visible form. This tube goes back into the micro centrifuge, and, this time, the DNA falls to the bottom. A pipette is used to remove the top liquid, and the DNA on the bottom is then ready for use or storage.

  
Electrophoresis

A gel is created by heating agarose along with a salt water solution/liquid buffer in a microwave. This is then added to a mold, and a comb in inserted to create small wells for the DNA samples at one end. It is allowed to cool, and the comb is removed. A buffer is added to an electrophoresis box to allow for conduction of current and to prevent drying out of the mold. The mold is placed into this box. Loading buffer, which allows DNA to be seen, is added to a DNA sample, which is then placed into one of the wells. Standard size DNA (a sample in which DNA length is already known) is added to another well for comparison. A current is then run through the box-the negative charge is at the end with the wells, and a positive charge is on the opposite end. DNA is repelled from the negative charge and begins to make its way to the positive charge. The gel itself is filled with small holes that the DNA travels through to get to the positive charge. Smaller strands are able to move more quickly. Eventually, strands of the same size end up together and clump, which makes them ultimately more visible. Once the process is complete, the mold is removed from the electrophoresis box and placed in an ethidium bromide solution, which stains DNA and makes it visible when it is placed under UV light (accomplished with a UV light box). The clumps appear as bands and their location can be compared with the bands of the known lengths of the standard DNA sample. This gives a rough estimate of the number of base pairs present at each band site.

Electrophoresis can be used to determine paternity. Because each person's DNA pattern is very unique, it shows up in electrophoresis as a unique set of bands, unlikely to be repeated by any but a close relative. Thus, parent and child can be identified by a close match of an electrophoresis reading. This is accomplished by taking the DNA of mother, child, and two possible fathers and putting them through gel electrophorisis. Every band of the child MUST MATCH at least one of the parents. Whatever bands don't match the mother must be found in the father's reading. Therefore, by process of elimination, paternity is determined.

 

Cell Reproduction and DNA

Chromosome 10

 1) IL2RA- interleukin 2 receptor, alpha protein encoding

      IL2RA is a protein that appears on the surface of certain immune cells, and is often found along with beta and theta versions of interleukin 2 receptor. Together, they respond to interleukin 2, a chemical that bonds with the various forms of IL2R, creating chemical signals that are then transferred into the interior of the cell.

2) VIM- vimentin

     This gene encodes a protein that helps to maintain the shape and integrity of a cell's cytoplasm, and is an integral part of the cytoskeleton. It also helps transport LDL (low-density lipoprotein) out of the cell.

3) ITBG1- integrin, beta 1

    This gene encodes a protein that is part of a group of proteins (made up of alpha and beta subunits) that form receptors on the cell membrane. These receptors as a whole help with cell adhesion and recognition, as well as tissue repair, hemostasis, and immune responses.

4) RET- proto oncogene

    RET encodes a cell-surface receptor that translates signals for cell-growth and differentiation. It plays an important role in embryonic development during the formation of the neural crest, one of the important stages of tissue differentiation in embryos.

5) MAPK8- mitogen-activated protein kinase 8

    The protein this gene encodes is part of a larger family of proteins that act as integration stations for multiple biochemical signals, and are therefore involved in many cellular activities. This particular one, once activated, leads to programmed cell death, which is important in preventing tumor proliferation or genetic mutation due to UV radiation.

6) CDK1- cyclin-dependent kinase 1

     This gene creates a kinase (an enzyme that helps create energy in a cell) that works during cell reproduction, helping a cell to move from one phase of cell reproduction to another. It also works as a regulator during cell cycles.

7) PLAU- plasminogen activator, urokinase

    This gene encodes an enzyme that breaks down protein bonds in the extracellular matrix. It specifically breaks down plasminogen into another enzyme called plasmin, which circulates in the blood and breaks down various blood proteins, including those that form clots.

8) FAS- fas cell surface death receptor

    This gene creates a protein that is a receptor protein that helps to convert signals in proliferating T cells and fibroblasts. It is pivotal in programmed cell death, and is implicated in the development of various diseases of the immune system. 

9) TCF7L2- transcription factor 7-like 2 (T cell specific, HMG box)

     This is involved in the transcription of DNA within the cell. It is involved in the stabilization and maintenance of healthy glucose levels in the blood.

10) FGFR2- fibroblast growth factor receptor 2

      This gene encodes a protein that interacts with various forms of fibroblast growth factors, helping to set in motion a cascade of events that lead to cell reproduction and differentiation. These processes are involved in wound healing and embryo development, among other things. 

Genetics Blueprint Critique
1) I often found the instructions unclear and lacking in specificity. The questions did not always relate directly to the information that was shared, and there were aspects to some of the interactive exercises that were not explained. I found myself feeling frustrated at points by what the program wanted.

2) I'm not sure I learned a whole lot more than I did from the textbook or from the above lab. There was a little more about history, but it was so brief and not adequately explained, that I don't think I retained much. It was informative to play around with building the molecules, but again, I'm not sure I would've figured it out without the emailed instructions of Ms. Oellers.

3) Visually, it was appealing. It was interesting to see DNA represented in a variety of forms. I would have actually liked a bit more time spent on this aspect.

4) The questions were phrased in an odd way that made it difficult to figure out which answers were being sought. I don't feel like the writing was the best.

5) The idea of the lab is nice- going through a version of historical experiments, and then playing around with molecules and building DNA helps make things more clear. I just think it could be done better. 

Digestion and Nutrition

My Daily Menu

1. Explain why the menu of food choices you made is nutritious, meaning the food choices follow the government's recommendations for Calories and nutrients.
   
   I tried to make choices that are relatively reflective of what I eat. I am vegan, so substitute tofu and tempeh, etc for the dairy and meat choices (I think this would've helped with the sodium issue, as well, as these food are much lower in sodium then cottage cheese, etc). I got enough calories pretty easily, as well as proteins and carbohydrates, and stayed under the DV for fat, etc, although in my own diet I like to add healthy fats like avocado and Flax oil.
   

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2. How does a 2 000 Calorie daily menu compare to your recommended Calorie intake amount on the chart? What would you do to your 2 000 Calorie menu to make it better match your recommended Calorie intake?
   
   My recommended intake is between 2000 and 2200, which is about where I ended up. However, I feel like when we are listening to our bodies, our caloric needs will vary day-to-day.
   

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3. How can you use the Nutrition Facts label to make healthy decisions?
   
   The labels help to make very clear how much sodium and sugar are in various products. I also look closely to see how many ingredients are listed, and how many of those ingredients sound like they were produced in a chem lab.
   

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4. Did the menu you created contain the foods you like to eat? Explain why or why not.
   
   Yes, however my own diet has much more variety.
   

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5. Do any of the items you selected provide more than 30% in any nutritional area? If so, which foods and which nutrients? Why did you choose those foods?
   
   The grains added a lot. In a veggie diet, eating a wide variety as opposed to one big chunk of a particular food, seems more helpful, though. However, use of nutrient-dense foods such as unsalted nuts and whole grains such as quinoa helps us to meet our bodies' nutritional requirements.
   

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6. What did you notice about the nutrients in fast foods?
   
   I didn't even go there. I haven't eaten at a fast food retaurant in ten years. Seriously. That stuff'll kill you.
   

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7. What did you notice about the food group icons in your Daily Meals area? Did you favor a specific food group? Explain.
   
   I went for fruits and veggies a lot. They allow us to eat quite a bit and feel full without adding unnnecessary calories. They also add important vitamins and minerals.
   

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Nervous System

Nerve Histology

 Nervous tissue has a similar arrangement to muscle tissue. Each axon is surrounded by connective tissue, and then bundled into a larger group, a fascicle, which itself is surrounded by connective tissue. This is then arranged together into the larger working nerve unit, also surrounded by connective tissue. The endoneurium surrounds each nerve cell, the perineurium surrounds each fascicle, and the epineurium surrounds each nerve as a whole. In muscle tissue, these connective tissue sheaths are the fascial surroundings of the muscle fiber, fascicle, and whole muscle.
Nerves connect to the spine via thicker ganglia areas. As the nerve travels through the body, various neurons innervate specific areas and the bundle as a whole thins.
Multiple Sclerosis is a serious disease that affects the myelin sheathing of neurons. The ability to transmit signals becomes progressively impaired. People with MS have symptoms that can vary day-to-day, but generally include sensitivity to temperature extremes, muscle weakness, vision problems, and difficulty coordinating movements.

 Anatomy of a Neuron

 
 Sensory neurons carry information from their specialized receptors to the CNS. They are located in the skin, and throughout the body, including within organs and around blood vessels. They can sense pressure, temperature, pain, etc. and are the information-carriers that keep the CNS apprised of our internal and external environments. Motor neurons innervate skeletal and smooth muscle, and carry the CNS's instructions and reactions to all parts of the body. Interneurons integrate the information between the two, ensuring that the appropriate response is matched with the appropriate stimulus. They are also the coordinators of spinal reflexes.

Reaction Time Rulers

	Visual Stimulus		Auditory Stimulus		Tactile Stimulus
	Person 1	Person 2	Person 1	Person 2	Person 1	Person 2
Trial 1	R-3”  L-2.25”	R-3” L-6”	R-2” L-1”	R-5”  L-2”	R-13.5”  L-10”	R-3”  L-6”
Trial 2	R-1” L-3.25”	R-5” L-14”	R-2”  L-5.75”	R-3.25”  L-2.5”	R-2”  L-8”	R-2.5”  L-3.75”

                                                                                                                                                                                                
My hypothesis had been that visual stimuli would process faster as a large part of our brains are dedicated to visual processing. Visual stimulus did work well for me, but not so much with my partner, who has some visual impairments (possible explanation). My quickest reaction times were visual and audio, and she did well with audio and tactile. To be honest, beyond her slight visual issues, I have no explanation for why we differed. Perhaps genetic tendencies, or training of certain stimuli over the years makes a difference. We also might both be different about how we administered the test. Having a third person test both of us might have given better data.
Nerves take a bit to create a reaction because the sensory input must travel biochemically along a sensory nerve to the CNS, where it is processed by interneurons and sent either back out to the appropriate muscles (a reflex) or up to the brain and then back down the spinal cord and out. All this is done through action potentials. While they do not take long to generate, they do take some time, and therefore we don't have instantaneous reactions, even with reflexes.
My hypothesis was not supported, and this data would lead me to create a more definite experiment.

                                                Somatic division                                                                              Autonomic division

		Sympathetic	Parasympathetic
Function	Works with Skeletal Muscles	Arousal, mental acuity, flight-or-fight	Relaxation, repair, digestion and basic functions
Neurotransmitter	Acetylcholine	Norepinephrine	Acetylcholine