Chemical Biology DNA Molecular Engineering

DNA

Deoxyribonucleic acid (DNA) can be considered the hereditary "code of life" because it possesses the information that determines an organism's traits and is transmitted from one generation to the next. DNA can be compared to a recipe or a list of instructions about how to create and maintain a specific living thing. The DNA in an individual's cells contains unique genetic instructions about how to make and operate that individual.

DNA can be removed from organisms through a common and useful scientific procedure called DNA extraction. In order to understand this process, it is useful first to identify the basic structures that hold DNA molecules within living things.

Cells

DNA is located inside the cells of all species. However, different organisms are made up of different types of cells. Members of the Animal, Plant, Protist (algae, amoebas, paramecia, etc.), and Fungi (mushrooms, yeasts, molds, etc.) kingdoms are comprised of eukaryotic cells. This means that these cells have a true nucleus, a membrane bound organelle within which the DNA is contained. The nucleus of eukaryotic cells is the "control center" that directs all cellular activities. Members of the kingdom Monera (bacteria and cyanobacteria) are comprised of prokaryotic cells that do not have nuclei. In these cells, DNA exists as a long loop coiled loosely within the cytoplasm of the cell.
The nucleus of eukaryotic cells is surrounded by a nuclear membrane (also called a nuclear envelope) and the entire cell is bound by a cell membrane (also called a plasma membrane). These barriers are both made up of two layers of fatty, oily compounds called lipids. The most abundant types of membrane lipids are phospholipids. These molecules have hydrophilic ("water-loving") heads linked by a phosphate group to two hydrophobic ("water-hating") tails. The formation and stability of cell membranes is based on the orientation of phospholipid molecules in an aqueous (watery) environment. In such surroundings, phospholipids form a barrier of two rows with their hydrophobic tails facing each other (away from water) and their hydrophilic heads pointed outward (in contact with the aqueous environment). This two-layered structure is known as a phospholipid bilayer Protein and carbohydrate molecules are also imbedded within the phospholipid bilayer of cell membranes to transport particular molecules into and out of the
cell, and to conduct cellular messages. (see Figure below).

All eukaryotic cells have a nuclear membrane that encircles the nucleus, as well as a cell membrane that encases the entire cell. However, plant cells (and some bacterial, fungal, and protist cells) have an additional barrier surrounding the cell membrane called a cell wall. Animal cells do not have cell walls. Plant cell walls are made of cellulose, which is a sturdy polysaccharide material comprised of glucose units. Cellulose gives plants their rigidity and provides a tough barrier that enables plant cells to hold a great deal of fluid without bursting.

The DNA of eukaryotic cells is about 100,000 times as long as the cells themselves. However, it only takes up about 10% of the cells' volume. This is because DNA is highly convoluted (folded) and packaged as structures called chromosomes within cell nuclei. A chromosome is a bundle of tightly wound DNA coated with protein molecules. An organism's chromosomes bunch together within the nucleus like a ball of cotton, but during cell division (mitosis) they become individually distinct (human mitotic chromosomes are X- shaped) and can be observed as such with microscopes. DNA is not visible to the eye unless it is amassed in large quantity by extraction from a considerable number of cells.

A simulated view of the three-dimensional architecture of genetic material as it appears in a human cell nucleus. Colors signify different chromosomes.
Image courtesy of Tobias A. Knoch, Erasmus Medical Center

When chromosomal DNA is unfolded and the proteins coating it removed, the structure of DNA is exposed as a twisted ladder called a double helix. The sides of the ladder form the DNA backbone with alternating sugar and phosphate molecules linked by covalent bonds. The rungs of the ladder are comprised of pairs of nitrogenous bases [adenine (A) with thymine (T) and cytosine (C) with guanine (G)] joined by hydrogen bonds. Although the structure of DNA is well known and clearly defined, even the most powerful microscopes cannot visualize the DNA double helix of chromosomes.

All living things are dependent on DNA, and the structure of DNA is consistent among all species. However, the particular sequence of nitrogenous bases within DNA molecules differs between organisms to create explicit "blueprints" that specify individual living things. This sequence of base pairs is what makes an organism an oak tree instead of a blue jay, a male instead of a female, and so forth.

DNA Extraction From Plant Cells

The DNA of a plant cell is located within the cell's nucleus. The nucleus is surrounded by a nuclear membrane and the entire cell is encased in both a cell membrane and a cell wall. These barriers protect and separate the cell and its organelles from the surrounding environment. Therefore, in order to extract DNA from plant cells, the cell walls, cell membranes and nuclear membranes must first be broken. The process of breaking open a cell is called cell lysis. Physical actions such as mashing, blending, or crushing the cells cause their cell walls to burst. The cell membranes and nuclear membranes may then be disrupted with a detergent-based extraction buffer. just as a dishwashing detergent dissolves fats (lipids) to cleanse a frying pan, a detergent buffer dissolves the phospholipid bilayer of cell membranes. It separates the proteins from the phospholipids and forms water-soluble complexes with them. Once the cell wall and cell membranes are degraded the cell contents flow out, creating a soup of DNA, cell wall fragments, dissolved membranes, cellular proteins, and other contents. This "soup" is called the lysate or cell extract.

DNA molecules are then isolated away from the cell debris in the lysate. For this purpose, the detergent-based extraction buffer also includes salt. The salt causes some of the cellular debris in the soup to precipitate out of solution while the DNA remains dissolved. This means that the cell debris become suspended particles that can be seen. The cell extract is then filtered through layers of cheesecloth. The cheesecloth traps the precipitated cell debris while the soluble DNA passes through. DNA is soluble in the aqueous cellular environment and in the presence of the extraction buffer, but is insoluble in alcohol (such as ethanol and isopropanol). Applying a layer of ethanol on top of the filtered lysate causes the DNA to precipitate out of the solution, forming a translucent cloud of fine, stringy fibers at the point where the alcohol and cell extract meet. Cold ethanol works best to precipitate DNA to wound onto a wooden stick in a process known as "spooling" the DNA.

Strawberry cells are excellent sources of DNA for extraction and visualization. They are multicellular and octoploid. This means they have eight copies of their seven chromosomes in each of their many cells. Therefore, just one berry will yield enough DNA to be easily seen and spooled. Strawberries are also a soft fruit, which makes them easy to mash. Mashing the berries breaks down the strawberry tissue, releases the individual cells, separates the seeds from the cells, and breaks the cell walls. In addition, ripe strawberries produce pectinase and cellulase—enzymes that contribute to the breakdown of cell walls.

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Experimental Procedure:

View:
(The experimental method and materials in the following video are specific to the NC Community Colleges. They are very similar to those that you will actually use, which follow. The protocol is slightly different in the procedural steps with variations in some materials and the relative amounts. However, the chemical-biological principals and types of reagents/materials are identical in performance.)

https://www.youtube.com/watch?v=usaE_XZx-a8&feature=iv&src_vid=vPGKv53zSRQ

Before beginning the experiment, complete the background reading and view the YouTube video using the above link. Afterwards, working with your group perform the following procedure.

Procedure:

• Obtain one fresh strawberry and remove the green sepals (tops) from the berry.
• Place the strawberry in a re-sealable plastic bag
• Close the bag slowly, pushing all the air out of the bag as you seal it.
• Being careful not to break the bag, thoroughly mash the strawberry with your hands for two minutes .
• Pour 10 mL of extraction buffer into the bag with the mashed strawberry. Reseal the bag.
• Mash the strawberry for one additional minute.
• Place a funnel into a 50 mL centrifuge tube. Place the cheesecloth in the funnel to create a filter. The cheesecloth may overlap the edge of the funnel.
• Pour the strawberry mixture into the funnel, filtering the contents through the cheesecloth and into the 50 mL centrifuge tube.
• Carefully pour 2 mL of the filtered contents from the 50 mL centrifuge tube into a clean 15 mL tube. Use the lines on the side of the 15 mL tube to help measure the amount added.
• Hold the 15 mL tube at an angle. Using a plastic dropper carefully add 5 mL of cold ethanol by running it down the inside of the tube. Add the ethanol until the total volume is 7 mL (use the lines on the side of the tube to measure). You should have two distinct layers. CAUTION: Do NOT mix the strawberry extract and the ethanol!
• Watch closely as translucent strands of DNA begin to clump together where the ethanol layer meets the strawberry extract layer. You may see tiny bubbles in the ethanol layer appear where the DNA precipitates.
• Slowly and carefully rotate the wooden stick in the ethanol directly above the extract layer to wind (or "spool") the DNA. Remove the wooden stick from the tube and observe the DNA.
• Rinse and return all plastic containers. All solutions can be disposed of down the drain.

(report form)

DNA Applications:

Solve the mystery: Who stole the "magic holographic strawberry" lollipop? http://www.pbslearningmedia.org/asset/tdc02_int_creatednafp2/

Forensics: With a degree of certainty, DNA profiles can determine whether two biological samples - one from a crime and one from either a suspect or victim - came from the same individual by marking a small number of segments of DNA in one sample and then checking for the presence or absence of those same segments in the other sample. Enzymes cut the long strands of DNA into much smaller segments in precise repeatable ways. Each segment has a specific length, but all share the same repeating sequence of bases (or nucleotides). For example, one segment might be ATCATCATCATCATC, while another might be ATCATC. Gel electrophoresis is a method used to separate these repeating segments according to length. Next, a small set of radioactive "markers" are added to the sample. These markers are segments of DNA of known length, with bases that complement the code of, and bind to, the sample segments of the same length. The sample segment above (ATCATCATCATCATC), for example, would be tagged by a marker with the complementary code TAGTAGTAGTAGTAG. Markers that do not bind to sample segments are then rinsed away, leaving in place only those markers that bound to complementary sample segments. Photographic film, which darkens when exposed to the radioactive markers, identifies the location of all marked sample segments. This film, then, becomes the DNA "fingerprint" that forensic investigators analyze. The final step is simply lining up the sample profiles side by side and comparing them for the presence or absence of segments with particular lengths. The more segments the two samples have in common, the higher the probability that the samples came from the same person.

Post Lab Questions:

1. Describe the physical appearance of the DNA that you have extracted. Is it a single molecule? How does it fit in the nucleus of the cell? Briefly explain.
   
   
2. Strawberries are octoploid (8 sets of chromosomes). If you were to extract DNA from a diploid banana (2 sets of chromosomes, which humans have as well), how much DNA would you expect to get relative to strawberry, assuming the chromosome sizes are approximately the same?
   
   
3. Describe three applications of how a DNA sample can produce useful biological-genetic information. Use the examples in the video that you viewed
   
   Challenge Question (Bonus #1): The DNA in a strawberry cell is ~100,000 times as long as the diameter of a strawberry cell itself (assuming that the cell is spherical). Using strawberry cell research data: relative density & number of cells published in G.W. Cheng & P.J. Breen, J. Amer. Soc. Hort. Sci., 117(6):946-950. 1992 (http://journal.ashspublications.org/content/117/6/946.full.pdf), calculate the DNA length in centimeters and feet. Show your calculation on a separate sheet and attach.
   
   
4. Match the following:
   
   
   
   
5. Complete the following table:
   
   
   

   DNA antisense	DNA sense	mRNA	Name of Amino Acid Coded
   T
   G
   C
   A
   C
   C

   
   
6. Reading the antisense genetic base sequence, TGCACC, from left to right, which direction in relation to the deoxyribose saccharide-phosphate direction, 3’ to 5’ or 5’ to 3’ is the sequence going? Briefly explain the basis of your selection.
   
   
7. Write (draw) a structural formula that correctly shows the arrangement of all the atoms for the dipeptide formed from the 2 coded amino acids. (Use either a Lewis structure, or condensed structure, or line structure.)
   
   
8. What is the molecular formula and molar mass of the dipeptide?
   
   
9. How many Calories would be provided from eating 10. grams of the dipeptide? (Assume that it is equivalent to the caloric content of a protein.) Show your calculation.
   
   Challenge Question (Bonus #2): If all of the energy (Calories) were transferred to 5.5 liters of water @ 25.0 oC (about room temperature & approximately the amount of H2O in your body) what would be the new temperature of the water?
   
   
10. Who stole the “magic holographic strawberry” lollipop? (Refer to the reading and links to answer this “Who-done-it?” question.)

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http://chemwiki.ucdavis.edu/Wikitexts/Diablo_Valley_College/DVC_Chem_106%3A_Rusay/Amino_Acids

• The four structural levels of proteins are:

• • 1^o (primary): The sequence of amino acids, eg. ARDV: Ala.Arg.Asp.Val.
  H₂N- on the left and on the right -COOH

• • 2^o (secondary): Structures which include, folds, turns, alpha-helices, and beta-sheets held in place by hydrogen bonds.

• • 3^o (tertiary): 3-d arrangement of all atoms in a single polypeptide chain.

• • 4^o (quaternary): Arrangement of all polypeptide chains into a functional protein, eg. hemoglobin with 4 strands: 2 pairs of 2 chains, one "alpha" and one "beta"

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