Axolotl genomic DNA was isolated and purified by cell lysis, organic extraction, and isopropanol precipitation. Agarose gel electrophoresis was performed with the axolotl genomic DNA to determine if it was successfully isolated and purified, as well as determining the concentration of it. Then, a serial dilution of a concentrated solution of a 700 bp region of the ampicillin-resistant gene located on the pGEM vector was performed. Additionally, the cox3 gene of the Axolotl genomic DNA that was previously isolated and purified, was subjected to PCR amplification, along with the serial dilution samples of the pGEM vector. Finally, agarose gel electrophoresis was performed to determine PCR sensitivity and PCR amplification of the Axolotl genomic DNA.
Deoxyribose nucleic acid is a polymer made from nucleotides covalently bonded together by phosphodiester linkages. Each nucleotide has a nitrogenous base, a 5-carbon sugar, and a negatively charged phosphate. The four nitrogenous bases include thymine, cytosine, guanine, and adenine, which encode information for cell structure and function. A nitrogenous base is bonded to the 1’ carbon of the sugar, deoxyribose, and the phosphate molecule is bond to the 5’ carbon of the same sugar. Multiple nucleotides are connected by phosphodiester linkages, which bond the phosphate of one nucleotide to the 3’ carbon of another nucleotide. This single stranded polymer, running from 5’-3’, then interacts with a complementary strand, called the antiparallel strand, running from 3’-5’ (Sinden 1994). These strands are held together by hydrogen bonds between the nitrogenous bases, which cause a double helix shape (in bDNA this causes one 3600 turn every 12 base pairs) with a negatively charged sugar-phosphate backbone (Booker 2009). Additionally, there are many different processes and factors which can impact the shape of DNA and therefore its expression. For example, if DNA is in a high salt environment and has a large GC content it will transform into zDNA, where there are 10 base pairs per 3600 turn. (Booker 2009). Furthermore, the DNA in eukaryotes is found within the nucleus of the cell, and in Prokaryotes, the DNA is found within the nucleoid region. (Booker 2009). DNA found in both form a complex with proteins, called chromatin. These proteins are able to change the structure of DNA, which impacts expression. Chromatin, named euchromatin, is termed open and access to transcription proteins, heterochromatin is not. Changing the compression of chromatin can be accomplished through modifications such as phosphorylation and methylation, depending upon the species. (Booker 2009).
The first step of DNA isolation is to lyse open the cellular and nuclear membranes. Both membranes are made from a phosphor lipid bilayer. (Branton 1972). In order to accomplish this task Triton X, Tris-Cl pH 8.0, EDTA pH 8.0 are added. Triton X incorporates itself into the cellular membrane, in doing so it destabilizes the membrane opening up holes of which contents leak out and contribute to the cell lysing open. (Schnaitman 1971). Tris-Cl pH 8.0 also has a similar function to Triton X, it accomplishes lysing open the cell by binding to lipopolysaccharides in the cell membrane which destabilize the membrane and cause it to lyse open. (Philips 1995). EDTA is a chelator. Chelators are molecules which grab onto metal ions and remove them from solutions. There are ions needed to maintain membrane structure, by binding to these ions EDTA is able to impair membrane structure and contribute to it lysing open. (Philips 1995). SDS binds to and denature the proteins on the cell membrane which destabilizes the cell membrane contributing to lysing the cell open. (Juodka 1995). ~~~~Another factor of opening up the cell is also ensuring that the DNA extracted from the cell is neither denatured nor degraded. In order to ensure that neither of these events occur EDTA pH 8.0, Tris-Cl pH 8.0, and NaCl are added. EDTA has a second purpose in this solution, some of the ions that it binds to are needed for the function of DNase enzymes, without these ions present DNase enzymes are incapacitated to do any work and are unable to degrade DNA. (Philips 1995). Tris-Cl pH 8.0 and EDTA pH 8.0 also have another function of acting as a buffer to maintain a constant pH, this is necessary to not denature DNA as DNA when exposed to a high or low pH becomes denatured. (Philips 1995). NaCl interact with the phosphate group of DNAs (specifically the Na+ ion) this interaction aids in the prevention of denaturing of DNA. (Philips 1995). The next step is to isolate and purify DNA from the solution. In order to accomplish this proteinase K, RNase, and ammonium acetate are added and organic extractions are performed. Proteinase K is a protein which is added to solution to break down other cellular proteins. (Juodka 1995). RNase is a protein which is added to solution to break down RNA. (Philips 1995). Ammonium Acetate is used to precipitate proteins out of solution. Ammonium acetate decreases the solubility of proteins by interacting with the proteins much alike the Na+ ion does with DNA, this causes the proteins to become less polar and precipitate out of solution; nonpolar molecules do not dissolve in water. (Philips 1995). The next step is to perform organic extractions. In this process chloroform, phenol and isoamyl alcohol are added into the solution. Chloroform and Phenol are nonpolar molecules both denser than water. Phenol turns proteins into nonpolar molecules by irreversible denaturing them, this precipitates them out of the top aqueous layer of water and dissolves them in the bottom organic layer containing chloroform and phenol. (Green 2017). Isoamyl alcohol serves the function of reducing foaming of the interphase layer, making for a cleaner separation between aqueous and organic layers. (Green 2017). The second extraction of chloroform and isoamyl alcohol serves the function of removing any removing phenol from solution as phenol can negatively impair following procedures. (Green 2017). The next step is to precipitate DNA out of the solution. NaCl has a second function to aid in the precipitation of DNA through a process called “salting out”. This is completed by adding chilled isopropanol. Isopropanol decreases the dielectric constant of the solution. This allows the Na+ ions (added in earlier steps for another purpose) to more intensely interact and bond with the phosphate group of DNA; in doing so DNA becomes a nonpolar molecule and precipitates out of the solution. (Philips 1995). The purpose of using chilled isopropanol is because it allows for greater efficiency and increase the effect of lowering the dielectric constant. The final step is to complete an ethanol wash. The ethanol dissolves and washes away remaining salt ions from previous steps. (Philips 1995).
PCR amplification allows the synthesis of hundreds of millions of copies of a target sequence of DNA in under 2 hours. Materials needed for the PCR process include dNTPs, taq Polymerase, primers, and DNA mixed in a buffer. Buffers serve the function of enhancing PCR amplification, for example the buffer used below contains MgCl2, this salt is a cofactor which enhances taq Polymerase activity. (Lalam 2004). By placing all of the needed material into a solution and running it through the PCR process millions of copies of the target sequence will be made. The process of PCR amplification is as follows. In order to completely sperate the strands of DNA into single strands the solution containing the materials is heated to around 94 for 30 seconds to 5 minutes, depending on the DNA in question. Then the cycle of PCR can occur in the order of denaturation, initiation, and elongation. During denaturation, the hydrogen bonds between double strands of DNA are broken, in order to accomplish this the solution is held at around 940C for 30 seconds or longer. During initiation, the primers hybridize with the DNA sequence, this is achieved by bring the solution down to the annealing temperature commonly is between 40 and 60. Finally, the elongation step can occur in which taq polymerase synthesizes a complementary strand of the target sequence it binds to, in order to achieve this the solution is raised to approximately 720C, the optimal temperature for taq polymerase. (Van Pelt-Verkuil 2008). After the desired number of cycles are finished it is common to allow the solutions to rest at 72 for around 5 minutes to conclude the process. Each cycle doubles the target sequence until there is a limiting material. Usually after 40 cycles there is a limiting reactant and the rate of synthesis plateaus. With all of that being said there are some limits of PCR. The most important limit in relation to the following exercise is PCR sensitivity. PCR sensitivity describes the minimum concentration of target sequence available for PCR to function. If the concentration is too low, then the primers will not bind to the DNA primer sequence and the target sequence will not be replicated.
Gel electrophoresis is a means of separating charged molecules based on size and charge. This can be accomplished by placing charged molecules in a well of a medium then adding an anode and a cathode to opposing sides of the medium. A buffer is needed to allow the electric current to flow through the medium from electrode to electrode. Agarose gel is a medium which contains a matrix of very small pores. The pore size is determined by the concentration of agarose, higher concentration creates small pores, lower concentration creates larger pores. (Johansson 1972). In order to make agarose gel, agarose is dissolved in a hot buffer, once the solution cools it stiffens and creates this matrix of pores. The charged material placed in the wells will migrate through this matrix towards the oppositely charged electrode. If the charged material is larger then it will take a longer time to migrate through the pores or will not even be able too. If the charged molecule is smaller then it will migrate through the pores quickly. By observing which direction, the charged material has moved and how far it has moved in that time, one is able to determine both the charge (either positive or negative) and size of the material. Charged molecules can be a multitude of different things, in this exercise it is DNA. After the charged material has been moved and separated it then must be imaged. By adding SyberSafe to the agarose gel, when the gel is exposed to UV light the DNA becomes fluorescent by a process explained below.
**These processes listed above were completed in order to achieve three goals. The first goal of is to isolate and purify Axolotl DNA. In order to achieve this step to isolate and purify DNA were preformed and then electrophoresis was ran on the resulting sample to confirm its presence of isolated and purified Axolotl DNA. The second goal was to assess PCR sensitivity through the amplification of 700 bp region of the ampicillin resistant (ampr) gene located on a pGEM vector. In order to achieve this first a serial dilution was preformed, then the PCR process was ran on the resulting solutions, and electrophoresis was ran to display PCR sensitivity. These results proved that the isolated Axolotl DNA was in a sufficient concentration to preform PCR amplification. The third goal was amplification of a 500 bp region of the Axolotl cox3 gene. Electrophoresis was then ran to confirm PCR success of the isolated DNA region. The remain solution containing an amplified 500 bp region of the Axolotl cox3 gene can be used in later studies for a variety of uses. One of such uses may be transcribing and translating this specific region of the encoded protein in order to use it to preform work in later procedures.
**Special equipment used throughout this procedure include TBE buffer, 6X loading dye, Triton X, and Sybersafe. TBE buffer contains tris, borate, and EDTA. Its function is to serve as a buffer allowing for the migration of nucleic acids in gel electrophoresis. (ThermoFisher 2019). 6X loading dye contains bromophenol blue, xylene cyanol FF, and EDTA. (ThermoFisher 2019) 6X loading dye serves the function of increasing the density of the sample and providing an approximate location of DNA within the gel throughout the electrophoresis process. Triton X serves the function of lysing open the cell membrane by implanting itself between phospholipids in the membrane. Since it is not a rectangular molecule it causes surrounding phospholipids to curve which open up small holes within the membrane which cause destabilization and then breaking of the cell membrane. (Schnaitman 1971). Sybersafe when in contact with DNA fluoresces under UV light. Sybersafe is dispersed throughout the agarose gel, when it comes into contact with DNA they bind causing the structure to change where light at the 509nm wavelength is absorbed and the 524nm wavelength is emitted. (Johansson 1972).
Agarose gel electrophoresis showed that there was successful isolation and purification of genomic axolotl DNA. The PCR sensitivity was determined by amplifying pGEM vector with a 700 bp region of the Ampr gene, which was subjected to agarose gel electrophoresis to determine the sensitivity. Finally, using this isolated axolotl genomic DNA, the cox3 gene was targeted during PCR amplification, which was shown to be successful through the results for agarose gel electrophoresis.
The axolotl tissue was placed in a weigh boat and weighed with a scale. After recording the mass, DNA Extraction Buffer A (2 % Triton X100/100 mM NaCl/10 mM Tris-Cl ph 0.8/25 mM EDTA ph 0.8/0.5 % SDS/0.1 mg/mL proteinase K) was added until the scale read 5 grams. Once added, the tissue within the weigh boat was quickly sliced into small pieces with a dissecting knife. This solution was transferred into the chilled dounce homogenizer and mashed 5 times with the plunger in a twisting motion. This process was repeated until the tissue was completely homogenized within the solution. Then, 600 uL of this solution was transferred into four 1.5 mL microcentrifuge tubes, totaling 2.4 mL of homogenized axolotl tissue. Each filled microcentrifuge tube was then incubated at 50C for 45 minutes. During this time the tubes were mixed every 10-minute interval. After removing the tubes from the incubation chamber, an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) as there was homogenate (600 uL) was added to each tube. Each microcentrifuge tube was then vortexed for 25 seconds. Next, the samples were placed into the microcentrifuge at 14,000 rpm for 8 minutes. Then, the aqueous layer (top most) was transferred into new 1.5 mL microcentrifuge tubes, each original four tubes with new corresponding four tubes. The volume of each tube was measured by using a micropipette. To these new tubes, an equal amount of chloroform/isoamyl alcohol (24:1) as there was aqueous layer was added. Each tube was then vortexed for 30 seconds. These samples were placed into the microcentrifuge at 14,500 rpm for 8 minutes. After centrifuging the aqueous layer (topmost) was transferred into new 1.5 mL microcentrifuge tubes, each original four tubes with new corresponding four tubes. The volume of each tube was measured by using a micropipette. For each corresponding tube, 50 % of this measured volume was added of 7.5 M ammonium acetate and mixed. Next, 60 % of the volume measured before the addition of 7.5 M ammonium acetate of chilled isopropanol was added to each corresponding tube. Each tube was then vortexed for 20 seconds, stored at -20C for one week, and reassessed. The samples were then centrifuged for 10 minutes at 14,500 rpm. The remaining supernatant was removed with a micropipette, leaving the pellet inside of each tube. Next, 500 uL of 70% ethanol was added to the microcentrifuge tube. The microcentrifuge tube was then vortexed briefly (between 1 and 5 seconds). The tubes were then incubated at room temperature for 3 minutes, followed by centrifugation at high speed for 5 minutes. The supernatant was removed using a micropipette, leaving the pellet within each tube. The four remaining microcentrifuge tubes were then gently placed open side down on a paper towel for 7 minutes. Next, 50 uL of 1X TE solution (10 mM Tris-Cl/1 mM EDTA ph 0.8) and 1ug/uL of RNase was added to each tube. The pellets were then dissolved gradually using gentle mixing techniques, this includes inverting, swishing, ect. The solutions within these tubes were then combined using a micropipette into one tube, this should total a 200 uL solution and will be referred to from now on as “concentrated genomic DNA”. Next 7 uL of concentrated genomic DNA was added to a new microcentrifuge tube and to this tube 63 uL of 1X TE was added, this tube will be referred to from now on as “1/10 genomic DNA dilution.” Next, 7 uL of 1/10 genomic DNA dilution was added to a new microcentrifuge tube and to this tube 63 uL of 1X TE was added, this tube will be referred to from now on as “1/100 genomic DNA dilution.”
A 0.9 % agarose gel was made with 30 mL of 0.5X TBE, 0.27 g of agarose, and 3 uL of SyberSafe for the purpose of agarose gel electrophoresis. To prepare these samples for electrophoresis, 10 uL of each solution (concentrated genomic DNA, 1/10 genomic DNA dilution, 1/100 genomic DNA dilution) were mixed with 3 uL of 6X loading dye, then each solution was added to the agarose wells. Mixing was completed by placing 10 uL of solution onto parafilm with a micropipette, then micropipetting the loading dye into this drop, and this was mixed together with the micropipette. Agarose gel A was placed into the electrophoresis chamber (with wells nearest to the cathode), filled with 0.5X TBE. To well 1, 10 uL of 1 kb size standard was added. To well 2, 10 uL of 2 ng/uL concentration standard was added. To well 3, 10 uL of 6 ng/uL concentration standard was added. To well 4, 10 uL of 18 ng/uL concentration standard was added. To well 5, 13 uL of the concentrated genomic DNA and 6X loading dye mix was added. To well 6, 13 uL of the 1/10 genomic DNA dilution and 6X loading dye mix was added. To well 7, 13 uL of the 1/100 genomic DNA dilution and 6X loading dye mix was added. Well 8 was left blank. The electrophoresis chamber was turned on and ran at 150 V for 35 minutes. The electrophoresis chamber was then checked for bubbles, monitored to ensure that the DNA was running towards the positive pole, and that the DNA was not running off the gel. After the 35 minutes elapsed, the electrophoresis machine was turned off and the gel was taken out of the chamber. The gel was then imaged with a UV transilluminator within 20 minutes of the gel being removed.
Table 1. Serial Dilution of 40 ng/uL Concentrated Stock Solution
| Tubes | dH20 (uL) | Amount of DNA Vector (uL) | Total Volume (uL) | Concentration (ng/uL) |
|---|---|---|---|---|
| A | 92.5 | 7.5 | 100 | 3 ng/uL |
| B | 495 | 5 | 500 | 30 pg/uL |
| C | 495 | 5 | 500 | 300 fg/uL |
| D | 495 | 5 | 500 | 3 fg/uL |
| E | 495 | 5 | 500 | 0.03 fg/uL |
There was 40 ng/uL of concentrated stock solution (vector) and distilled water that were transferred to a 1.5 mL eppendorf tube labeled “A” and was vortexed for 30 seconds (Table 1). Four more 1.5 mL eppendorf tubes were labeled “B”, “C”, “D”, and “E” and a serial dilution was performed where 495 uL of distilled water was added to tubes “B”, “C”, “D”, and “E” (Table 1). In tube “B”, 5 uL of the solution in tube “A” was added (Table 1). This was carried out for tubes “C”, “D”, and “E”, where 5 uL of the solution before it was added and then the solution was vortexed for 30 seconds before it was used for a dilution (Table 1). Tubes “A-E” were then placed on ice.
Table 2. 100 Fold Dilution of Tubes “A-E”