Chem 40.1 FR 8 and 9

Extraction of DNA from Live Organism and Agarose Gel Electrophoresis of DNA Natalie B. Nisce Department of Food Science and Nutrition, College of Home Economics, University of the Philippines Diliman, Quezon City, Philippines ABSTRACT The following experiment describes the process and principles behind extracting DNA from live shrimps and assessing the DNA extract’s concentration and purity, based on the detection of its basic unit, nucleic acid. This experiment aims to obtain an extract with high nucleic acid purity and concentration. The extraction process involves cell lysis, the denaturing of proteins and destruction of protein-DNA complexes. The purity of the nucleic acid obtained was assessed using a double beam UV-vis spectrophotometer at wavelengths 260nm and 280nm. The recorded absorbance values were 0.6598 and 0.4757 for 260nm and 280nm, respectively. The % nucleic acid of the extract was 10% and the concentration of DNA was computed to be 3.299 . To further evaluate the purity of the extracted DNA, the obtained sample was subjected to agarose gel electrophoresis. However, due to possible old reagents, the gel did not impart clear bands. Apart from the agarose gel electrophresis, it can be concluded that the methods used in this experiment are significant in the extraction and overall study of DNA.

INTRODUCTION All living organisms contain the genetic carriers, DNA. With the exception of identical siblings, each organism carries a different set of DNA, making each one unique from the other. This is because DNA is responsible for coding most of the genetic information in an organism and is expressed in an organism’s physical appearance, personality, and behavior. With the enormous amount of information that completes an organisms genetic make up, it is astounding to discover how it all fits within the DNA molecule. The answer lies within the DNA’s double-stranded helix structure. The basic units of DNA are nucleotides. Nucleotides are made up of three covalently bonded parts, a nitrogenous base (purines: adenine and guanine, pyrimidines: thymine and cytosine), a deoxyribose sugar, and a phosphoric acid residue. An N-glycosidic bond links the C-1’ carbon of the deoxyribose sugar to the N-9 nitrogen in purines or the N-1 nitrogen in pyrimidines.

Figure 8.1 DNA Nucleotide structure

Nucleotides form nucleic acids through polymerization. The repeating 3’-5’ phosphodiester bonds form in the DNA backbone between phosphoric

acid and the 3’ and 5’ carbons of adjacent deoxyribose sugars.

Figure 8.2 Phosphodiester bond connecting two nucleotides The four nucleotide bases combine in different sequences to form the essential information of genes. The nucleotide bases from a single DNA strand form hydrogen bonds with their complementary nucleotide bases from the second strand to form the doublestranded structure. Adenine forms two hydrogen bonds with thymine while guanine forms three hydrogen bonds with cytosine.

The goal of this experiment is to extract DNA from the muscle tissue of live shrimp and to rationalize the procedure employed in the extraction. The characterization of the DNA extract will be determined by estimating the concentration and purity through UV spectroscopy and agarose gel elctrophoresis. EXPERIMENTAL DETAILS The experiment was divided into two parts, the first part was where the DNA was extracted from the live shrimp and purified and the second part was the analysis of the purified DNA sample. Figure 8.3 Nucleotide base pairing between the DNA double helix The mentioned structure can be found in DNA’s ideal functioning form. Unfortunately, genes may mutate and cause severe and even fatal diseases. Unlocking the secrets behind DNA codes would be beneficial to understanding the causes and functions of many diseases caused by genetic mutations. Human genetic engineering and gene therapy may be the key to creating cures and more importantly, preventive medicine. To study DNA, first it must be extracted and isolated.

In the first part, the DNA sample was acquired from fresh shrimp. The shrimp head, tail, legs, and exoskeleton were removed and discarded. The muscular meat was obtained and cut into smaller pieces over ice. The sample, along with liquid nitrogen, was pulverized through grinding in circular motion.

DNA molecules are large and fragile thus, extracting a pure, undamaged, large yield needs to follow a carefully calculated procedure set within ideal conditions to keep extracted DNA stable. The ideal conditions that must be considered include pH, temperature, ionic strength, cellular conditions, and mechanical stress. Upon extraction and isolation, double-beam UV-vis spectrophotometry can be used to determine the concentration and purity of the DNA extract. The aromatic nucleotide bases found in DNA absorb UV light, allowing this method to detect the presence of DNA. Aside from UV light absorption, further assessment of the purity of the DNA extract was performed through electrophoretic methods. Electrophoresis is an experimental method based on the differential movement of charged molecules in an electric field and can be used to purify and analyze many biomolecules. The movement of molecules takes place in a polymerized gel matrix, which is used because it is more stable as compared to a free solution. Applying electric current causes migration of the different molecules across the rigid gel matrix. The difference in movement of molecules is influenced by molecule size, shape, charge, and chemical composition. Agarose was chosen as the gel medium for the analysis of DNA. In agarose gel electrophoresis, the DNA molecules move through the electric field due to charge but because the molecules have an equal charge to mass ratio, the basis of separation depends on the size and shape of the DNA molecules. Nucleic acids migrate at a rate inversely proportional to its size.

Figure 8.4 Grinding of shrimp sample with liquid nitrogen The pulverized sample was placed inside two centrifuge tubes, one tube containing 0.34g and the other containing 0.31g of the sample. Then, the samples were suspended in 10mL of pre-heated 0.05 M Tris-HCl buffer, pH 8.0 at 55 . Next, around 1mL 10% SDS was added dropwise to the sides of the tube to get a final concentration of 1% SDS. The centrifuge tubes were incubated by submerging the tubes in a heated water bath at 55 for 45 minutes, while gently shaking the tubes every 10 minutes. Chloroform was added slowly, dropwise to the sides of the tubes. The tubes were shaken then, centrifuged twice, five minutes each time. A wide-tipped Pasteur pipette was used to collect the aqueous layer and transferred to a small beaker. 5 M NaCl was then added to the collected aqueous layer. 95% ethanol was added to the beaker. The solution was put inside tubes and centrifuged for 5 minutes. DNA appeared as fibrous white precipitate. The solution was decanted and the precipitate was airdried.

In preparing the sample for DNA extraction, the sample was cut into small pieces over ice. Ice was needed to prevent DNA degradation by nuclease enzymes. The sample was cut into small pieces to aid in cell lysis to release the DNA from the cell. Liquid nitrogen was also used for the purpose of destroying the cell membrane and keeping the sample at a very cold temperature. Upon adding liquid nitrogen, the shrimp sample hardened up and froze, making it easier to grind the sample.

Figure 8.5 Isolated DNA extract Lastly, the DNA was dissolved using 10 mL 0.05 M Tris-EDTA buffer at pH 8.0. The concentration in % (w/v) of the stock solution was obtained. For the second part of the experiment, 40 μL of the sample was pipetted out and diluted to 5.0 mL using the Tris-EDTA buffer at pH 8.0. The absorbance of the solution was read at 260 and 280 nm against the Tris-EDTA buffer pH 8.0 as blank. From this, the ratio of A260 and A280 was calculated and the % DNA purity was obtained. The DNA concentration was also estimated by following a specific formula. Agarose gel electrophoresis was employed to further analyze the DNA extract’s purity. The gel was prepared from 0.32 g gel powder, which was mixed in 32 mL of 1X TAE buffer. The mixture was heated and mixed constantly but prevented from boiling. The agarose solution was removed from heat once it became completely transparent. It was allowed to cool to 37 ° C. 300 μL of ethidium bromide was added and swirled to mix. The solution was poured carefully and smoothly into the gel tray to prevent air bubbles from forming. The comb was placed over the gel but was not allowed to touch the bottom of the gel. Once the gel solidified after 20-30 minutes at room temperature, the comb was removed from the gel in one fluid motion.

Tris-HCl buffer pH 8.0 was added to the sample to set the proper pH conditions for DNA. The bonds stabilizing the DNA molecule are all stable at pH 8.0. H-bonds are stable within pH 4-10, phosphodiester linkages are stable within pH 3-12, and glycosidic bonds are stable at pH 4 and up. Aside from stabilizing DNA, higher pH levels also deactivate nucleases, which in turn prevent DNA from being degraded. The buffer was also heated to 55 At this temperature, most proteins will denature whereas DNA can remain intact up to 80 . Like proteins, DNA structure can be viewed in levels. The primary structure is the order of the bases in the polynucleotide sequence. The secondary structure is the 3D conformation of the backbone. The tertiary structure is the supercoiling of the double strands into a helix. Supercoiling occurs to make all the information found in DNA fit into a very compact space. In eukaryotic DNA, supercoiling occurs by formation of protein-DNA complexes called chromatin. These complexes form through electrostatic interaction between negatively charged phosphate groups found in the DNA backbone and positively charged histone proteins. When isolating DNA, DNA must be freed from these complexes. At pH 8.0 electrostatic interactions between DNA and histones are reduced. Sodium dodecyl sulfate (SDS), an anionic detergent further breaks protein-DNA complexes by reducing the positive character of histones. SDS disrupts ionic interactions between the positively charged histones and the negatively charged phosphates on the backbone of DNA. SDS also denatures deoxyribonucleases and other proteins.

The wells were flushed with 1X TAE buffer before the samples were added. The sample was prepared by adding 30 μL loading buffer into an eppendorf tube before pipetting 70 μL DNA sample into the same tube. A pipette tip was used to mix the solutions. 20 μL of the solution was loaded into the well. Care was made not to puncture the bottom of the gel with the pipette tip or spill sample out of the wells.

Denatured proteins were removed from the sample solution by adding chloroform. Chloroform promotes separation of organic and aqueous phases. Denatured proteins stay in the organic phase while DNA remains in the aqueous phase. To further initiate separation of layers, the sample solutions were centrifuged.

The gel chamber was filled with running buffer until the gel containing the sample was completely immersed. The power supply was set to 60 V. The apparatus was turned off once the tracking dye reached 80% of the gel length.

After centrifugation, the aqueous layer containing the DNA was collected. 5M NaCl, a high concentration salt, was added to remove bound cationic amines and to dissociate any leftover proteins. The salt weakens ionic interactions between DNA and cations.

RESULTS AND DISCUSSION

Lastly, ethanol, an organic solvent, was added to precipitate DNA. Ethanol works to precipitate DNA by making the aqueous medium less polar. DNA precipitate is threadlike in appearance due to its long standed supercoiled double helix structure. The DNA obtained was resuspended in 0.05M Tris-EDTA buffer pH 8.0 to keep the sample in stable conditions. UV spectroscopy was used to determine the nucleic acid concentration and purity of the extracted and isolated DNA sample. Nucleic acids contain aromatic nucleotide bases adenine, guanine, thymine and cytosine. These nucleotide bases can absorb UV light due to the rich amount of electrons found in their aromatic rings, carbonyl groups, and nitrogen and oxygen atoms. Nucleic acids absorb UV light at a maximum of 260nm. UV absorption was also measured at 280nm to account for the possible absorption of proteins still present in the DNA solution. Table 8.1 UV absorbance readings of shrimp DNA sample Wavelength UV absorbance 260nm 0.6598 280nm 0.4757

Besides direct measure of UV spectroscopy, thermal denaturation is another method that can be used to characterize DNA. In thermal denaturation, the DNA solution is treated with denaturing agents and then UV absorbance is measured. The UV absorbance shows notable increase after addition of denaturing agents and increase in temperature. In denatured DNA, there is minimal base-to-base interaction, which alters the resonance behavior of the aromatic rings found in the bases and thus, absorption increases. The midpoint in the absorption increase is called melting temperature. Each DNA has a characteristic melting temperature value. The advantage of this method is that it can identify unknown DNA samples by matching it with the known meting temperature values. A disadvantage is that the DNA samples will be denatured and cannot be recovered in its native form.

DNA molecules are negatively charged at neutral pH due to the presence of phosphate groups. The negative charge of the DNA molecules cause them to move toward the positive electrode at the opposite end of the gel. Each nucleotide residue contributes to the overall negative charge of the molecule due to the amount of phosphate groups. More phosphate groups means more negative charges but this also means that the molecule is larger and heavier. This makes the charge to mass ratio nearly the same for each molecule thus, without the charge playing a role in separation, the molecule size and shape are the only separating factors. The smaller the molecule, the easier for it to navigate through the cross-linked agarose gel. The concentration of the agarose gel also influences the mobility of the DNA molecules. Agarose polymers form a network of bundles whose pore sizes depend on the agarose concentration. A lower concentration of agarose, around 0.3%, allows for the sieving of DNA molecules within 5-60 kilobase pairs. Tris-Acetate-EDTA (TAE) was the electrophoretic buffer used because of its near neutral pH, which allows for the negative DNA molecules to migrate to the anode at the opposite end of the gel. TAE buffer is commonly used to separate large DNA because it interacts with the agarose gel resulting in larger pore size and lower field strength. These interactions lead to a decrease in gel smearing. After obtaining the gel after electrophoresis proper, ethidium bromide was used to detect the bands formed. Ethidium bromide is a fluorescent assay commonly used because of its convenience, sensitivity, and versatility. However, ethidium bromide is highly toxic and must be handled with utmost precaution. To limit the amount of ethidium bromide used and its contact to apparatus, it was administered “in-gel”, before the gel solidified. Purines and pyrimidines have weak fluorescence spectra. Ethidium bromide inserts between stacked base pairs in nucleic acid and enhances fluorescence twenty-five fold, making bands in the gel visible. CONCLUSION AND RECOMMENDATION

Another method used to assess the purity of extracted DNA is agarose gel electrophoresis. Agarose gel electrophoresis is chosen as a gel medium to analyze larger fragments, such as DNA.

Figure 8.6 Agarose gel electrophoresis

The resulting shrimp DNA solution was calculated to be 3.4% (w/v). There may have been some product loss due to an error in the execution of the procedure. Instead of slowly adding ethanol, a large amount was added all at once thus, the solution needed to be centrifuged for product to be collected. For future experiments of the same nature, it is recommended that the procedures be followed carefully and strictly as to not commit careless mistakes that may lead to product loss and worse, laboratory accidents. Based of the UV absorbance readings, the percent purity and estimated DNA concentration of the sample was calculated to be 10% and 3.299 μg/mL respectively. 10% nucleic acid is a relatively low percentage considering the steps performed to isolate

and purify the DNA sample. This may be due to the error in procedure mentioned. This percentage is only based on the nucleic acid to protein ratio and does not include other possible contaminants thus, the true percent nucleic acid may be even less if the other contaminants were put into consideration. In calculating for percent nucleic acid, a formula, which takes other contaminants into consideration, should be included in future experiments that require the same method so that the most accurate amount of nucleic acid present can be calculated. Agarose gel electrophoresis is a method often used in DNA separation and assessment due to its rapid and simple process, ease of separation, sensitive staining procedures, high resolution, and ability to analyze a wide range of molecular weights. However, when performed in this experiment, the resulting gel was not successful. This is probably due to old agarose gel powder and reagents. New reagents and materials should be used when performing agarose gel electrophoresis so that a gel with visible bands may be produced. Other possible sources of error in DNA extraction include improper handling of reagents, and unwanted cleavage of DNA fibers by nucleases that may not have been denatured. Further study about the structure of the DNA extracted may also be added to the experiment such as the determination of the conformation and structure of the DNA sample. REFERENCES (1) Boyer, R. (2012). Biochemistry Laboratory Modern Techniques and Theories. New Jersey: Pearson Education, Inc. (2) Campbell, M. K., Farrell, S.O. (2012). Biochemistry 7th Edition. USA: Cengage Learning. (3) Lee, P.Y., Costumbrado, J, Hsu, C.Y., Kim, Y.H. “Agarose gel electrophoresis for the separation of DNA fragments.” Journal of visualized experiments 10.62 (2012): 37913923. 4 Nov 2014.

APPENDIX Weight centrifuge tube: 6.68g Weight centrifuge with sample: a.) 7.02g Weight sample: a.) 0.34g b.) 0.31g

b.) 6.98

Conc. (10%)(x)=(1%)(10mL+x) x=0.909mL x100= 3.4% w/v

=

=1.3870 -> 10% nucleic acid

dsDNA concentration=50

x

x dilution factor

=50

x0.6598x

= 3.299