Extraction and Characterization of Bacterial DNA

ABSTRACT:

In this experiment, deoxyribonucleic acid (DNA) was extracted from Micrococcus lysodeikticus, a bacteria with a high guanine and cytosine content. The standard method of chloroform-isoamyl alcohol extraction was used, and the DNA was solubilized in Tris buffer. The DNA was then quantified and qualified using UV spectrophotometry. The DNA was determined to have been extracted in relatively high amounts, but the purity was lacking, especially in regards to ribonucleic acid contamination. The hyperchromic effect was utilized to gauge the purity of the DNA. Future studies will focus on the ecologically safer and more efficient methods of DNA extraction.

Key Words: Ribonuclease, RNA, DNA, DNA structure, bacteria. Deoxyribonucleic acid (DNA) is the genetic material inside of a cell. The code of nucleotide base pair sequences allows for the cell to translate messages into proteins that can be used in all parts of the cell. DNA is a double helix that is composed of rungs being the base pairs that are hydrogen bonded together, and a sugar phosphate backbone that is covalently bound together1. This structure gives the DNA the strength yet flexibility it needs in order to be able to unzip so that the genetic material it holds can be accessed2.

The complementarity of the DNA molecule is truly what sets the molecule apart from others. Even if the DNA is denatured and the hydrogen bonds come undone, because each base pair binds to only one specific type of other base pair, this phenomena allows the DNA to spontaneously come together again. The base pairs will line up with their respective pair due to the energetic favorability of having as many hydrogen bonds as possible2.

Because of DNA's importance as a biological storage molecule, accessing the DNA trapped inside of the cells has become a main avenue of research for scientists. Depending on the organism, different solvents and methods can be used to yield the highest amounts and most pure DNA.

Even within the same types of organisms, there may be different challenges that arise from a specific species. Specifically in bacteria, different strains can have higher or lower G+C content3. Because the guanine and cytosine nucleotides are bound together by three hydrogen bonds as opposed to the 2 that bind adenine and thymine together, the G+C bonds are more difficult to pull apart. This means that harsher solvents may have to be used in order to adequately extract and solubilize the DNA.

When the DNA is extracted, it can be denatured as a way to assess its purity. The hyperchromic effect is a phenomena that occurs when DNA is denatured so that the two strands of the double helix come apart and assume a formation that is random and coiled4. Because there is much more surface area in the randomly coiled DNA than the uniform and compact double helix form, the DNA will absorb substantially more UV light at a wavelength of 260nm.

In this experiment, the DNA of Micrococcus lysodeikticus was extracted using a chloroform-isoamyl alcohol method and was precipitated using cold ethyl alcohol. Following this partial purification of the DNA, the DNA was quantified and qualified using UV spectroscopy. The hyperchromic effect was employed to ascertain the purity of the DNA by denaturing the molecule using high tempertaures.

EXPERIMENTAL PROCEDURES

The experiment was conducted as described by Boyer2.

To begin the experiment, a set amount of freeze dried bacterial cells of the species Micrococcus lysodeikticus were massed out and suspended in a set volume of saline-EDTA in a 50mL Erlenmeyer flask. An aliquot of 10mg/mL lysozyme was added to the bacterial solution and was incubated at 37?°C for 30 minutes.

After the incubation, an aliquot of 25% sodium dodecyl sulfate was added to the mixture and was heated at 60?°C for 10 minutes, after which the mixture was cooled to room temperature. The mixture was then added to a Nalgene centrifuge tube.

An aliquot of 5M sodium perchlorate solution was added to the solution, followed by a generous portion of 24:1 chloroform-isoamyl alcohol. This mixture was then gently shook for 20 minutes with gloves on so that the proteins in the mixture could be separated out. The solution was then placed in a refrigerated centrifuge at 7800rpm for 5 minutes. The aqueous upper layer of the mixture was then carefully transferred into another container, where ice cold ethyl-alcohol was gently poured over it in generous amounts in order to precipitate the DNA. The nucleic acids were then spooled and transferred to a set volume of Tris buffer. Once a sufficient amount of DNA had been spooled, the aqueous DNA mixture was split into two portions. In one sample, RNAse was added and inverted to mix. Both samples were then stored in a 4?°C refrigerator for one week, and the rest of the procedure was conducted.

Both the DNA with and without RNAse were used to make separate dilutions so that the Absorbance at 260nm was approximately 0.400. From both of the dilutions, 2mL aliquots were added to microcentrifuge tubes, and the two different types of DNA solutions were treated in the same fashion. One sample was kept at room temperature, one tube was denatured in boiling water for 10 minutes and then left to cool to room temperature slowly, and the other tube was denatured in boiling water for 10 minutes and then placed in an ice bath to cool to room temperature quickly.

Absorbance values were gathered from the room temperature samples at 260nm and 280nm. The absorbance for the room temperature sample at 260nm was recorded again at the end of the experiment. The sample that was heated and allowed to cool slowly had the first absorbance recorded immediately after it was taken out of the boiling water. Another 260nm absorbance value was taken after the sample had cooled down to room temperature. The sample that was heated and cooled in an ice bath had its absorbance measured after it was at room temperature.

To analyze the data gathered from this experiment, the following two equations were used to determine how much DNA was isolated.

Equation 1. Determining the concentration of the DNA solution.

Equation 2. Determining the amount of DNA isolated.

As a point of comparison, the theoretical amount of DNA that could be isolated from the mass of cells used was determined using the following.

Equation 3. Determining the theoretical mass of DNA.

Next, the following equation was used to determine the percent of the cell's weight that is composed of DNA.

Equation 4. Percentage of cell weight that is DNA

Then the purity of the DNA could be determined given the ratio of absorbance at 260nm to the absorbance at 280nm.This also lends insight as to what possible impurities could be in the DNA, such as protein or RNA (ribonucleic acid). The following equation demonstrates the change in the absorbance values at 260nm to reflect what occurred in terms of the hyperchromic effect.

Equation 5. Percentage of change in the absorbance values.

DNA mass (??g)

Theoretical DNA mass (??g)

Percent Cell Weight (%)

A260/A280

Percent Change A260 for Hot & RT (%)

Percent Change A260 for Hot-RT & RT (%)

Percent Change A260 for Hot-Ice-RT & RT (%)

DNA

1004

1787

0.75

2.71

12.5

-4.44

6.77

DNA with RNAse

794

4.51

31.2

-6.98

25.9

Table I. Summary of results from the spectrophotometry data collected regarding the DNA and DNA with RNAse. Equations 1 through 5 were used to determine the values listed in the table below.

RESULTS

The results of the experiment are listed in Table I. The calculations were performed for both the DNA samples and the DNA with RNAse samples.

DISCUSSION

In this experiment, various quantities surrounding the DNA sample were determined so that the amount of DNA and the purity of the DNA could be assessed. This was possible due to the scientific knowledge of the structure of DNA.

The theoretical and actual masses of the DNA were very close to each other, indicating that a sufficient amount of DNA was isolated and the isolation process was performed accurately. Additionally, the percent cell weight was the same as the theoretical cell weight of 0.75%. However, the ratio of A260/A280 values lend insight as to the purity of the DNA, and the ratio is much higher than the pure DNA value of 1.90. This suggests that the RNAse was ineffective at removing an adequate amount of the RNA. However, the A280 value used seems to be skewed, so that it unnecessarily inflates the ratio. Further spectrophotometry would have to be done using the same DNA in order to determine the most accurate ratio for the sample.

The hyperchromic effect took hold for both the sample that was boiled and quickly cooled and the sample that was boiled and the absorbance value was immediately taken. There appeared to be a large effect from the speed cooling versus the slow cooling, which makes logical sense considering that the more time given to the DNA to arrange itself back into its complementary sequence, the more precise and accurate job that the intermolecular forces will do. However, if the DNA is cooled quickly, it does not have time to rearrange precisely. Rather, the DNA could get stuck in tangles and therefore absorb more UV light that the normal double helix.

While the chloroform isoamyl procedure that was used in this experiment was effective, it is important to always strive for more efficient and/or more ecologically friendly alternatives to hazardous chemicals. In a study by Cheng and Jiang, the researchers developed a method of DNA extraction that allowed to cut the time it takes to extract DNA from microbes in more than half, and also eliminated the need for a separate cell wall lysing agent5.

Another reason to be on the lookout for different procedures for DNA extraction is due to the blossoming field of microbiome research. In a study by Knudsen et al, the choice of DNA extraction methods influenced the data that was garnered about the population in the microbiome6. Therefore, microbiome researchers must be cognizant of how experiments early on in the project could affect assumptions down the line. Reasons such as these mean that science must always be advancing into newer and better techniques.

REFERENCES

(1) Yakovchuk, P.; Protozanova, E.; Frank-Kamenetskii, M. D. Nucleic Acids Res. 2006, 34 (2), 564“574.

(2) Boyer, R. Modern Experimental Biochemistry, 3rd ed.; Roberts, B., Lake, J., Prescott, M., Eds.; Benjamin/Cummings: San Francisco, 2000.

(3) Sanders, C. A.; Yajko, D. M.; Hyun, W.; Langlois, R. G.; Nassos, P. S.; Fulwyler, M. J.; Hadley, W. K. J. Gen. Microbiol. 1990, 136 (2), 359“365.

(4) Lara Castellazzi, C.; Orozco, M.; Amadei, A. J. Phys. Chem. B 2013, 117, 8697“8704.

(5) Cheng, H. R.; Jiang, N. Biotechnol. Lett. 2006, 28 (1), 55“59.

(6) Knudsen, B. E.; Bergmark, L.; Munk, P.; Lukjancenko, O.; Prieme, A.; Aarestrup, F. M.; Pamp, S. J. bioRxiv 2016, 1 (5), 064394.

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