This experiment aimed to extract DNA from cheek cells and determine the length of an unknown DNA fragment using gel electrophoresis. The DNA was extracted by swishing Gatorade in the mouth, followed by mixing with detergent, pineapple juice, and isopropyl alcohol, which precipitated the DNA. The extracted DNA was visualized at the interface of the alcohol and solution. For gel electrophoresis, DNA samples, including a dye mixture, unknown DNA, and DNA markers, were loaded into an agarose gel, and an electric field was applied to separate the fragments by size. The distance migrated by the DNA markers allowed for the estimation of the unknown DNA fragment’s length. The results showed that the unknown DNA fragment was approximately 2.7 kilobases (kb) long, based on the semi-logarithmic standard curve. This experiment successfully demonstrated DNA extraction and electrophoresis, providing insights into molecular biology techniques for DNA analysis.

Introduction

Gel electrophoresis separates DNA, RNA, or proteins by size (Hames et.al, 1990). Each DNA molecule is a double helix made up of two complementary nucleotide strands held together by hydrogen bonds between the base pairs guanine (G)-cytosine (C) and adenine (A)-thymine (T). DNA consists of negatively charged phosphate backbone causes migration toward the positive electrode under an electric field (Alberts et. al, 2002). Dyes like ethidium bromide and SmartGlow Pre Stain (a non-carcinogenic alternative) visualize DNA by fluorescing under UV light. Molecules move through the gel’s pores at rates inversely proportional to their length; smaller fragments migrate faster (Mika et. al, 2024).

Agarose gels are primarily used in gel electrophoresis, where an electric field is applied to separate biomolecules based on their size and charge. The gel matrix acts as a molecular sieve, allowing smaller molecules to migrate faster than larger ones. Agarose gel is a gel-like substance derived from agar; a polysaccharide obtained from red algae. It is widely used in molecular biology for the separation and analysis of nucleic acids (DNA and RNA) and proteins. Gel concentration affects pore size and resolving power, and migration distances can be plotted against the logarithm of molecular length. DNA markers or ladders, containing known fragment sizes, allow for accurate size estimation of unknown samples by comparing migration distances. By creating a standard curve from known-length molecules, the lengths of unknown DNA can be calculated, typically expressed in kilobases (kb) or base pairs (bp) (Lee et. al, 2012).

The objective of this experiment is to measure the length of an unknown DNA fragment by analyzing its electrophoretic migration in comparison to standard DNA fragments of known lengths.

If the unknown DNA fragment migrates similarly to a standard DNA fragment of known length, then its size can be estimated based on that comparison.

Materials and Methods

DNA Extraction from Cheek Cells

DNA extraction from cheek cells involved swishing 5 ml of Gatorade in their mouth for 2 minutes, then transferring the solution to a test tube. After adding 2 ml of dishwashing detergent and swirling the tubes to mix, 2 ml of pineapple juice was added to the solution. The tubes were inverted to mix. Next, 2 ml of ice-cold isopropyl alcohol was gently added, and the tube was left for 10 minutes to precipitate the DNA (Mika et al., 2024).

Preparation of Agarose gel

For the next experiment, Agarose gels were prepared in advance and stored with the gray plastic comb in place. This comb was carefully removed after the gel solidified and saved for reuse. Each student was assigned a specific gel and electrophoresis unit, with the chamber designed to accommodate one gel. When loading samples, it was advised to skip the end wells to prevent contamination, and students were encouraged to load two samples of each DNA type if desired.

Agarose Gel Electrophoresis

Each gel was loaded with three types of samples: a dye mixture, unknown DNA, and a DNA marker, with each well holding approximately 25 µL of sample.

The DNA samples were prepared and frozen prior to the experiment. To load the samples, a micropipette equipped with a disposable pipet tip was used. The tip was submerged in the sample solution, and by pressing the thumb button, 25 µL of the sample was drawn into the pipet tip. Care was taken to check for and expel any air bubbles before carefully directing the pipet tip into the submerged well of the gel. Each well was filled to its 25 µL capacity without overfilling, and a new pipet tip was used for each sample to avoid cross-contamination. After loading, the sample vials were returned to the freezer.

Once the DNA samples were loaded, the gels were positioned in the electrophoresis chamber with the wells closest to the black (negative) electrode, ensuring that DNA would migrate toward the red (positive) electrode. The buffer solution (0.04 M Tris-Acetate EDTA, pH 8.0) was prepared and chilled in advance, and approximately 200 mL of this cold buffer was added to cover the gels completely, eliminating any trapped air bubbles. The orange lid was placed gently on top, adjusting as necessary.

An 8 cm clear ruler was placed on the lid, aligned with the wells to monitor DNA migration visually. The gel electrophoresis unit was then connected to a power source, and the settings were adjusted to run at 100 volts for 35 minutes. The timer was set, and the start button was pressed once all parameters were confirmed. The progress of DNA migration was observed by turning on the blue LED light underneath the chamber, which illuminated the bands as they moved away from the wells.

Electrophoresis continued until the dye mixture had migrated to within 2-3 mm of the gel’s end. After the run, the unit was turned off, and the cables were disconnected. The gels were visualized under the blue LED light, and photographs were taken using a black imaging box with an orange filter taped to the lid. Care was taken to align the camera with the ruler for accurate measurement of bands.

Data Analysis

Following imaging, the distance migrated by DNA Marker Fragment was measured in mm using ruler. Results were recorded in a designated table for analysis. This distance was plotted on a semi-logarithmic graph against the known sizes of the DNA markers to estimate the unknown fragment’s length in kilobases (kb). The distance migrated by the unknown DNA fragment was measured from the well to the leading edge of the band. Using the standard curve, the corresponding value on x-axis of graph, which is the length of unknown DNA fragment (Mika et al., 2024).

Simplified protocol

DNA Extraction from Cheek Cells:

1. Swish 5 mL of Gatorade in mouth for 2 minutes.
2. Transfer the solution to a test tube.
3. Add 2 mL of dishwashing detergent and swirl to mix.
4. Add 2 mL of pineapple juice and invert the tube to mix.
5. Gently add 2 mL of ice-cold isopropyl alcohol.
6. Leave the tube for 10 minutes to precipitate the DNA.

Agarose Gel Electrophoresis:

1. Prepare agarose gels in advance, storing them with a gray plastic comb.
2. Remove the comb carefully after the gel solidifies for reuse.
3. Assign specific gels and electrophoresis units to each student.
4. Skip end wells to avoid contamination when loading samples.
5. Load three types of samples into the wells: dye mixture, unknown DNA, and DNA marker (25 µL per well).
6. Prepare and freeze DNA samples prior to the experiment.
7. Use a micropipette with a disposable pipet tip to load samples into the wells.
8. Ensure no air bubbles in the pipette tip and use a new tip for each sample.
9. Ensure the gel is positioned in the electrophoresis chamber with the wells near the black (negative) electrode.
10. Prepare and chill 200 mL of buffer solution (0.04 M Tris-Acetate EDTA, pH 8.0) in advance.
11. Add the chilled buffer to cover the gels completely, removing air bubbles.
12. Place the orange lid on the electrophoresis chamber, adjusting if necessary.
13. Use an 8 cm clear ruler to monitor DNA migration visually, aligned with the wells.
14. Set the electrophoresis unit to 100 volts for 35 minutes.
15. Observe the DNA migration using the blue LED light underneath the chamber.
16. Continue electrophoresis until the dye mixture is 2-3 mm from the end of the gel.
17. After the run, turn off the unit and disconnect the cables.
18. Visualize the gels under blue LED light and take photographs using a black imaging box with an orange filter.
19. Align the camera with the ruler for accurate measurement of bands.

Data Analysis:

1. Measure the distance migrated by the DNA Marker Fragment in mm using a ruler.
2. Record the measurements in a table for analysis.
3. Plot the migration distance on a semi-logarithmic graph against the known DNA marker sizes.
4. Estimate the length of the unknown DNA fragment in kilobases (kb) using the standard curve.

Results

The DNA extracted from cheek cells was successfully precipitated and became visible at the interface of the alcohol and the solution in the test tube.

The distance migrated by DNA marker fragment was measured. The distances were plotted in a semi-log graph.

Table 1: DNA Marker Fragment Length

The distance migrated by unknown DNA fragment was measured and the fragment length was identified by comparing with the corresponding value of marker in semi-log paper.

Table 2: Unknown DNA Fragment Length Determination

Discussion

The DNA was extracted from cheek cells by adding various solutions, which facilitated its precipitation and made it visible at the interface of the alcohol and the solution in the test tube. DNA molecules are too small to be visualized and can only be seen using electron microscope, but clumping makes it visible (Mika et. al, 2024).

The gel electrophoresis experiment effectively demonstrated the separation of DNA fragments based on size, utilizing agarose gel as the medium (Hames et. al, 1990). The 0.04 M Tris-Acetate EDTA buffer at pH 8.0 facilitated the movement of negatively charged DNA toward the positive electrode (Alberts et. al, 2002). The constructed standard curve from known DNA markers allowed us to estimate the unknown fragment’s length at approximately 2.7 kilobases (kb).

The distinct separation of the dye mixture confirmed the integrity of the electrophoresis process, indicating that the gel was functioning properly. Visualization under blue light enabled clear imaging of DNA bands, and careful alignment with a ruler ensured accurate distance measurements.

Overall, this experiment successfully illustrated the principles of gel electrophoresis, emphasizing the importance of clean sample preparation and experimental conditions for obtaining reliable results. Future studies could investigate different agarose concentrations or incorporate additional controls to enhance the analysis further.

References

1. Hames, B. D., & Rickwood, D. (1990). Gel Electrophoresis of Nucleic Acids: A Practical Approach. Oxford University Press.
2. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. The Structure and Function of DNA. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26821/
3. Mika, T. A., Klein, R. J., Bullerjahn, A. E., Connour, R. L., Swimmer, L. M., White, R.
4. E., Gosses, M. W., Carter, T. E., Andrews, A. M., Maier, J. L., & Sidiq, F. (Eds.). (2024). Anatomy and physiology BIO 211 laboratory manual (3rd ed.). Owens Community College.
5. Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Journal of visualized experiments : JoVE, (62), 3923. https://doi.org/10.3791/3923