Garam Lee, Seoyun Jeong
Abstract
Gene therapy technologies, such as CRISPR gene editing, are rapidly advancing alongside biotechnology and medical advancements using DNA. Consequently, treatments like stem cell therapy and personalized genetic therapy are no longer in a distant future. What enabled these advancements in DNA-based therapies and various technologies? It was the ability to analyze DNA structures through techniques such as DNA electrophoresis. We conducted experiments to extract and analyze the structure and function of DNA. Although the experiments were not completely successful, through literature research in papers and books, we have gained an understanding of the function of DNA, its extraction process and scientific principles, and the process and principle of electrophoresis.
Keywords: DNA, DNA extraction, gel electrophoresis
I. Introduction
1. Research Purpose and Necessity
a. Research Purpose
Since the Industrial Revolution, technology has advanced rapidly, allowing humanity to enjoy a prosperous life. Alongside this, biotechnology benefiting all life on Earth has also advanced. Despite this progress, many diseases remain untreatable with current medical or biotechnological methods. While some diseases' causes are still unknown, genetic diseases can be more easily traced to abnormalities in chromosome numbers, structures, or DNA itself. Understanding the structure or characteristics of DNA can reveal the causes of genetic diseases. By using electrophoresis, a chemical phenomenon, DNA can be separated and its structure can be determined, significantly contributing to DNA-related research.
b. Necessity of the Research
Analyzing the base sequences within human cells' DNA can uncover the functions of all genes, enabling the identification and treatment of genes causing genetic diseases. Additionally, DNA testing is fundamental for paternity tests and identifying suspects from saliva or hair samples. DNA is intricately connected to our daily lives. Research has shifted from structural analysis of DNA to functional analysis and understanding the interrelationships between genes. Applying this knowledge in the field of DNA research requires substantial technical support, with DNA analysis being essential. High-resolution separation, rapid analysis, and sensitive detection of specific DNA fragments from other DNA strands are crucial for advancing DNA research and its applications.
c. Expected Effects
Analyzing DNA's structure and properties through research can promote the development of treatments for genetic diseases, CRISPR gene editing, paternity testing, and other DNA-related research and technologies.
2. Research Methods
a. Literature Review
To ensure comprehension at the student's level, a literature review was conducted to gain a theoretical understanding of the experimental content before conducting the DNA electrophoresis experiment.
b. Experimental Activities
Both DNA extraction and DNA electrophoresis experiments were conducted.
3. Previous Studies
a. Development of the Electrophoresis Phenomenon
In the 1930s, Swedish biophysicist Arne Tiselius, while researching serum proteins, discovered that when a protein mixture is placed between buffer solutions in a tube and an electric field is applied, the sample components move in one direction at a rate determined by their charge and mobility. He devised an analytical method using electrophoresis, which contributed to the advancement of molecular biology, chemistry, and other scientific fields. In 1948, Arne Tiselius was awarded the Nobel Prize in Chemistry for his contributions.
b. Discovery of the DNA Double Helix Structure by Watson and Crick
DNA research gained momentum in 1953 when James Watson and Francis Crick published a paper in 'Nature,' revealing that DNA has a double helix structure. Watson and Crick conducted their research using X-ray photographs, and Watson, realizing that DNA had a double helix structure, worked with Crick to build a model of the DNA molecule, eventually presenting the double helix structure to the world.
II. Body
1. DNA
a. Structure of DNA
DNA (Deoxyribonucleic Acid) is a high molecular weight compound composed of two long strands of nucleotides twisted into a double helix structure. Each nucleotide, the basic unit of DNA, consists of a phosphate group, a sugar, and a base. These nucleotides are positioned along the outside of the double helix in a repeating pattern. The bases include adenine (A), thymine (T), guanine (G), and cytosine (C), with adenine pairing complementarily with thymine and guanine pairing with cytosine. These complementary base pairs are located on the inside of the double helix structure. DNA contains phosphate groups, which give DNA its negative charge.
2. DNA Extraction
a. DNA Extraction Process and Principles
(1) DNA Extraction Process
Experimental Materials: Broccoli/Banana, 2g of salt, 150ml of distilled/purified water, 7ml of surfactant, electronic scale, weighing paper, measuring spoon, pipette, mortar and pestle, beaker, strainer, wooden stick, ethanol, glass rod.
Add 2g of salt to 150ml of distilled/purified water and mix, then add 7ml of surfactant and mix using a pipette.
Let the salt-surfactant solution sit at room temperature for 10 minutes, then mash the broccoli/banana using a mortar and pestle.
Mix the mashed broccoli/banana with the salt-surfactant solution from step 1.
Strain the mixture through a strainer to remove small particles, then stir the filtered solution with a glass rod.
Using a pipette, carefully pour cold ethanol along the wall of the beaker.
When a white, thread-like substance appears at the ethanol layer, collect it by winding it around a wooden stick.
Figure 1. This is the result of the DNA extraction experiment. The DNA samples are, in order, from broccoli and banana.
(2) Principles
In step 1, the salt causes the DNA to clump together, while the surfactant helps release the DNA from the cells. In step 2, mashing the broccoli/banana breaks down the cell walls and membranes. In step 5, adding cold ethanol causes the DNA to precipitate out because DNA is not soluble in ethanol, unlike in water, allowing it to be collected.
3. DNA Electrophoresis
a. Definition of Electrophoresis Phenomenon
Electrophoresis refers to the movement of charged particles through a solution under the influence of an electric field. Electrophoresis is a separation method that utilizes the differing movement of particles within a uniform electric field based on the properties of the fluid. This method was developed after observing that clay particles dispersed in water moved under an electric field in a uniform electric field environment.
b. Types of Electrophoresis Methods
(1) Capillary Electrophoresis
This method, performed in very fine tubes (capillaries), separates substances based on their charge and molecular weight.
(2) Gel Electrophoresis
Gel electrophoresis, while having lower resolution, can separate large molecules from 200bp to 50kb. It is the most commonly used electrophoresis method due to its simplicity and ease of handling. Based on the type of gel used, it is divided into 'Agarose Gel Electrophoresis' and 'Acrylamide Gel Electrophoresis'. For DNA electrophoresis, agarose gel is used for fragments that are not too small (1-300bp) or too large (>40kb). To perform DNA electrophoresis, DNA must be separated based on size and shape. Since there are countless DNA molecules with similar sizes and shapes, conventional electrophoresis cannot achieve this separation. Gel electrophoresis overcomes this limitation by causing DNA to be cut by the gel loaded into wells, resulting in separation by size. This method is used primarily in laboratories to separate macromolecules based on size. For example, it can be used to separate proteins by making them negatively charged and moving them toward a positive charge, analyzing proteins, DNA, and RNA, and analyzing plasmids (DNA that can replicate independently of chromosomes) in antibiotic-resistant bacteria.
c. Chemical Principles of Electrophoresis Phenomenon
The principle of electrophoresis is related to chemical principles. When an electric current is passed through a solution, the charged particles in the solution move towards the anode or cathode. In a field where there is a voltage difference, negatively charged particles move towards the anode, and positively charged particles move towards the cathode.
During this process, like charges repel each other, and opposite charges attract. The force acting between two electrically charged objects is directly proportional to the product of the charges and inversely proportional to the square of the distance between them, known as Coulomb's law. The amount of charge on an object is measured in Coulombs (C).
In a buffer solution, macromolecules become charged. The charge on these molecules depends on the pH of the buffer solution, which plays a crucial role in electrophoresis. The overall charge of the molecules determines their movement speed within the electric field, causing separation based on the total charge or size of the molecules. If the total charge of a molecule in an electric field is qqq, the force FFF acting on the molecule is a function of the charge and the strength of the electric field. This relationship can be expressed mathematically as:
where E is the potential difference between the electrodes, and d is the distance between them. The term E/d represents the electric field strength.
According to Stokes' law, the relationship between the force FFF, the size and shape of the molecule, and the viscosity of the solution is given by:
where r is the radius of the spherical particle, η is the viscosity of the solution, and v is the velocity of the particle.
Based on this, the equation we can derive is:
And manipulating this equation is:
In other words, the velocity vvv of a molecule is directly proportional to the electric field strength and the total charge, but inversely proportional to its size and the viscosity of the solution.
d. Buffer (Buffer Solution) Usage
A buffer is a solution that stabilizes pH. In electrophoresis experiments, a buffer is necessary to transport DNA, providing the ions needed to carry the DNA molecules. Buffers are primarily divided into TAE Buffer and TBE Buffer.
(1) Comparison of TAE and TBE Buffers
Figure 2. The left-side photo shows Tris (Trisaminomethane), and the right-side photo shows EDTA (Ethylenediaminetetraacetic acid).
Tris provides cations and attracts the negatively charged DNA. EDTA prevents DNA degradation and helps maintain its negative charge. TAE Buffer contains acetate, while TBE Buffer contains borate. Tris has a high pH, close to 11, and acetate, with a relatively lower pH, helps lower the pH of Tris. Additionally, TAE Buffer is used in agarose gel electrophoresis for large DNA molecules, whereas TBE Buffer is used for smaller DNA molecules and is commonly used in DNA sequencing.
e. Experimental Procedure and Results
Extract DNA (can be easily extracted using broccoli/banana, etc.).
Treat DNA with restriction enzymes to cut it into smaller fragments.
If necessary, perform loading dye treatment using EtBr (ethidium bromide).
Place the DNA samples in the electrophoresis apparatus and connect the electrodes.
After electrophoresis is complete, treat with fluorescent dye and observe under ultraviolet light.
Figure 3. The photo above shows that the DNA samples have migrated. (The left-side photo shows the typical experimental results, while the right-side photo displays the results of the failed experiments conducted during the research process.)
f. Chemical Considerations of Experimental Principles
DNA contains phosphate groups, which carry negative charges, making DNA exhibit a negative charge in an aqueous solution. Thus, when DNA is placed in an electric field, it moves towards the positive electrode. This property allows DNA to be separated by size. Electrophoresis works on the principle of moving DNA through a porous support matrix (gel) after loading the DNA sample and applying voltage. DNA's migration speed depends on the molecule's size, the gel's concentration, and the DNA's structure. Larger molecules move slower, while smaller ones move faster through the gel's pores. As the concentration of the agarose gel increases, the porous structure becomes denser, causing DNA molecules to move more slowly. Additionally, DNA can exist in supercoiled, linear, or open circular forms, with the migration speed determined by the cross-sectional area. Supercoiled DNA, having the smallest cross-sectional area, moves the fastest. Other factors affecting migration speed and results include the current intensity and buffer concentration.
III. Conclusion
1. Research Conclusion
The final report indicates that the DNA extraction experiment, despite multiple failures, ultimately succeeded. Failures occurred due to using tap water instead of distilled/purified water, using non-cold ethanol, and incorrect salt concentration, but DNA was successfully extracted from both banana and broccoli. The DNA electrophoresis was attempted twice but failed both times due to equipment issues. Proper electrophoresis equipment and tools like micropipettes, EtBr, gel casts, and gel combs were unavailable. Time constraints also affected the results; the agarose gel was not used immediately after cooling, and the broccoli's freshness was compromised. These factors were considered the limitations of the study.
2. Suggestions
a. Seoyun Jeong
It was surprising to see that DNA from living organisms like broccoli and bananas could be extracted without lengthy, complicated procedures. Adjusting the salt concentration to clump the DNA and deciding which water type to use were challenging. After many failures, successfully extracting DNA from bananas was very rewarding. Although the DNA electrophoresis experiment failed, using previously unfamiliar tools and materials like gel trays, agarose gel, and TAE buffer was highly beneficial. I hope to use micropipettes and gel combs in future experiments to extract DNA from broccoli and bananas, separate proteins with a centrifuge, and perform DNA electrophoresis with proper equipment.
b. Garam Lee
This study helped me understand the principles and types of electrophoresis, a chemical phenomenon, and the principles of DNA extraction using food. The broccoli extraction experiment was conducted nine times, with only one success, and the electrophoresis literature review involved unfamiliar formulas and terms. To overcome these difficulties, I performed the same experiment with bananas, used online resources to understand electrophoresis results, and borrowed books to fully grasp the concepts. This research, tied to my career interests, taught me chemical methods for understanding DNA structure, how to address experimental failures, and how to write a scientific report, making it a meaningful and educational experience.
IV. References
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