4.2: DNA Extraction

Background Information

Within the chromosomes of organisms, specific sections of DNA, called genes, code for specific proteins. A gene is “read” through a process of transcription to generate a sequence of mRNA. The mRNA is then used as the blueprint instructions to generate a specific protein during a process called translation. The process of generating a protein from a gene is often referred to as gene expression (Figure 1). A change, or mutation, in DNA can generate a protein that has a different amino acid sequence and therefore an altered or malfunctioning protein. Mutations in DNA of human chromosomes that cause malfunctioning or absent proteins are often the cause of genetic diseases. This lab discusses biotechnological advances that are used to detect and treat genetic diseases, advance agriculture or treat infectious diseases.

Figure 1. Gene expression. The process of transcription generates mRNA from a gene. mRNA undergoes translation to generate a protein. A change in the DNA sequence can alter the amino acid sequence of the protein and therefore can change or stop the function of the protein.

The first step in working with nucleic acids (DNA and RNA) is to remove the molecules from inside the cell. Different types of cells need to be processed differently in order to release nucleic acids. All cells have a cell membrane, a phospholipid bilayer that separates the internal environment of the cell from the external environment. In eukaryotes, DNA is housed inside the nucleus of the cell which is surrounded by the nuclear membrane, a second double-layered membrane, also composed largely of lipid molecules. When extracting DNA from plant cells, the cell wall must also be considered; some types of plant tissue require grinding or flash-freezing in order to break the tough cell wall.

In the DNA extraction procedure, plant cell walls and cell membranes are broken down by blending or mashing the cells. There are two key ingredients in the DNA extraction buffer aside from the water: dish soap (detergent) and salt. The detergent in the extraction buffer solution acts to dissolve phospholipids that form the cell membrane and the nuclear membrane. When the phospholipid membranes are dissolved, the cell lyses and releases the cellular contents, including DNA. The salt has two functions in the extraction process. It helps to neutralize the charge on the sugar-phosphate backbone, making DNA less soluble in water and allows it to more easily precipitate when the alcohol is added. The salt also helps to remove the proteins that are bound to the DNA and to keep the proteins dissolved in the lysis solution. The technique of filtration uses a medium, in this case, cheesecloth, to separate solids from liquids. The resultant material is referred to as filtrate. When cold ethanol is added to the filtrate, DNA precipitates at the water/ethanol interface. Although an individual DNA molecule is not visible with the naked eye, DNA isolated from large quantities of cells can be observed.

This protocol for extraction of DNA is based largely on the principle of solubility. Solubility refers to the ability of one substance (the solute) to dissolve in another substance (the solvent). Polar substances dissolve easily in polar solvents, but do not dissolve easily in nonpolar solvents, a phenomenon commonly referred to as “like dissolves like.” Water is a polar solvent, and molecules that dissolve easily in water are referred to as hydrophilic. DNA molecules are hydrophilic because the sugar-phosphate backbone of the molecules is highly polar. This means that DNA dissolves in water, so in this experiment, the DNA that is released when the cells are crushed dissolves in the juice/extraction buffer mixture.

Although the chemical reactions described above are all happening when you add the buffer and crush the food (strawberries), they are not visible with the naked eye. However, the addition of the cold ethanol caused a much more dramatic result! Ethanol is a nonpolar solvent, and when it is added to the juice extract, the DNA precipitates out of the solution. A precipitation reaction is a chemical reaction that causes a solid substance to emerge from a liquid solution. In this experiment, the addition of ethanol to the reaction forces the DNA to precipitate out of solution, which we can then spool onto the wooden stirrer or glass rod.

Exercise 1: DNA Extraction

Materials needed

1. Isopropyl alcohol 91% (rubbing alcohol) or 95% ethanol, Chilled in the freezer
2. Graduated cylinders
3. Salt
4. DI Water
5. Cheesecloth
6. Dishwashing liquid (preferably, Dawn)
7. Wide-mouth glass test tubes
8. Funnel
9. Disposable plastic cups
10. Microcentrifuge tubes
11. Wooden stirrer or glass rod
12. Resealable plastic bag
13. 100 or 200 mL beaker
14. Ice buckets with ice
15. A DNA source (about 1 cubic inch of food)
    1. strawberries
    2. bananas
    3. ground flax seed
    4. ground wheat germ
    5. peas
    6. broccoli
    7. spinach

Preparation

• Have isopropyl alcohol (rubbing alcohol) or ethanol cooled in an ice bucket ● Prepare your food item if needed (i.e. remove green strawberry tops, peel banana, grind wheat germ, etc)
• If not already made, prepare DNA extraction (lysis) buffer by combining the following: • 45 mL DI water
• 5 mL liquid dish soap
• 0.75 g NaCl (table salt)

Procedure

1. Place the food item (i.e 2 large strawberries) into the plastic zipper bag. Seal it and gently smash the food with your hands for about 2 minutes. Completely crush the food (strawberries) to disrupt the cells.
2. Open the plastic zipper bag and add 10 mL of the DNA extraction (lysis) buffer. Squeeze the bag to remove all air and seal the bag tightly.
3. Gently (to prevent over-sudsing or excessive foaming), but thoroughly, continue to crush the food (strawberries) inside the bag for about one minute or until it is a slushy consistency.
4. Completely line the funnel with a layer of cheesecloth. Place the funnel into the wide-mouth test tube.
5. Pour the food juice/DNA extraction buffer mixture into the funnel so the juice passes through the cheesecloth and into the test tube. Use the cheesecloth to strain the mixture so that only the juice flows into the tube and the pulp is retained in the cheesecloth.
6. Discard the cheesecloth and the pulp. Remove the funnel from the tube. The glass tube now contains a liquid called “filtrate”.
7. Carefully and slowly pipette an equal volume of ice-cold ethanol on top of the filtrate in the test tube using the plastic transfer pipette. The alcohol is less dense than the filtrate and will float as a layer on top of the filtrate. Do not mix or stir!
8. Hold the tube still at eye level and observe what happens at the interface of the alcohol and the filtrate. DNA will precipitate at the alcohol-lysis buffer interface. This means it will come out of solution into a “solid” form and appear as fluffy white cotton or cloudy material. Verify with your instructor that you have isolated DNA.
9. (optional) Use your wooden stirrer or glass rod to transfer your extracted DNA into a microcentrifuge tube. Add a small amount of ethanol to the tube to prevent your DNA from drying out.

Questions for Review

1. What reagent in the extraction buffer functions to dissolve membranes?
2. What reagent in the extraction buffer precipitates DNA so that it can be seen with the naked eye?
3. What reagent in the extraction buffer functions to remove proteins from DNA?