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Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) are the two nucleic acids found in living beings. DNA is the genetic material that is found in most organisms. On the other hand, RNA acts as a genetic material in some viruses that acts as a messenger adapter, structural, and in some cases as a catalytic molecule. The process of extracting Deoxyribonucleic Acid (DNA) from various samples is known as DNA Isolation. The methods which are used to isolate DNA differ depending on the sample’s source, age, and size.
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Key Terms: DNA, RNA, Nucleic Acids, Genetic Material, Green Peas, Enzyme, Ethyl Alcohol, DNA Isolation
Aim
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To isolate DNA from available plant materials such as spinach leaves, green pea seeds, papaya or any other plant materials available.
Apparatus
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- Any available plant material (For this experiment– Spinach Leaves)
- Distilled Water
- Mortar and pestle
- Test Tubes
- Beakers
- Ethanol
- Enzymes (Cellulase, ribonuclease, lipases, protease)
- Spool
Procedure
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During homogenization, the cell walls and membranes are damaged, and the resultant substance disperses in the buffer solution.
- Either manually rupturing the cells or chemically lysing them with enzymes produces the cell extract.
- Around 0.5 g of spinach leaf tissue should be crushed with a pestle in a mortar by using the enzyme cellulase to process the material.
- Afterward, a protease breaks down the peptide links in the plant material's proteins. In other words, the histone proteins that are bound to the DNA are eliminated by the enzyme.
- Use ribonuclease to dissolve the RNA.
- To dissolve lipids, use lipase.
- To allow the DNA to precipitate, add cooled ethanol. Basically, the DNA concentration is increased.
- To extract the precipitated DNA, use spooling. The process of spooling involves winding the DNA's tiny threads onto a reel.
DNA Extraction
Also Read:
Precautions
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- The weight of the leaf sample should be between 0.5 and 0.6 grams. There is a positive association between the DNA that was recovered and the leaf weight, which is between 0.5 and 1.0 g.
- Wash the plant material well with distilled water, pat it dry, and weigh it to get rid of any dust particles.
Observation
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We can observe that the DNA is visible on the spool as white precipitates of fine thread.
Theoretical Explanation of the Experiment (Extracting DNA From Anything Living)
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We will also see how to extract the DNA from any living thing. Finding something with DNA is the first step. Since DNA is the blueprint for life, everything that is alive has DNA.
We prefer to use green split peas for this test. But there are also many other DNA sources, including
- Spinach
- Broccoli
- Papaya
Step 1: We blend the green pea seeds.
- We put the following things in a blender
- ½ cup of green split peas (100 mL)
- 1/8 teaspoon salt
- 1 cup of cold water (200 mL)
- And blend them for 10 – 15 seconds.
- The blender will help in separrating the pea cells from each other.
Read More: Structure of DNA
Step 2: Soapy Peas
- We pour the pea-cell mixture from the previous step into another container. And add 2 tablespoons of liquid detergent (20-30 mL) and mix it well.
- And we let the mixture set for 10 minutes.
- After the mixture, is fully set we pour it into a test tube.
Peas Mixture
Step 3: Adding Enzyme
- Now, add a pinch of any enzyme to each test tube and stir it very gently.
- Stirring the solution excessively could break up the DNA and will make it harder to see.
- We can use a meat tenderizer for enzymes. If it is not available, we can also use pineapple juice or a cleaning solution for contact lenses.
- Meat tenderizer acts as an enzyme to cut off the proteins from the DNA.
Read More: Test for Protein
Step 4: Separating Alcohol
- To create a layer of alcohol on top of the pea mixture, tilt the test tube and slowly pour alcohol (ethyl alcohol) into the tube down the side. We pour until the alcohol content of the tube is almost equal to the amount of the pea mixture.
- As alcohol is less dense than water, it floats on top.
- After this, where the layers of water and alcohol converge, there should be clusters of white stringy material.
White Stringy Substance
Read More: Physical and Chemical Properties of Alcohol
Explanation of White Stringy Substance
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Deoxyribonucleic Acid (DNA) is a long, stringy molecule. It holds together better because of the salt that we added in step one. As a result, the clusters of DNA molecules we perceive are intertwined.
When salty DNA comes in contact with the alcohol, it loses its ability to remain dissolved in water. We refer to this as precipitation. More DNA strands are drawn into the alcohol as it rises due to the physical force of the DNA clumping together when it precipitates.
To gather the DNA, we can use a wooden stick or a straw. Our DNA can be transferred to a tiny alcohol-filled container if we want to preserve it.
Read More: Preparation of Alcohols
Things to Remember
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- DNA Isolation is defined as the process of extracting DNA from various samples.
- In order to isolate DNA for available plant materials such as green peas, there are four major steps.
- First, blend the peas with cold water and a bit of salt on high for 15 seconds.
- Then, add 2 tablespoons of liquid detergent and mix. Wait 5-10 minutes and pour the mixture into test tubes or other small glass containers, each about 1/3 full.
- Now, add a pinch of enzymes to each test tube and stir the mixture gently.
- Pour down the mixture into test tubes or containers, each about 1/3 full.
- Lastly, tilt the test tube and slowly pour rubbing alcohol into the test tube down the side so that it forms a layer on top of the pea mixture.
- DNA is a long, stringy molecule. More DNA strands are drawn into the alcohol as it rises due to the physical force of the DNA clumping together when it precipitates.
Previous Year Questions
- Mutation in plant cells can be induced by… [NEET 2021]
- If the total amount of adenine and thymine in a double-stranded DNA is
- Complete the flow chart on central dogma...[NEET 2021]
- If Adenine makes
- Purines found both in DNA and RNA are ;...[NEET 2019]
- The length of one turn of the helix in a
- The codons UUU and UUC codes for phenylalanine only. this feature of genetic code is called… [KCET 2016]
- Read the statements regarding structure of polynucleotide chain and choose the correct option.
- 1.A nitrogenous base is linked to the pentose sugar through a
- phospho-diester linkage.
- 2.Two nucleotides are linked through 3'-5' N-glycosidic linkage to
- form a dinucleotide.
- A phosphate group is linked to 5'-OH of a nucleoside through a N- glucosidic linkage to form a nucleotide.
- The name chromatic coined by:
- The back bone of RNA consists of which of the following sugar?
Sample Questions
Ques. What can I do to improve my DNA yield? (3 Marks)
Ans. Give each step additional time to finish. Be sure to wait at least five minutes after adding the detergent. The DNA will get stuck to the base layer if the nuclear and cell membranes are still intact. Alternately, try allowing the test tube of alcohol and pea mixture to sit for 30 to 60 minutes. Over time, you can notice more DNA precipitate into the alcohol layer.
Keep it cold. You can obtain more DNA by using ice-cold alcohol and water. Enzymes that could damage the DNA are slowed down by the cold water, protecting it. The DNA precipitates (solidifies and manifests) more quickly with the aid of the cold alcohol.
Make sure you had enough DNA at the beginning. A lot of food sources for DNA, such as grapes, also have high water content. There won't be enough DNA to assess if the combined cell soup is too watery. Go back to the first step and add less water to correct this. You shouldn't be able to see through the cell soup because it should be opaque.
Ques. What type of enzyme does the meat tenderizer contain? (3 Marks)
Ans. Bromelain and papain are the two enzymes that are most frequently found in meat tenderizers. These two enzymes are derived, respectively, from papaya and pineapple. They both tear down proteins since they are proteases. Proteases are included in enzymatic cleaning solutions for contact lenses to eliminate protein buildup. These proteases include Pancreatin and Subtilisin A, which were taken from bacteria (extracted from the pancreas gland of a hog).
Ques. Why does the DNA clump together? (3 Marks)
Ans. Alcohol does not cause DNA to disintegrate; instead, it causes DNA to precipitate. When there is a lot of it, this makes the DNA congregate. And cells typically have a lot of it.
For instance, the human body has 46 chromosomes in each cell (or 46 DNA molecules). A single cell would have six feet of DNA if those DNA molecules were lined up end to end! Our bodies have more than a billion miles of DNA if the human body is composed of approximately 100 trillion cells, each of which carries six feet of DNA.
Ques. How can we be so sure that the white stringy substance is DNA? (3 Marks)
Ans. Nucleic acids can be stained using a specific process, however, an adult or teacher must handle the chemical in question. So, for the time being, you'll just have to put your faith in the nucleic acid molecules that are precipitating in the alcohol.
Ques. How will the experiment differ when animal cells are used instead of plant cells? (3 Marks)
Ans. In terms of structure, all living things, including plants and animals, share the same DNA molecule. However, depending on whether the product was extracted from a plant or an animal, the end result of this extraction technique may have a somewhat different appearance. The DNA may appear less string-like because there are more impurities present (proteins, carbs, etc.), or the amount of DNA that precipitates may vary.
Ques. In what ways is cold water better than warm water for extracting DNA? (3 Marks)
Ans. Using cold water during the extraction process helps preserve the DNA. Enzymatic reactions slow down with cooling. This defends DNA against damaging enzymes.
To annihilate the DNA of viruses that can penetrate our cells and cause us harm, these enzymes are located in the cytoplasm of the cell rather than the nucleus. The nuclear membrane of a cell typically shields its DNA from such enzymes (known as DNases), but the addition of detergent breaks down the membrane.
Ques. Can a microscope be used to see the extracted DNA? (3 Marks)
Ans. Unfortunately, using a microscope will prevent you from seeing the DNA molecule's double helix structure. All you will see is a huge jumble of clumped-together DNA molecules. In reality, the DNA double helix's breadth is just one billionth of a meter wide! Even with the most potent microscope, this is much too small to be seen. Instead, an image of the DNA molecule can be created using a method called X-ray crystallography. James Watson and Francis Crick were able to determine the appearance of the DNA molecule by examining such a photo.
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