Class 12 Biology Notes Chapter 6 (Chapter 6) – Lab Manual (English) Book

Detailed Notes with MCQs of the practical aspects relevant for your government exam preparation, specifically looking at Experiment 6 from your Lab Manual, which deals with DNA isolation. Understanding the 'why' behind each step is crucial.

Experiment 6: Isolation of DNA from Plant Material (e.g., Spinach, Green Pea Seeds, Papaya)

1. Aim:
To isolate Deoxyribonucleic Acid (DNA) from a given plant sample.

2. Principle:
The isolation of DNA from plant cells involves several key steps based on the chemical and physical properties of DNA and cellular components:

• Cell Disruption: Plant cells possess a rigid cell wall (primarily cellulose) and cell membrane, along with a nuclear membrane enclosing the DNA. The first step is to break these barriers. Mechanical action (grinding) disrupts the cell wall, while chemical treatment targets the membranes.
• Membrane Lysis: Detergents (like Sodium Dodecyl Sulphate - SDS, often found in liquid dish soap or specific lab buffers) are amphipathic molecules that disrupt the lipid bilayer of the cell and nuclear membranes, releasing the cellular contents, including DNA. Detergents also help denature proteins, including enzymes like DNases that could degrade DNA.
• Protein and RNA Removal: DNA in the nucleus is associated with proteins (like histones). RNA is also present in the cell. While simple protocols might rely on precipitation differences, more refined methods might use enzymes:
  • Protease (e.g., Proteinase K): Degrades proteins.
  • RNase (Ribonuclease): Degrades RNA.
    In many classroom protocols, the detergent and salt concentration help in denaturing and partially removing proteins. Filtration removes larger debris.
• DNA Precipitation: DNA is soluble in aqueous solutions due to its charged phosphate backbone. However, it is insoluble in alcohols like ethanol or isopropanol, especially in the presence of salt (like NaCl).
  • Role of Salt (NaCl): The positive sodium ions (Na+) neutralize the negative charges on the phosphate groups of DNA. This reduces the repulsion between DNA strands and makes the DNA molecule less hydrophilic (less soluble in water).
  • Role of Chilled Ethanol: Adding chilled ethanol (usually 70-95%) causes the DNA to precipitate out of the aqueous solution. Ethanol dehydrates the DNA molecule, further decreasing its solubility. Using chilled ethanol enhances the yield of precipitation and significantly slows down the activity of any residual DNase enzymes, protecting the DNA from degradation.
• Collection: The precipitated DNA appears as a mass of fine, white, thread-like fibres at the interface between the aqueous layer and the alcohol layer. It can be collected by spooling onto a glass rod or loop.

3. Requirements:

• Biological Material: Fresh plant source (e.g., 5g spinach leaves, 10-15 green pea seeds, small piece of ripe papaya).
• Chemicals: Lysis buffer (containing detergent like liquid soap/SDS and NaCl), chilled ethanol (95% or absolute), distilled water. (Optional: Protease, RNase).
• Glassware/Apparatus: Mortar and pestle, beakers, test tubes, funnel, cheesecloth or muslin cloth, glass rod or inoculation loop. (Optional: Water bath, centrifuge).

4. Procedure Outline (Simplified Classroom Protocol):
1. Homogenization: Take the plant material, cut it into small pieces, and grind it thoroughly in a mortar and pestle with a small amount of distilled water or buffer containing salt (NaCl). This breaks the cell walls.
2. Lysis: Transfer the paste to a beaker. Add the lysis buffer containing detergent (e.g., 10 ml of liquid soap solution with NaCl). Mix gently but thoroughly. Avoid vigorous mixing which can shear the long DNA molecules. (Optional: Incubate at 60°C for 15 mins to enhance lysis and denature DNases).
3. Filtration: Filter the mixture through 2-3 layers of cheesecloth/muslin cloth into a clean test tube or beaker. Squeeze gently to collect the filtrate, which contains DNA, proteins, RNA, and other soluble components. Discard the solid debris.
4. Precipitation: Take the filtrate in a test tube. Gently pour chilled ethanol down the inner side of the tube, allowing it to form a distinct layer on top of the filtrate. Use approximately double the volume of ethanol compared to the filtrate. Do not mix.
5. Observation: Allow the setup to stand undisturbed for a few minutes. Observe the interface between the lower aqueous layer (filtrate) and the upper ethanol layer.
6. Collection (Spooling): DNA precipitates out as fine, white threads. These threads can be gently wound (spooled) onto a clean glass rod or inoculation loop by slowly rotating it at the interface.

5. Observation:
Fine, white, thread-like precipitate aggregates at the junction of the aqueous filtrate and the overlying chilled ethanol layer.

6. Conclusion:
The thread-like precipitate observed is DNA, confirming its isolation from the plant material.

7. Key Precautions for Optimal Results:

• Always use chilled ethanol for precipitation.
• Add ethanol gently along the sides of the tube to form a layer and allow DNA precipitation at the interface. Avoid disturbing the layers.
• Grind the plant material thoroughly to ensure maximum cell disruption.
• Avoid vigorous shaking or mixing after adding detergent to prevent mechanical shearing of DNA.
• Use clean glassware to avoid contamination.

Key Concepts for Government Exams:

• Understand the function of each reagent: Grinding (mechanical disruption), Detergent (membrane lysis, protein denaturation), Salt (neutralizes DNA charge), Chilled Ethanol (DNA precipitation, DNase inhibition).
• Know the principle of DNA precipitation: DNA is insoluble in alcohol, especially in the presence of salt and at low temperatures.
• Recognize the appearance of isolated DNA: White, thread-like precipitate.
• Understand why low temperature (chilled ethanol) is preferred.

Multiple Choice Questions (MCQs):

1. What is the primary role of detergent (like liquid soap or SDS) in the DNA isolation process from plant cells?

   • a) To break the cellulose cell wall
   • b) To precipitate the DNA from the solution
   • c) To dissolve cell and nuclear membranes by disrupting lipids
   • d) To specifically bind and remove RNA molecules

2. Why is chilled ethanol added as the final step in this DNA isolation protocol?

   • a) To dissolve the cell membranes more effectively
   • b) To cause DNA, which is insoluble in alcohol, to precipitate out
   • c) To break down proteins associated with DNA
   • d) To stain the DNA making it visible

3. During the DNA isolation experiment, the DNA typically appears as:

   • a) A clear, transparent liquid
   • b) Small green pellets at the bottom of the tube
   • c) Fine white threads or fibres precipitating at the ethanol-filtrate interface
   • d) A bright yellow band suspended in the solution

4. The addition of Sodium Chloride (NaCl) during the DNA extraction process primarily helps to:

   • a) Act as the main lysing agent for the cell wall
   • b) Neutralize the negative charge on the DNA backbone, aiding precipitation
   • c) Directly digest contaminating proteins
   • d) Keep the DNA dissolved in the aqueous solution

5. Which initial step is essential for breaking the rigid cell wall of plant cells before chemical lysis?

   • a) Adding chilled ethanol
   • b) Filtering the mixture through cheesecloth
   • c) Mechanical grinding or homogenization using a mortar and pestle
   • d) Incubating the sample at 60°C

6. DNA precipitates when chilled ethanol is added because:

   • a) Ethanol increases the negative charge on DNA
   • b) DNA is largely insoluble in alcohol compared to the aqueous buffer
   • c) Ethanol specifically breaks the hydrogen bonds within the DNA helix
   • d) Ethanol dissolves cellular debris, leaving pure DNA behind

7. If RNase enzyme were to be added during the DNA isolation procedure, its specific function would be to:

   • a) Break down the plant cell wall
   • b) Degrade RNA molecules, thus purifying the DNA sample
   • c) Help in precipitating the DNA more efficiently
   • d) Degrade proteins bound to the DNA

8. Why should the chilled ethanol be added gently down the side of the test tube containing the filtrate?

   • a) To ensure the ethanol mixes rapidly and completely with the filtrate
   • b) To prevent the ethanol from freezing the sample
   • c) To form a distinct layer above the aqueous filtrate, allowing DNA to precipitate clearly at the interface
   • d) To activate the detergent present in the filtrate

9. The principle of separating DNA from other soluble cellular components using ethanol relies on:

   • a) Differences in electrical charge
   • b) Differences in molecular weight
   • c) Differences in solubility in alcohol versus water
   • d) Differences in density

10. The filtrate obtained after grinding the plant material and filtering through cheesecloth contains:

    • a) Only cell wall fragments
    • b) Only pure DNA
    • c) DNA, RNA, proteins, and other soluble cellular components
    • d) Only intact nuclei

Answer Key for MCQs:

1. c) To dissolve cell and nuclear membranes by disrupting lipids
2. b) To cause DNA, which is insoluble in alcohol, to precipitate out
3. c) Fine white threads or fibres precipitating at the ethanol-filtrate interface
4. b) Neutralize the negative charge on the DNA backbone, aiding precipitation
5. c) Mechanical grinding or homogenization using a mortar and pestle
6. b) DNA is largely insoluble in alcohol compared to the aqueous buffer
7. b) Degrade RNA molecules, thus purifying the DNA sample
8. c) To form a distinct layer above the aqueous filtrate, allowing DNA to precipitate clearly at the interface
9. c) Differences in solubility in alcohol versus water
10. c) DNA, RNA, proteins, and other soluble cellular components

Study these notes well, focusing on the rationale behind each step. Good luck with your preparation!