Stepwise DNA Extraction Procedure for Students
The extraction of deoxyribonucleic acid (DNA) from various sources is known as deoxyribonucleic acid (DNA) isolation. The methods used to isolate DNA vary depending on the sample's source, age, and size.
For genetic analysis, which is utilized for scientific, medicinal, or forensic objectives, DNA isolation is required. DNA is used in a variety of applications by scientists, including the introduction of DNA into cells, animals, and plants, as well as diagnostic purposes.
Isolation of DNA: Practical Procedure
Aim:
DNA extraction from plant materials such as green pea seeds, spinach, papaya, and soon.
Requirements:
Distilled water
Spinach leaves
Tris
SDS
EDTA
Isopropanol
Sodium acetate
Chloroform
Isoamyl alcohol
Phenol of the analytical grade
Procedure:
The cell wall and membranes are ruptured during homogenization, and the resulting product disperses in the buffer solution.
The cell extract is prepared by either manually rupturing the cells or chemically lysing them with enzymes.
In a mortar, grind around 0.5 gm of spinach leaf tissue with a pestle, then homogenise it with 2 ml of extraction buffer (100 mM tris, 20 mM EDTA, 0.5M NaCl, 7M Urea, 0.1 per cent - Mercaptoethanol, and 2% SDS make up the extraction buffer (pH 8.0).
After crushing, the tissue's long fibres are to be maintained, and the homogenate is to be transferred to a 2 mL microfuge tube.
The tubes are to be filled with an equal volume of phenol-chloroform (isoamyl alcohol 25: 24: 1) and gently shaken to blend properly.
Before beginning, the experiment tubes are to be centrifuged for 15 minutes at 15000 rpm at room temperature.
In a fresh tube, the upper aqueous phase is to be collected.
An equivalent volume of chloroform (isoamyl alcohol 24: 1) is stirred in.
The top aqueous phase is transferred to a new tube after centrifugation at room temperature for 10 minutes at 15000 rpm.
By adding 0.1 volume of 3M Sodium acetate pH 7.0 and 0.7 volume of isopropanol to the solution, the DNA is precipitated.
The tubes were centrifuged at 4'C for 15 minutes at 15000 rpm after 15 minutes of incubation at room temperature.
After that, the DNA pellet must be washed in 100 per cent ethanol and to be air-dried.
TE is to be used to dissolve the DNA (Tris-cl 10 nM, pH 8.0, EDTA 1mM).
RNA (10 mg/ml) is added to the DNA to get rid of RNA 5ul.
Ethanol precipitation in the presence of Na and at a temperature of - 20'C or below can be used to concentrate DNA samples.
The acquired DNA can be used for PCR, DNA fingerprinting, genome mapping, and recombinant DNA.
Observation
The DNA appears as white precipitates of fine thread on the spool.
Precautions
The leaf sample should weigh between 0.5 and 0.6 grams. The leaf weight in the range of 0.5 gm to 1.0 gm and the DNA recovered to have a favourable correlation.
Standard pharmaceutical companies are to be taken into consideration for the chemicals required for DNA isolation.
To remove any dust particles, wash the plant material well with distilled water, wipe dry, and weigh it.
Recombinant DNA Technology Process
Recombinant DNA technology is a process that modifies the phenotypic of an organism (host) through the introduction and incorporation of a genetically modified vector into the host's genome.
As a result, the procedure comprises inserting a foreign DNA fragment into the genome that contains the target gene.
The introduced gene is referred to as a recombinant gene, and the procedure is referred to as recombinant DNA technology. It's not as simple as it sounds to embed a gene of interest into the host's genome.
Process of Recombinant DNA Technology
In recombinant DNA technology, the desired gene is chosen for injection into the host, followed by the perfect vector into which the gene must be incorporated, and so recombinant DNA is created.
The recombinant DNA must next be injected into the host. Finally, it must be preserved in the host and passed down to the offspring.
Fragmentation of DNA
Restriction endonucleases are used to cleave the DNA into pieces once it has been extracted and purified.
The restriction enzymes used in recombinant DNA technology are critical for detecting the precise position where the desired gene is inserted into the vector genome.
Restriction endonucleases cut DNA at certain locations and are sequence-specific, usually palindrome sequences.
They look at the length of the DNA and trim it at specific spots called restriction sites.
To obtain the complementary sticky ends, the appropriate genes and vectors are snipped by the same restriction enzymes.
As a result, ligases will have an easier time connecting the necessary gene to the vector.
Conclusion:
Isolating DNA from plant material is not just a scientific experiment; it's a journey of discovery. It allows us to appreciate the intricate dance of molecules within living cells and marvel at the power of DNA as the carrier of life's instructions. With each extraction, we gain a renewed sense of wonder for the diversity and complexity of the living world, and perhaps, even a glimpse into the fascinating story of our own existence.
So, the next time you bite into a juicy papaya or munch on a handful of green peas, remember the invisible blueprint hidden within each cell. With a little scientific curiosity and these simple techniques, you too can unlock the secrets of life, one DNA molecule at a time.
FAQs on How to Isolate DNA from Green Peas, Spinach Seeds, and Papaya
1. What is the main objective of the experiment to isolate DNA from plant material like peas or papaya?
The main objective is to successfully extract Deoxyribonucleic Acid (DNA) from plant cells in a visible form. This experiment demonstrates the fundamental biochemical principles of cell lysis, membrane disruption, and DNA precipitation using common materials, making the genetic blueprint of an organism tangible.
2. What are the three main stages in the process of isolating DNA from a plant cell?
The isolation of DNA from a plant cell, as per the CBSE curriculum for 2025-26, involves three primary stages:
- Lysis: This is the first step, where the tough plant cell wall and membranes (cell membrane and nuclear membrane) are broken down to release the cellular contents, including the DNA. This is typically achieved through mechanical grinding and chemical treatment.
- Separation: In this stage, the DNA is separated from other cellular components like proteins, lipids, and polysaccharides. This is done by using a buffer solution and filtering the mixture.
- Precipitation: Finally, the DNA is made visible and separated from the solution. By adding chilled ethanol, the DNA, which is not soluble in alcohol, clumps together and precipitates out as a thread-like mass.
3. Why is a detergent or soap solution used in the DNA extraction buffer?
A detergent or soap solution is crucial because its molecules disrupt the cell membrane and the nuclear membrane. These membranes are primarily made of lipids (fats). The detergent breaks down these fatty layers, similar to how soap washes away grease, causing the membranes to rupture and release the DNA into the solution.
4. What is the specific role of chilled ethanol in the precipitation of DNA?
The role of chilled ethanol is critical for DNA precipitation. DNA is soluble in water but insoluble in alcohol. When chilled ethanol is added to the aqueous solution containing the extracted DNA, it forces the DNA to clump together and separate from the solution. The cold temperature reduces the activity of any remaining enzymes (like DNases) that could degrade the DNA and enhances the precipitation process, making the white, thread-like DNA strands visible.
5. Why do we need to mechanically grind or blend the plant material (like spinach or peas) at the beginning?
Plant cells have a rigid cell wall made of cellulose, which provides structural support. This wall is a major barrier that must be broken to access the cell's contents. Mechanically grinding or blending the plant material physically tears apart these tough cell walls, a process called maceration. This initial step is essential for the subsequent chemical steps (like using detergent) to effectively reach and break down the cell and nuclear membranes to release the DNA.
6. How does the DNA isolated in this school-level experiment appear, and is it pure?
In this experiment, the isolated DNA appears as a cloudy or stringy, white, thread-like precipitate at the interface between the aqueous solution and the alcohol layer. While it is visibly DNA, it is considered a crude extraction and is not pure. It is often contaminated with RNA and some proteins that might have precipitated along with the DNA. Professional laboratory techniques would include additional steps with enzymes like RNase and protease to obtain highly purified DNA.
7. Why are fruits like papaya or banana often recommended for this DNA isolation experiment?
Fruits like papaya and banana are excellent choices for several reasons. Firstly, they are soft and easy to mash, which simplifies the mechanical breakdown of cell walls. Secondly, papaya contains an enzyme called papain, and bananas contain similar enzymes, which are natural proteases. These enzymes help in breaking down the proteins (like histones) that are tightly wound around the DNA, leading to a cleaner extraction and a better yield of DNA.
8. Besides genetic engineering, what are some real-world applications of isolating plant DNA?
Isolating plant DNA has numerous important applications beyond just lab experiments. Key examples include:
- Agriculture and Crop Improvement: Scientists study plant DNA to identify genes for desirable traits like drought resistance, high yield, or disease immunity, which helps in developing improved crop varieties.
- Conservation Biology: It is used to assess the genetic diversity within endangered plant species, helping to formulate effective conservation strategies.
- Forensics: Plant DNA can be used to trace the origin of illegal timber, solve crimes by linking suspects to a location through plant material, or verify the authenticity of agricultural products.
9. Why is salt (like NaCl) added to the extraction solution?
Salt, such as Sodium Chloride (NaCl), plays a vital role in the extraction process. DNA molecules have a negative charge due to their phosphate backbone, which causes them to repel each other. The positive sodium ions (Na+) from the salt are attracted to these negative charges, effectively neutralising the DNA's charge. This allows the DNA strands to come closer together and clump, which is essential for the precipitation step when alcohol is added.
10. How does DNA isolation relate to the broader field of recombinant DNA technology?
DNA isolation is the foundational and first step in recombinant DNA technology (RDT). RDT involves manipulating genes by cutting a specific gene from one organism and inserting it into another. To do this, scientists must first isolate the DNA from the source organism to access the desired gene. Without a reliable method to extract DNA, subsequent steps like gene cloning, creating genetically modified organisms (GMOs), or producing medicines like insulin through bacteria would be impossible.
