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:
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.
Precautions
-
The leaf sample should weigh between 0.5 and 0.6 grammes. 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.