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DNA extraction; Tissue; CTAB buffer; Centrifugation

Introduction

The CTAB (Cetyltrimethylammonium bromide) method is the most established one for removing DNA from plants. A CTAB-based buffer is used in this method to help dissolve the plant cell walls and let the DNA out. The process involves grinding the plant tissue, adding CTAB buffer, centrifuging, and DNA precipitation, among other steps. Due to its effectiveness and capacity to isolate DNA of high quality, the CTAB method has been widely used for DNA isolation in plants [1]. The DNeasy Plant Mini Kit is another widely used method for isolating DNA from plants. The spin column-based method used in this kit makes it possible to effectively isolate DNA from plant tissues. The plant tissue is ground up, a lysis buffer is added, and the lysate is put into a spin column. After that, the column is centrifuged, and an elution buffer is used to elute the DNA. Using the DNeasy Plant Mini Kit, you can quickly and effectively isolate DNA from a variety of plant tissues. Another method for separating DNA from plants is the modified CTAB method. PVP (Polyvinylpyrrolidone) and NaCl (Sodium Chloride) are two additional reagents that are utilized in this modified version of the CTAB method. The PVP assists with eliminating the auxiliary metabolites and colors, while NaCl assists with hastening the DNA. Because it makes it possible to efficiently isolate DNA of high quality in plants, the modified CTAB method is widely used for DNA isolation. Another method for separating DNA from plants is the Chelex method. This technique includes the utilization of Chelex pitch, which assists with chelating the metal particles present in the plant tissue, consequently delivering the DNA [2, 3]. The DNA is released by heating the mixture, adding Chelex resin, and grinding the plant tissue. The Chelex method is a quick and simple way to isolate DNA from a variety of plant tissues.

Method

For DNA isolation from plants: Sample Preparation: Collect plant tissue, such as leaves or young shoots, and finely grind or chop it to increase the surface area and facilitate the release of DNA.

Cell lysis: Add a lysis buffer containing a combination of detergents, enzymes (e.g., cellulose, pectinase), and salts to break down the cell walls and release the cellular contents. The choice of lysis buffer depends on the plant species and tissue type [4].

Protein removal: Add protein precipitation reagents (e.g., phenolchloroform) to the lysate to remove proteins, polysaccharides, and other contaminants. Centrifuge the mixture to separate the aqueous DNA-containing phase from the organic phase.

DNA precipitation: Transfer the aqueous phase to a clean tube and add cold ethanol or isopropanol along with a salt (e.g., sodium acetate). The DNA will precipitate out of the solution due to the alcohol and salt combination. Centrifuge the mixture to collect the DNA pellet.

DNA wash: Wash the DNA pellet with a solution containing ethanol to remove any residual salts or contaminants. Centrifuge the mixture again and carefully remove the supernatant without disturbing the DNA pellet.

DNA resuspension: Dissolve the DNA pellet in a suitable buffer, such as Tris-EDTA (TE) buffer, to obtain a concentrated DNA solution. The DNA can be stored or used directly for downstream applications. For DNA isolation from single-cellular organisms (e.g., bacteria, yeast): Sample preparation: Collect the single-celled organisms by centrifugation or filtration and pellet them.

Cell lysis: Add a lysis buffer containing detergents (e.g., SDS, Triton X-100) and enzymes (e.g., lysozyme, proteinase K) to disrupt the cell membrane and release the DNA. The lysis buffer composition may vary depending on the organism [5, 6].

Protein removal: Similar to plant DNA isolation, use protein precipitation reagents (e.g., phenol-chloroform) to remove proteins and contaminants. Centrifuge the mixture to separate the DNA-containing phase.

DNA precipitation: Add cold ethanol or isopropanol along with a salt (e.g., sodium acetate) to the DNA-containing phase. Centrifuge the mixture to collect the DNA pellet.

DNA wash: Wash the DNA pellet with ethanol to remove any residual salts or contaminants. Centrifuge the mixture, remove the supernatant, and let the pellet air-dry briefly [7].

DNA re suspension: Dissolve the DNA pellet in a suitable buffer, such as TE buffer, to obtain a concentrated DNA solution. The DNA can be stored or used for downstream applications.(Table 1)

Table 1: The key steps involved in DNA isolation from plants and single-cellular organisms.

Discussion

Researchers in genetics, microbiology, and molecular biology all rely on DNA isolation as a crucial technique. DNA is extracted from cells or tissues and then purified for use in other applications. DNA isolation is a well-established method for working with organisms with multiple cells [8, 9], but it presents unique difficulties when working with organisms with just one cell. Organisms with a single cell are referred to as single-cell organisms or unicellular organisms. Bacteria, archaea, and some types of algae are examples of organisms with one cell. Because they provide insight into fundamental biological processes like gene expression, cell division, and evolution, these organisms are an important focus of scientific research. Due to the small cell size and low DNA content, DNA isolation from single-cell organisms is difficult. Additionally, it is challenging to gain access to the DNA in single-cell organisms due to their rigid cell walls or membranes. However, there are a number of approaches that have been developed for isolating DNA from single-cell organisms, each of which has its own set of benefits and drawbacks [10, 11]. Lysis, in which the DNA is extracted from a single cell organism by breaking it open, is a common method for isolating DNA from single-cell organisms. Chemical disruption, such as detergents or enzymes, or mechanical disruption, like sonication or grinding, can both lead to lysis. The DNA can be purified in a variety of ways after the cell is lysed [12], such as precipitation or column-based purification. Whole-genome amplification (WGA) is another method for isolating DNA from single-cell organisms [13, 14]. WGA is a method that lets researchers amplify a single cell's entire genome. Multiple displacement amplification (MDA) is the process of synthesizing multiple DNA copies with a DNA polymerase. WGA can be used to generate enough DNA for sequencing or PCR to use in the future [15]. Methods based on microfluidics have recently emerged as a promising strategy for separating DNA from single-cell organisms. High-throughput analysis of single-cell genomics is made possible by these strategies, which make use of microscale devices to manipulate and isolate individual cells. Droplet-based microfluidics [16], for instance, can be used to enclose individual cells in droplets so that they can be lysed and their DNA amplified with WGA.

Conclusion

In conclusion, DNA isolation from single-cell organisms is necessary for studying fundamental biological processes, despite the unique difficulties it presents. For isolating DNA from single-cell organisms, lysis, whole-genome amplification, and microfluidics-based methods have been developed. Researchers must carefully consider which method is best suited for their specific research question because each of these methods has its own advantages and disadvantages. The study of single-cell genomics stands to make significant contributions to our comprehension of biology and evolution if these techniques are further developed and refined. Various applications of molecular biology, such as PCR, gene cloning, and sequencing, all require the isolation of DNA from plants. Several techniques for efficiently isolating DNA from plant tissues have been developed as a result of advancements in molecular biology techniques. The type of plant tissue, the quantity and quality of the required DNA, and the applications that follow are all important considerations when choosing a method for DNA isolation. The CTAB method, the DNeasy Plant Mini Kit, the modified CTAB method, and the Chelex method are the most frequently used methods for isolating DNA from plants.

Acknowledgement

None

Conflict of Interest

None

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