Nucleic acid extraction is a crucial step in the analysis of microorganisms such as fungi, yeast, and bacteria. It involves the isolation of DNA and/or RNA from cells for further study and analysis. The extracted nucleic acids can be used for a variety of

Nucleic acid extraction is a crucial step in the analysis of microorganisms such as fungi, yeast, and bacteria. It involves the isolation of DNA and/or RNA from cells for further study and analysis. The extracted nucleic acids can be used for a variety of applications, such as PCRbased diagnostics, gene expression analysis, and sequencing

Cell Structure Diversity: A Challenge for Extraction

Fungi, yeast, and bacteria differ in their cell structure and composition, and these differences affect the efficiency and specificity of nucleic acid extraction. Fungi have complex cell walls made of chitin and other polysaccharides, which can make lysis and extraction challenging. Potential low number of fungal cells can cause false negative results. Meanwhile, Yyeast cells have a relatively simple cell wall , but some strains may have tough cell walls that require specific enzymes for lysis. Bacterial cells have a single cell wall made of peptidoglycan, which varies in composition and thickness depending on the species. While it is crucial to select the optimal primers and probes during analysis, maximizing nucleic acid extraction efficiency requires a careful approach. The methods for nucleic acid extraction must be optimized for each type of microorganism to ensure the highest quality and quantity of nucleic acids for downstream applications. The success of the extraction process depends on the selection of appropriate lysis conditions, the removal of contaminants, and the preservation of the integrity of the nucleic acids. In this context, it's important to have a comprehensive understanding of the differences and similarities between fungi, yeast, and bacteria, and to develop and optimize nucleic acid extraction protocols that are specific to each type of microorganism

Nucleic Acid Extraction from Fungi, Yeast, and Bacteria

In general, the steps involved in nucleic acid extraction from all three organisms are similar. These include cell lysis, removal of cellular debris and contaminant, and purification of nucleic acid. However, the methods used for cell lysis and purification may differ depending on:

• Specific experiment requirement

• Type of organism

• Quality and quantity of nucleic acid desired

• Downstream applications

Let’s understand the steps involved in nucleic acid extraction of these organisms:

Sample preparation: Collect sufficient amount of starting material (Exp: mycelium, spores, yeast strains, or bacterial cells) and homogenize the sample in a buffer solution (e.g., PBS or TE buffer).

Lysis: The microbial cells are lysed to release their nucleic acids. This can be done through physical methods (such as grinding using Fastprep - 24 5G, Cat No. 116005500) or chemical methods (such as using lytic enzymes or detergents). MP Biomedicals supplies different lysing matrices using physical force to break open the cell walls and release the cellular contents. The bead-cell mixture is then centrifuge to separate the beads from the lysed cells with the supernatant containing the released nucleic acids. The size and rigidity of the beads contained in our lysing matrices have been optimized for efficient lysis of fungi, yeast, or bacteria.

Pro Tips: Some yeast strains may have tough cell walls that require the addition of specific enzymes (such as zymolyase Cat No. 08320921) to facilitate lysis. Addition of specific enzyme (such as lysozyme Cat No. 02100831-CF) could be used to facilitate lysis of bacteria cell with tough cell walls.

Nucleic Acid Precipitation

After the cells have been lysed, the nucleic acids can be precipitated by adding an equal volume of isopropanol or ethanol and incubating the mixture at room temperature or in the freezer. The nucleic acids will form a pellet after centrifugation, which can then be washed with 70% ethanol and air-dried.

Resuspension: The dried pellet is resuspended in a buffer solution containing RNase inhibitors to prevent RNA degradation.

Purification: The nucleic acids can be further purified using spin-columns (SPINeasy DNA Pro Kit for Soil Cat No. 116546050 or SPINeasy RNA Kit for Bacteria Cat No. 116541050), which remove salts, proteins, and other contaminants.

Pro Tips: It's important to note that different microbes may have varying degrees of cell wall and membrane rigidity, which can affect the efficiency of lysis. In some cases, additional steps such as cell wall digestion or enzymatic treatment may be necessary to obtain highquality nucleic acids. Additionally, some bacteria contain endonucleases that can degrade their own DNA, so it's important to take precautions to avoid contamination with such enzymes

In all cases, it's important to optimize the lysis conditions to achieve high-quality nucleic acids, since contaminants such as proteins, salts, and cell debris can negatively impact downstream applications such as PCR, sequencing, or expression analysis.

References:

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5. Bacteria:

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