Fungal and Plant Tissue DNA: A Comparative Study of In Situ Extraction Methods

1. Significance of In Situ DNA Extraction

### 1. Significance of In Situ DNA Extraction

In situ DNA extraction is a crucial technique in the fields of molecular biology, genetics, and biotechnology, offering a range of benefits and applications that extend beyond traditional DNA extraction methods. The term "in situ" is derived from Latin, meaning "in place," and in the context of DNA extraction, it refers to the process of extracting DNA directly from tissues or cells without physically removing or isolating them from their natural environment.

1.1 Preservation of Spatial Information
One of the primary advantages of in situ DNA extraction is the preservation of spatial information. This allows researchers to study the genetic material in the context of its original location within the organism, which is particularly important for understanding gene expression patterns and the spatial distribution of genetic variants.

1.2 Minimization of Contamination
In situ extraction methods reduce the risk of contamination that can occur during the transfer of samples between different laboratory environments. By extracting DNA directly from the tissue, researchers can minimize the exposure of the sample to external contaminants, thus enhancing the reliability of the results.

1.3 Enhanced Sensitivity and Specificity
The in situ approach often provides enhanced sensitivity and specificity in detecting and analyzing DNA. This is especially valuable when working with rare or trace amounts of DNA, such as in forensic investigations or when studying endangered species.

1.4 Suitability for Different Tissues
In situ DNA extraction is applicable to a wide variety of plant and fungal tissues, including those that are difficult to process using traditional methods. This versatility makes it an invaluable tool for researchers working with diverse biological samples.

1.5 Integration with Other Techniques
The extracted DNA can be readily integrated with other molecular techniques, such as polymerase chain reaction (PCR), sequencing, and microarray analysis. This allows for a comprehensive study of the genetic material, providing a more complete understanding of the biological processes at play.

1.6 Contribution to Basic and Applied Research
In situ DNA extraction contributes significantly to both basic and applied research. In basic research, it aids in understanding the fundamental mechanisms of gene regulation and expression. In applied research, it can be used for disease diagnosis, crop improvement, and environmental monitoring.

1.7 Ethical Considerations
From an ethical standpoint, in situ DNA extraction can be less invasive than other methods, particularly when working with living organisms. This is particularly relevant for studies involving endangered or protected species, where minimizing harm is a priority.

In conclusion, the significance of in situ DNA extraction lies in its ability to provide detailed, context-specific genetic information while minimizing the potential for contamination and preserving the integrity of the sample. As we delve deeper into the subsequent sections, we will explore the specific methods used for plant and fungal tissue DNA extraction, compare their efficacy, and discuss the challenges and solutions associated with these techniques.

2. Plant Tissue DNA Extraction Methods

2. Plant Tissue DNA Extraction Methods

DNA extraction from plant tissues is a fundamental process in molecular biology, genetics, and genomics. It allows for the analysis of genetic material, gene expression, and the identification of specific plant species or strains. Here, we discuss various methods used for the extraction of DNA from plant tissues in situ.

2.1 Traditional Extraction Methods

Traditional methods for DNA extraction from plant tissues involve several steps, including:

- Homogenization: Plant tissues are finely ground using liquid nitrogen or mechanical homogenizers to release cellular contents.
- Lysis: Cell membranes are disrupted using detergents or enzymatic treatments to release DNA.
- Isolation: DNA is separated from other cellular components through techniques such as centrifugation or filtration.
- Purification: DNA is purified using organic solvents or affinity chromatography to remove proteins and other contaminants.

2.2 Cetyltrimethylammonium Bromide (CTAB) Method

The CTAB method is a widely used technique for plant DNA extraction. It involves:

- Extraction Buffer Preparation: A buffer containing CTAB is used to solubilize the nucleic acids and precipitate proteins.
- DNA Precipitation: DNA is precipitated out of the solution using isopropanol or other alcohols.
- Washing and Purification: The DNA is washed to remove excess salts and contaminants and then resuspended in a suitable buffer.

2.3 SDS-Protease K Method

This method utilizes the detergent SDS (sodium dodecyl sulfate) and protease K to break down proteins and release DNA:

- Incubation with SDS and Protease K: Plant tissue is incubated with a mixture of SDS and protease K to digest proteins and disrupt cell membranes.
- Phenol-Chloroform Extraction: The mixture is then extracted with phenol-chloroform to separate DNA from proteins and other impurities.
- Ethanol Precipitation: DNA is precipitated using ethanol and resuspended in TE buffer.

2.4 Commercial Kits

Commercial DNA extraction kits offer a convenient and often more efficient alternative to traditional methods. These kits typically include:

- Pre-prepared Buffers: Ready-to-use buffers that facilitate cell lysis and DNA binding.
- Binding Matrix: A matrix that selectively binds DNA, allowing for easy separation from contaminants.
- Elution: DNA is eluted in a small volume of buffer, resulting in a concentrated DNA sample.

2.5 Microfluidic Devices

Advancements in microfluidics have led to the development of devices that can perform DNA extraction in a highly controlled and automated manner:

- Integration of Steps: Microfluidic devices can integrate multiple steps of the extraction process into a single system.
- Miniaturization: These devices require minimal sample and reagent volumes, making them suitable for high-throughput applications.

2.6 Enzymatic Digestion

Enzymatic methods involve the use of enzymes to digest proteins and other cellular components, leaving DNA intact:

- Enzyme Selection: Specific enzymes, such as proteinase K or lysozyme, are chosen based on their ability to degrade proteins without damaging DNA.
- Incubation: The tissue is incubated with the enzyme, allowing for the breakdown of cellular components.
- DNA Recovery: DNA is recovered through subsequent purification steps.

2.7 Magnetic Bead-Based Extraction

Magnetic bead-based methods offer a rapid and efficient way to extract DNA:

- Binding to Beads: DNA binds to magnetic beads coated with specific ligands.
- Separation: Beads are separated using a magnetic field, allowing for the removal of contaminants.
- Elution: DNA is eluted from the beads in a clean buffer.

Each of these methods has its advantages and limitations, and the choice of method often depends on the specific requirements of the research, such as the type of plant tissue, the amount of DNA needed, and the downstream applications of the extracted DNA.

3. Fungal Tissue DNA Extraction Methods

3. Fungal Tissue DNA Extraction Methods

Fungal tissue DNA extraction is a critical process for the study of fungal genetics, taxonomy, and molecular biology. Unlike plant tissues, fungal cells have a cell wall made primarily of chitin, which requires specific protocols for effective DNA extraction. Here are some common methods used for the extraction of DNA from fungal tissues:

3.1 Traditional Extraction Methods

Traditional methods for fungal DNA extraction often involve mechanical and enzymatic lysis. The steps typically include:

- Sample Collection and Preparation: Fungal samples are collected and prepared by washing with sterile water to remove contaminants.
- Cell Disruption: The fungal cell wall is disrupted using mechanical methods such as bead beating or enzymatic treatments with lytic enzymes like lysing enzymes or chitinases.
- Nucleic Acid Extraction: After disruption, the cell lysate is treated with detergents and high salt to precipitate proteins and other cellular debris, followed by nucleic acid extraction using phenol-chloroform or other organic solvents.
- DNA Purification: The extracted DNA is further purified using methods like ethanol precipitation or column-based purification kits.

3.2 Modified CTAB Method

The Cetyltrimethylammonium bromide (CTAB) method is a widely used technique for fungal DNA extraction due to its effectiveness in binding and precipitating nucleic acids:

- CTAB Treatment: The fungal tissue is treated with a CTAB buffer, which helps in the solubilization of nucleic acids and precipitation of proteins.
- Chloroform Extraction: An equal volume of chloroform is added to the CTAB-treated sample to separate the aqueous phase containing the nucleic acids from the organic phase containing proteins and lipids.
- DNA Precipitation: The nucleic acids are precipitated using isopropanol or ethanol, followed by washing with 70% ethanol to remove any remaining contaminants.

3.3 Commercial Kits

Commercial DNA extraction kits have been developed to streamline the process, offering a more rapid and often cleaner extraction of DNA from fungal tissues:

- Kit Selection: Choose a kit that is specifically designed for fungal DNA extraction or one that has been optimized for hard-to-lyse organisms.
- Sample Processing: Follow the manufacturer's instructions for sample processing, which usually includes cell lysis, binding of DNA to a matrix, washing steps to remove impurities, and elution of purified DNA.

3.4 Innovative Techniques

Innovative techniques such as microwave-assisted lysis, sonication, and the use of novel enzymes are being explored to improve the efficiency and yield of fungal DNA extraction:

- Microwave-Assisted Lysis: This method uses microwave energy to rapidly heat fungal cells, leading to cell wall rupture and efficient DNA release.
- Sonication: Ultrasonic waves can disrupt fungal cells, making the DNA more accessible for extraction.
- Novel Enzymes: The use of newly discovered enzymes that target specific components of the fungal cell wall can improve the lysis process.

3.5 Considerations for In Situ Extraction

In situ DNA extraction from fungal tissues presents unique challenges, such as the need to preserve the tissue's spatial structure while extracting DNA. This requires careful optimization of the extraction conditions to avoid tissue degradation or DNA contamination.

3.6 Quality Assessment

After extraction, the quality and quantity of the DNA are assessed using methods such as agarose gel electrophoresis, spectrophotometry, or fluorometry to ensure the DNA is suitable for downstream applications.

In conclusion, the extraction of DNA from fungal tissues is a multi-step process that requires careful consideration of cell wall composition and the choice of appropriate lysis and purification methods. Advances in technology and the development of novel techniques continue to improve the efficiency and reliability of fungal DNA extraction for various applications in research and diagnostics.

4. Comparison of Plant and Fungal DNA Extraction

4. Comparison of Plant and Fungal DNA Extraction

In situ DNA extraction from plant and fungal tissues, while sharing some common principles, also exhibits distinct differences due to the unique structural and biochemical characteristics of these organisms. Here, we compare the methods and challenges associated with DNA extraction from plant and fungal tissues.

Structural Differences:
- Plant Tissues: Plants have rigid cell walls made primarily of cellulose, which provide structural support and protection. This necessitates the use of enzymes like cellulase to break down the cell walls during DNA extraction.
- Fungal Tissues: Fungi also possess cell walls, but they are composed of chitin, a polymer of N-acetylglucosamine, which requires different enzymatic treatments for degradation.

Chemical Composition:
- Plants: The presence of polyphenolic compounds and polysaccharides can interfere with DNA extraction, requiring additional steps for purification.
- Fungi: Fungal tissues may contain melanin and other secondary metabolites that can also complicate the DNA extraction process, often necessitating specialized protocols for removal.

DNA Extraction Methods:
- Plant DNA Extraction: Common methods include the CTAB (Cetyltrimethylammonium bromide) method, which is effective for breaking cell walls and binding DNA, and the SDS (Sodium dodecyl sulfate) method, which helps in cell lysis and DNA release.
- Fungal DNA Extraction: Methods often involve the use of lyticase or other enzymes that target chitin, followed by proteinase K treatment to digest proteins and release DNA.

Purity and Quality of DNA:
- Plant DNA: The high content of polysaccharides and polyphenols in plant tissues can lead to lower DNA purity, requiring additional purification steps such as gel extraction or column purification.
- Fungal DNA: The presence of melanin and other complex molecules can also reduce DNA purity, often necessitating the use of specialized kits or protocols designed for fungal DNA extraction.

Yield and Fragment Size:
- Plant DNA: The yield can be high, but the DNA may be sheared due to the mechanical disruption of cell walls during extraction.
- Fungal DNA: The yield may vary depending on the fungal species and tissue type, and the DNA may also be fragmented due to enzymatic treatments.

Challenges:
- Plants: Challenges include dealing with high levels of secondary metabolites, which can inhibit downstream applications such as PCR.
- Fungi: Challenges include the presence of complex cell wall structures and secondary metabolites, which can interfere with DNA extraction and purification.

Solutions:
- Plants: Solutions may involve the use of chaotropic agents, silica-based columns, or magnetic bead technology for purification.
- Fungi: Solutions may include the use of specific enzymatic cocktails for cell wall degradation and the application of affinity chromatography for DNA purification.

In conclusion, while both plant and fungal DNA extractions share the goal of obtaining high-quality, pure DNA, the methods and challenges differ significantly due to the distinct biological properties of these organisms. Understanding these differences is crucial for optimizing in situ DNA extraction protocols for various applications in plant and fungal research.

5. Challenges and Solutions in In Situ DNA Extraction

5. Challenges and Solutions in In Situ DNA Extraction

In situ DNA extraction, while offering numerous advantages, is not without its challenges. This section will discuss the common difficulties encountered during the process of in situ DNA extraction from plant and fungal tissues and the solutions that have been developed to address these issues.

5.1 Common Challenges

1. Cell Wall Barriers: The rigid cell walls of plants and the tough cell walls of fungi present a significant barrier to the penetration of enzymes and chemicals used in DNA extraction.

2. Polysaccharides and Proteins: The presence of complex polysaccharides and proteins in plant and fungal tissues can interfere with DNA purification, leading to low yields and impure DNA.

3. Nucleic Acid Degradation: The risk of nucleic acid degradation during the extraction process due to the presence of nucleases and other degradative enzymes is high.

4. Contamination: Contamination with exogenous DNA or PCR inhibitors can compromise the integrity of the extracted DNA, affecting downstream applications.

5. Low DNA Yield: In some cases, the yield of DNA extracted from in situ tissues may be low, which can limit the amount of material available for analysis.

5.2 Solutions

1. Physical Disruption: Utilizing mechanical methods such as bead beating or pressure cycling technology can effectively break down cell walls and facilitate DNA release.

2. Chemical Treatment: Employing chemical agents like cellulase, pectinase, or lysing enzymes can help degrade the cell wall components and release DNA.

3. Enzymatic Digestion: The use of proteases and other enzymes can help digest proteins and other contaminants, reducing the risk of interference with DNA purification.

4. DNA Stabilization: Incorporating DNA stabilization agents during the extraction process can help protect the DNA from degradation.

5. Purification Techniques: Advanced purification techniques such as affinity chromatography or magnetic bead-based methods can improve the purity and yield of extracted DNA.

6. Contamination Control: Implementing stringent laboratory practices, including the use of positive displacement pipettes, UV treatment, and DNase-free environments, can minimize contamination risks.

7. Yield Enhancement: Optimization of extraction protocols, including the use of multiple extraction rounds or the integration of additional purification steps, can enhance DNA yield.

8. Quality Assessment: Utilizing spectrophotometry, electrophoresis, or qPCR to assess the quality and quantity of the extracted DNA ensures that it is suitable for downstream applications.

9. Automation: The use of automated DNA extraction systems can standardize the process, reduce human error, and improve reproducibility.

By addressing these challenges with the appropriate solutions, researchers can improve the efficiency and reliability of in situ DNA extraction from plant and fungal tissues, thereby facilitating a wide range of applications in molecular biology and genetics.

6. Applications of In Situ DNA Extraction

6. Applications of In Situ DNA Extraction

In situ DNA extraction is a versatile technique with a wide range of applications across various scientific disciplines. Its ability to isolate DNA directly from tissues without the need for extensive purification processes makes it particularly useful in the following areas:

1. Molecular Taxonomy and Phylogenetics:
In situ DNA extraction is instrumental in molecular taxonomy, allowing researchers to study genetic variations and relationships among species. It is especially useful for endangered or rare species where obtaining fresh tissue samples may be difficult.

2. Environmental DNA (eDNA) Analysis:
eDNA analysis involves the detection of DNA fragments in the environment, which can provide insights into the presence of various organisms without the need for direct sampling. In situ DNA extraction techniques are critical for eDNA studies, enabling the identification of species from environmental samples such as soil, water, and air.

3. Disease Diagnosis and Plant Pathology:
In situ DNA extraction is used to diagnose diseases in plants and fungi by identifying the presence of pathogens within the tissues. This method is particularly useful for early detection and monitoring of infections, which is crucial for disease management strategies.

4. Conservation Genetics:
For conservation efforts, in situ DNA extraction facilitates the study of genetic diversity within populations, helping to identify unique or endangered genetic variants that may require protection.

5. Forensic Investigations:
In forensic science, in situ DNA extraction can be used to recover DNA from crime scenes, such as from plant material or fungal spores, which can provide valuable evidence for investigations.

6. Genetic Engineering and Breeding Programs:
In agricultural and horticultural breeding programs, in situ DNA extraction can be employed to screen for desirable traits in plant and fungal species, accelerating the development of new varieties.

7. Metagenomics:
In metagenomic studies, which involve the analysis of genetic material from environmental samples, in situ DNA extraction is essential for characterizing the complex microbial communities present in various ecosystems.

8. Bioprospecting:
For bioprospecting, where the goal is to discover new biologically active compounds, in situ DNA extraction can help identify organisms with potential for producing novel pharmaceuticals or other bioproducts.

9. Education and Research:
In educational settings and research institutions, in situ DNA extraction provides a hands-on approach to molecular biology, allowing students and researchers to perform DNA analysis on a variety of samples.

10. Biotechnology and Industrial Applications:
In the biotechnology industry, in situ DNA extraction can be used to screen for organisms with specific industrial applications, such as those involved in biofuel production or bioremediation.

In situ DNA extraction has revolutionized the way DNA is isolated and analyzed, opening up new possibilities for research and practical applications across various fields. As technology continues to advance, the applications of this technique are expected to expand even further.

7. Future Perspectives and Technological Advancements

7. Future Perspectives and Technological Advancements

The future of in situ DNA extraction from plant and fungal tissues holds great promise, with ongoing advancements in technology and methodology set to revolutionize the field. Here are some of the key future perspectives and potential technological advancements that could shape this domain:

1. Miniaturization and Automation: The development of miniaturized and automated systems for in situ DNA extraction could significantly reduce the time and labor required, making the process more efficient and accessible.

2. Integration with Microfluidics: Microfluidic devices, or "lab-on-a-chip" technologies, could be employed to perform in situ DNA extraction with minimal sample volumes, enhancing sensitivity and reducing reagent consumption.

3. Enhanced Sensitivity and Specificity: Improvements in molecular biology techniques, such as CRISPR-based methods, could offer higher sensitivity and specificity in DNA extraction, allowing for the detection of even trace amounts of DNA.

4. Non-destructive Extraction Techniques: The development of non-destructive methods for DNA extraction could be a game-changer, allowing researchers to study the same tissue multiple times and reducing the need for large sample sizes.

5. Portable Extraction Devices: Portable and field-ready devices for DNA extraction could enable on-site analysis, which is particularly useful for ecological studies and biodiversity assessments in remote locations.

6. Machine Learning and AI: The application of machine learning algorithms and artificial intelligence in the analysis of DNA extraction data could lead to more accurate and faster identification of genetic material, as well as the discovery of new patterns and relationships.

7. Nanotechnology: The use of nanotechnology in DNA extraction could provide new tools for enhancing the efficiency of the process, such as nano-sized extraction agents or nanosensors for detecting DNA.

8. Environmental DNA (eDNA) Analysis: The integration of eDNA analysis with in situ DNA extraction could open new avenues for studying the presence of organisms in their natural environments without the need for physical sampling.

9. Cross-disciplinary Applications: The intersection of in situ DNA extraction with other fields, such as materials science, could lead to the development of new materials that facilitate DNA extraction and preservation.

10. Ethical and Regulatory Considerations: As technology advances, there will be a growing need for ethical guidelines and regulatory frameworks to ensure the responsible use of in situ DNA extraction, particularly in the context of environmental and genetic privacy.

11. Educational and Outreach Programs: The future may see an increase in educational programs and public outreach initiatives aimed at teaching the public about the importance and implications of in situ DNA extraction.

12. Collaborative Research Networks: The establishment of international collaborative networks could facilitate the sharing of knowledge, resources, and expertise, accelerating the development of new in situ DNA extraction technologies.

As these advancements emerge, they will not only improve the efficiency and effectiveness of in situ DNA extraction but also expand the range of applications, from basic research to clinical diagnostics and environmental monitoring. The potential for innovation in this field is vast, and the coming years are likely to see significant strides in our ability to study and understand the genetic material of plants and fungi in their natural environments.

8. Conclusion

8. Conclusion

In conclusion, the extraction of DNA from plant and fungal tissues in situ is a critical technique in modern molecular biology, offering a wealth of opportunities for genetic analysis and research. The significance of in situ DNA extraction lies in its ability to provide direct and accurate genetic information from the tissues of interest, which is invaluable for studying gene expression, genetic diversity, and evolutionary relationships.

The methods for DNA extraction from plant and fungal tissues vary, with each having its own set of advantages and limitations. Plant tissue DNA extraction methods, such as the CTAB method and the SDS method, are effective for obtaining high-quality DNA from various plant tissues. Fungal tissue DNA extraction methods, including the mechanical disruption and enzymatic digestion, are tailored to overcome the challenges posed by the tough cell walls of fungi.

Comparing the DNA extraction methods for plants and fungi reveals that while both processes aim to isolate high-quality DNA, the specific techniques and conditions may differ due to the unique characteristics of each organism. The challenges faced in in situ DNA extraction, such as contamination, degradation, and low yield, can be addressed through careful sample preparation, optimization of extraction protocols, and the use of advanced technologies.

The applications of in situ DNA extraction are vast, ranging from forensic science to environmental monitoring, and from disease diagnosis to the study of plant-microbe interactions. As the demand for accurate and reliable genetic information grows, so does the importance of refining and advancing in situ DNA extraction techniques.

Looking to the future, technological advancements such as automation, nanotechnology, and the integration of artificial intelligence in DNA extraction processes are expected to further enhance the efficiency, accuracy, and scalability of in situ DNA extraction. These innovations will not only improve the quality of genetic research but also expand the scope of applications in various fields.

In summary, in situ DNA extraction is a powerful tool in the field of molecular biology, with ongoing research and technological advancements promising to unlock even greater potential in the study of plant and fungal tissues. As our understanding of these processes deepens, so too will our ability to harness the information they provide for the betterment of science and society.

9. References

9. References

1. Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
2. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., & Struhl, K. (2002). Current Protocols in Molecular Biology. John Wiley & Sons.
3. Doyle, J. J., & Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12, 13-15.
4. White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR Protocols: A Guide to Methods and Applications (pp. 315-322). Academic Press.
5. Raeder, U., & Broda, P. (1975). Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology, 1(2), 17-20.
6. Raper, K. B., & Fennell, D. I. (1965). The Genus Aspergillus. The Williams & Wilkins Co.
7. Kjellén, L., & Lindahl, U. (1993). In situ hyaluronan is a major component of the extracellular matrix in the pericellular environment of epithelial cells. Journal of Histochemistry & Cytochemistry, 41(1), 107-113.
8. Griffiths, D. J. (1992). In situ hybridization. In J. M. Walker & E. L. Rapley (Eds.), Molecular Genetics of Bacteria and Phages (pp. 77-96). Oxford University Press.
9. Koch, J., & Kjems, J. (2006). In situ detection of nucleic acids in cells and tissues. In J. M. Walker (Ed.), In Situ Hybridization Protocols (3rd ed., pp. 1-22). Humana Press.
10. Ausubel, F. M. (2005). In situ hybridization. Current Biology, 15(4), R147-R148.
11. Rost, T. L., & Ladd, A. J. C. (1988). In situ hybridization. In S. B. Radding, J. A. Nickol, & F. Hecht (Eds.), Nucleic Acid Hybridization: Applications to Plant Systems (pp. 95-122). Alan R. Liss.
12. Hahn, M. W., & Wägele, J. W. (2003). In situ hybridization to identify microorganisms in environmental samples. In E. Rosenberg (Ed.), Microbial Ecology and Evolution (pp. 57-72). Springer.
13. Stahl, D. A., & Amann, R. (1991). Development and application of nucleic acid probes. In E. Stackebrandt & M. Goodfellow (Eds.), Nucleic Acid Techniques in Bacterial Systematics (pp. 205-248). John Wiley & Sons.
14. Tebbe, C. C., & Vahjen, W. (1993). Interference of humic acids and DNA extraction from soil. Molecular Ecology, 2(2), 261-267.
15. Vogelstein, B., & Kinzler, K. W. (1999). The Genetic Basis of Human Cancer. McGraw-Hill Professional.
16. DeSalle, R., Egan, M. G., & Siddall, M. (2005). The unholy trinity: taxonomy, species concepts and DNA barcoding. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 360(1462), 1905-1916.
17. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. Journal of Molecular Biology, 3(3), 208-218.
18. Cullings, K. W., & Duniway, M. L. (1996). DNA extraction and PCR amplification from medullae of mature, decaying logs. Mycologia, 88(1), 58-62.
19. O'Donnell, A. G., & Clegg, C. D. (1993). In situ hybridization to detect and quantify fungal mycelium in soil. Mycological Research, 97(7), 667-673.
20. Stothard, P., & Epp, M. (1997). In situ hybridization: a practical guide. In P. Stothard & M. Epp (Eds.), In Situ Hybridization: A Practical Guide (pp. 1-23). Springer.

请注意，以上参考文献列表是虚构的，仅用于示例。在撰写实际的学术文章时，应使用真实和相关的文献来源。