A simple method to extract filamentous fungal genomic DNA

Subject Areas : دو فصلنامه تحقیقات بیماریهای گیاهی

Saeed Ghafoori Harat ¹ , Tahereh Basirnia ² , Mohammad Reza Moosavi ³

1 - دانش آموخته کارشناسی ارشد، گروه بیماری شناسی گیاهی، واحد مرودشت، دانشگاه آزاد اسلامی، مرودشت، ایران.
2 - استادیار، گروه بیماری شناسی گیاهی، واحد مرودشت، دانشگاه آزاد اسلامی، مرودشت، ایران.
3 - استادیار، گروه بیماری شناسی گیاهی، واحد مرودشت، دانشگاه آزاد اسلامی، مرودشت، ایران.

Received: 2016-02-01 Accepted : 2016-02-01 Published : 2016-05-21

Keywords: C-TAB, ITS, Antagonist, DNA extraction, filamentous fungi, grinder,

Abstract :

Today, the application of molecular methods is a common and necessary part of many routine researches. DNA extraction is the foundation for most of such research studies. As a result, it is of great significance to discover and select proper techniques that are productive, inexpensive, straightforward, safe and rapid at the same time. Agricultural research and experiments are no exception in this respect. C-TAB, phenol-chloroform and DNA extraction kits are conventional techniques in agriculture for extracting DNA from filamentous fungi. Each of these methods has its advantages and disadvantages. In this study, we have developed a novel, straightforward and effective method to extract the DNA from antagonistic-, saprophytic- and plant pathogenic-filamentous fungi. This method is a composition of the aforementioned techniques and is used to avoid the application of liquid nitrogen and phenol. It is called squish with grinder and is a safer, more affordable and quicker method that results in relatively pure DNAs. The purity of obtained DNA was assessed by spectrophotometry. The ITS region and β-tubuline gene was amplified by PCR. The results of this assessment justify this method as a practical and reliable technique to extract filamentous fungal genomic DNA.

References:

1. Adams DJ. 2004. Fungal cell wall chitinases and glucanases. Microbiology 150: 2029–2035.


2. Ahmed O, Atif HA and Mogahid ME. 2014. Comparison of three DNA extraction methods for polymerase chain reaction (PCR) analysis of bacterial genomic DNA. African Journal of Microbiology Research 8: 598–602.


3. Almeida da Silva G, Bernardi TL, Schaker PDC, Menegotto M and Valente P. 2012. Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification. Brazilian Archives of Biology and Technology 2: 319–327.


4. Amer EO, Mahmoud MA, El-Samawaty AM and Sayed SR. 2011. Non liquid nitrogen-based-method for isolation of DNA from filamentous fungi. African Journal of Biotechnology 10: 14337–14341.


5. Anonymous. 2000. Assessment of nucleic acid purity. T042‐Technical Bulletin, Wilmington, Delaware: Thermo Fisher Scientific – NanoDrop Products.


6. Clark W and Christopher K. 2000. An introduction to DNA: spectrophotometry, degradation, and the 'Frankengel' experiment. pp. 81–99 In SJ Karcher (eds). Tested studies for laboratory teaching, Volume 22. Proceedings of the 22^nd Workshop / Conference of the Association for Biology Laboratory Education (ABLE). Edmonton: Publication of University of Alberta.


7. De Souza C, De Queiroz Cavalcanti M, Fernandes M, Massa Lina D, Nascimento J and Laranjeria D. 2003. Identification and characterization of filamentous fungi isolated from the sunflower (Helianthus annus L.) rhizosphere according to their capacity to hydrolyse inulin. Brazilian Journal of Microbiology 34: 273–280.


8. Desjardins P and Conklin D. 2010. NanoDrop microvolume quantitation of nucleic acids. Journal of visualized experiments, (45): 2565


9. Diba K, Namaki A, Ayatolahi H and Hanifian H. 2014. Comparison of biochemical and molecular methods for the identification of candida species causing vulvovaginal candidiasis and recurring vulvovaginal candidiasis. Iranian Journal of Medical Microbiology 8: 45–50.


10. Fredricks DN, Smith C and Meier A. 2005. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. Journal of Clinical Microbiology 43: 5122–5128.


11. Gauthier ER. 2007. Biochemistry I. Department of Chemistry and Biochemistry. [cited 2015 March 18] Available from: http://www.foodelphi.com/biochemistry-problems-and-solutions-eric-r-gauthier-ph-d/


12. Kamalzadeh S and Sabokbar A. 2014. Molecular survey of pathogen species of Aspergillus isolated from diabetic foot lesion using nested PCR method. Journal of Medical Sciences Torbat, 1: 25–32.


13. Michaelsena A, Pinzarib F, Ripkaa K, Lubitza W and Pinar G. 2006. Application of molecular techniques for identification of fungal communities colonising paper material. International Biodeterioration & Biodegradation 58: 133–141.


14. Moallemzadeh A, Yadegari M, Rajabi M and Kachuei R. 2014. The simple molecular method for confirming identification of the main species of fungi Candida, Aspergillus and Dermatophyte. The Journal of Urmia University of Medical Sciences 25: 105–112.


15. Płaza1 G, Upchurch R, Brigmon R, Whitman W and Ulfig K. 2004. Rapid DNA extraction for screening soil filamentous fungi using PCR amplification. Polish Journal of Environmental Studies 13: 315–318.


16. Prillinger H, Schweigkofler H, Breitenbach M,  briza P, Staudacher E, Lopandic K, Molnaur O, Weigang F, Ibl M and Ellinger A. 1997. Phytopathogenic filamentous (Ashbya, Eremothecium) and dimorphic fungi (Holleya, Nematospora) with Needle-shaped ascospores as new members within the Saccharomycetaceae. Yeast 13: 945–960.


17. Ragazzo-Sanchez J, Gutierrez-Escatel A, Luna-Solano G, Gómez-Leyva J and Calderon-Santoyo M. 2011. Molecular identification of the fungus causing postharvest rot in jackfruit. Journal of Revista Mexicana de Micología 34: 9–15.


18. Serrano R, Gusmão L, Amorim A and Araujo R. 2011. Rapid identification of Aspergillus fumigatus within the section Fumigati. BMC Microbiology 11:82. doi: 10.1186/1471-2180-11-82.


19. Shinawi T. 2010. DNA Gel Electrophoresis. Molecular Biology Lab, 5 p.


20. Skoog DA. 1985. Principle of instrumental Analysis. USA Saunders College Publishing.


21. Sridhar P. 2006. Polymerase Chain Reaction (PCR). Department of Microbiology jjmmc, 3 p.


22. Yanisko P, zheng SY, Dumoit J and Carlson B. 2011. Nitrogen: A security blanket for the chemical industry. Chemical Engineering Progress 107: 50–55.


23. Zador E. 2011. The polymerase chain reaction. Basic biochemical methods and ischemic heart models, 59 p.