Establishment and evaluation of a method for rapid extraction of plant genomic DNA

Li, Lin; Luo, Linlin; Luo, Guangyu; Mo, Beixin; Liu, Lin

doi:10.3724/sp.j.1249.2020.01001

Cited by 1 publication

(1 citation statement)

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“…There are traditional methods for genome extraction of the filamentous fungi, such as the CTAB (cetyltrimethylammonium bromide) (Liu et al 2017), SDS method (Manian et al 2001, isopropanol method (Moslem et al 2010), quartz sand grinding method (Li et al 2006) and so on. Nowadays some new methods have been developed including enzymatic hydrolysis method, mechanical breakthrough combined with microwave method (Liu et al 2020), liquid nitrogen crushing and alkali cracking method (Li et al 2022), lysate heating method (Lin et al 2018), and plant genome kit extraction method (Bellemare et al 2018), reagent kit method (Serna-Domínguez et al 2018), vacuum filtration for genomic DNA extraction (Murigneux et al 2020). However, some problems still exist in the above methods.…”

Section: Introductionmentioning

confidence: 99%

A New Method for Rapid Extraction of Fungal Genome for PCR

Tan,

Liu,

Zhang

et al. 2023

Preprint

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Objectives The present work aimed to develop a new method for rapid extraction of the fungal genome for Polymerase Chain Reaction (PCR). Results(method) We used only 15 minutes to obtain an intact genome for PCR, due to cells were disrupted in the 0.5 mol/L NaOH solution. The quality and quantity of genomic DNA was verified by Nanodrop under OD260/OD280, even the density of dsDNA was measured. Firstly, we tried different lysis modes including enzymatic lysis and NaOH lysis. Finally the condition of NaOH cleavage method was selected and optimized for subsequent experiments. In conclusion, the optimal NaOH cleavage conditions were 100 μL of 0.5 mol/L NaOH to lysis the mycelium for 15 minutes at 25 ℃. The optimal growth time of mycelium was 48 hours, and the optimum amount to be used was 3.5 millimeter diameter. The method was applicable to different fungus and validation of multiple genes with diversable primers. Conclusions Compared to traditional gene extraction methods, the new method was less time-consuming, safer, and simpler.. This method was so fast that it took only 15 minutes to complete the preparation of Polymerase Chain Reaction (PCR) reaction templates. The quality and quantity of extracted fungal genomes were confirmed at OD260/OD280 in the range of 1.8-1.9, and density in the range of 150-350 ng/μL. The new method was successfully applied to Aspergillus niger JH101, Saccharomyces Y3, Aspergillus niger ATCC1015, Aspergillus oryzae 3.042, Ustilago maydis UM1, Trichoderma reesei SUS, and even genetically engineered A. niger strains, and the success rate was remarkably high reaching to 89.2 %. Thus this technology can significantly increase the efficiency of basic molecular biology procedures.

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