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Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing
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Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Published on: March 31, 2017

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High molecular weight genomic DNA mini-prep for filamentous fungi

Mi-Kyung Lee1, Hee-Soo Park2, Kap-Hoon Han3

  • 1Department of Bacteriology, The University of Wisconsin-Madison, Madison, WI 53706, USA.

Fungal Genetics and Biology : FG & B
|April 22, 2017
PubMed

Insights

We developed a simple mini-preparation protocol for high-quality genomic DNA (gDNA) from filamentous fungi. This method yields high molecular weight gDNA suitable for whole genome sequencing and other molecular applications.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Purifying high-quality genomic DNA (gDNA) from filamentous fungi for whole genome sequencing is typically a multi-step process.
  • Existing methods can lead to DNA shearing and loss, impacting downstream applications.
  • There is a need for a simplified, efficient protocol for obtaining high molecular weight gDNA from fungi.

Purpose of the Study:

  • To develop and present a simple, easy-to-follow mini-preparation protocol for high molecular weight (∼20kb) genomic DNA (gDNA) from filamentous fungi.
  • To provide a protocol suitable for various fungal species, including Aspergillus and Eurotium.
  • To ensure the purified gDNA is of sufficient quality for whole genome sequencing and other molecular analyses.

Main Methods:

  • A mini-preparation protocol involving inoculation, homogenization, and purification of gDNA was established.
  • A critical step involves 3-5 minutes of homogenization of freeze-dried mycelium using a motorized hand-held homogenizer.
  • A modified Qiagen DNeasy Plant Mini Kit procedure was employed, avoiding precipitation, drying, and resuspension steps.

Main Results:

  • The protocol yields 8 to 20μg of gDNA from approximately 20mg of fine mycelial powder.
  • Purified gDNA consistently exhibits a 260/280nm absorbance ratio of ∼1.9.
  • All 30 purified gDNA samples were of high molecular weight (∼20kb) and suitable for whole genome sequencing (160-260 X coverage).

Conclusions:

  • This mini-preparation protocol offers a simple and effective method for purifying high-quality, high molecular weight gDNA from filamentous fungi.
  • The protocol is suitable for whole genome sequencing, Southern blotting, and PCR-based amplification.
  • The method streamlines gDNA purification, reducing potential shearing and loss of valuable genetic material.
Keywords:
AspergillusFungiHigh molecular weightNGSQiagengenomic DNA

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