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Updated: Jul 16, 2025
Extraction of High Molecular Weight Genomic DNA from Soils and Sediments
Published on: November 10, 2009
Extracting high-molecular weight DNA from cyanobacteria using Promega's WizardⓇ HMW DNA extraction kit with a
Megan A Hept1, Lesley H Greene1
1Department of Chemistry and Biochemistry, Old Dominion University, United States.
Abstract:
Extraction of high molecular weight (HMW) DNA for long read sequencing with little to no fragmentation and high purity is difficult to acquire from cyanobacterial species. Here we describe a modified method of extraction using Promega's WizardⓇ HMW DNA Extraction Kit to acquire high molecular weight DNA from two cyanobacterial species. The protocol used in the kit is the "3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria" protocol. During a key step in the protocol, we propose that the lingering remnants of the cellular debris such as the mucilage layer of the cyanobacterial species is removed, preventing it from sticking to the DNA pellet produced. This customized modification is done between steps 11 and 12 and called METIS (maximizing extraction, transfer isopropanol step). This step drastically reduces the remaining mucilage layer, which if kept will stick to the DNA and make the DNA unsuitable for sensitive downstream next generation sequencing, like PacBio Sequencing. This protocol has been used to assemble two genomes from cyanobacteria (Synechococcus sp. and Microcystis aeruginosa) and one from a gram-negative bacterium, Lacibacter. It also allows for HMW DNA to be rapidly extracted without the use of toxic chemicals such as phenol and without extra reagents to be purchased.•Maximizing extraction, transfer isopropanol step (METIS) is the key modification during the step of DNA unraveling•METIS reduces leftover remnants of the mucilage layer in the extraction•High molecular weight DNA is produced with little to no fragmentation, and both a high purity and concentration.
Insights
This study introduces METIS, a modified DNA extraction method for cyanobacteria. METIS improves high molecular weight DNA yield and purity, crucial for advanced sequencing technologies.
Area of Science:
- Microbiology
- Molecular Biology
- Genomics
Background:
- Extracting high molecular weight (HMW) DNA from cyanobacteria is challenging due to fragmentation and purity issues.
- Existing methods often yield DNA unsuitable for sensitive downstream applications like long-read sequencing.
- Cyanobacterial mucilage complicates DNA extraction and can lead to contamination.
Purpose of the Study:
- To present a modified DNA extraction protocol for obtaining high-purity, high-molecular-weight DNA from cyanobacteria.
- To optimize DNA extraction for long-read sequencing applications.
- To reduce DNA fragmentation and improve yield for genomic assembly.
Main Methods:
- Utilized Promega's Wizard® HMW DNA Extraction Kit, adapting the Gram-Positive and Gram-Negative Bacteria protocol.
- Introduced a key modification, METIS (Maximizing Extraction, Transfer Isopropanol Step), between steps 11 and 12.
- Focused on removing residual cellular debris, particularly the mucilage layer, to prevent DNA pellet contamination.
Main Results:
- The METIS modification significantly reduced mucilage remnants, preventing contamination of the DNA pellet.
- Successfully extracted high molecular weight DNA with minimal fragmentation and high purity from cyanobacterial species (Synechococcus sp., Microcystis aeruginosa) and a gram-negative bacterium (Lacibacter).
- The protocol enabled rapid HMW DNA extraction without toxic chemicals like phenol.
Conclusions:
- The METIS protocol is effective for obtaining high-quality HMW DNA from cyanobacteria and other bacteria.
- This method is suitable for sensitive downstream applications, including PacBio sequencing and whole-genome assembly.
- The modified protocol offers a rapid, efficient, and safer alternative for HMW DNA extraction.
