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DNA Sampling and Extraction for Nanopore Sequencing

Introduction

Nanopore sequencing is a highly versatile technology capable of analyzing DNA directly from diverse sample types, from environmental sources to purified genomic extracts. However, the success of a sequencing experiment begins with DNA sampling and extraction—ensuring that high-quality DNA is obtained while keeping protocols accessible and cost-effective.

In this guide, we focus on DNA extraction methods suitable for classroom and teaching labs where simplicity, affordability, and safety are key considerations. We generally recommended approaches that:

  • Minimize toxic chemicals to ensure safety and ease of disposal.
  • Reduce reliance on specialized equipment to keep protocols accessible.
  • Lower costs by recommending practical and scalable methods.

In many cases, particularly when PCR amplification is used simpler homemade" DNA extractions can be sufficient, even when DNA integrity is not perfect. For applications that require long DNA reads such as whole-genome sequencing or structural variant analysis, we will highlight commercial extraction kits that give a better chance for high DNA integrity.

While specialized extraction methods may yield the highest-quality DNA, they often require additional steps, equipment, and reagents.

Basic principles of DNA extraction

PubMed lists more than 1600 articles on DNA extraction methods. Depending on your sample, you can find specialized methods, but we focus here on the most generalizable approaches knowing that many educators deploying in the classroom will want to focus on 1-2 methods that can cover the largest variety of samples. DNA extraction usually involves the following:

Cell lysis

A lysis buffer containing detergents, mechanical disruption, and heat are all commonly used tools. Mechanical disruption methods, such as bead beating, grinding with a mortar and pestle, or using liquid nitrogen for tough samples like plant tissue, help break open cell and nuclear membranes, allowing cellular material to be released into solution.

Protein digestion and removal

Once the cells are lysed, enzymes such as proteinase K are often added to degrade proteins that might be bound to the DNA or otherwise contaminate the sample. In addition to enzymatic digestion (e.g., lysozyme for bacterial samples), chemical agents (e.g., phenol or chloroform) can be used to separate proteins from the nucleic acids. This step helps ensure that the final DNA preparation is as free as possible from protein contaminants. We don't recommend any methods using phenol due to the increased safety hazard.

Precipitation

To isolate the DNA, an alcohol (usually ethanol or isopropanol) is added along with salts. The salts help neutralize the charges on the DNA, and the alcohol causes the DNA to precipitate out of solution. Centrifugation is then used to collect the DNA as a pellet. Most protocols will use isopropanol first since lower volumes are more efficient, and ethanol is used in washing since it has a lower evaporation point. Two important methods worth highlighting here:

  • Silica membrane-based purification: DNA binds to a silica column in the presence of chaotropic salts and is later eluted in water or buffer. Many commercial kits use this method and although spin columns add to the cost, column-based kits are fairly reliable and reproducible. A potential drawback is that columns may yield more fragmented DNA (i.e., DNA with lengths in the 1000's of basepairs rather than 10,000-100,000+ bp)
  • Magnetic bead purification: DNA attaches to magnetic beads, allowing for easy separation and cleanup. This method will frequently be used in Nanopore kits at various cleanup steps to purify DNA after enzymatic steps, rather than the initial DNA extraction.

Washing and Resuspension

The DNA pellet is washed (often with ethanol) to remove any residual salts or other impurities. After washing, the pellet is dried and then resuspended in a suitable buffer (commonly TE buffer) to stabilize the DNA for storage and subsequent applications.

Additional Reading

The Evolution of DNA Extraction Methods. Preetha J Shetty, The Evolution of DNA Extraction Methods. 2020 - 8(1). AJBSR.MS.ID.001234. DOI:10.34297/AJBSR.2020.08.001234.

DNA extraction recommendations by sample type and analysis endpoint

These are some selections we have tried and do not represent all possible or even the best possible approaches. Please give us feedback on new and/or lower-cost approaches you wish to share!

Note: All prices are estimates and may not include the total sample cost, for example including reagents or supplies not provided and/or potential education discounts.

Phage

A rapid competitive method for bacteriophage genomic DNA extraction

  • Format: Homemade
  • Sequencing endpoint: PCR amplification/16S
  • Costs: Low (<$1 sample)
  • Application notes: Protocol

Norgen Phage DNA Isolation

  • Format: Commercial kit
  • Sequencing endpoint: Whole genome
  • Costs: Midrange ($5 sample)
  • Application notes: Website

See Monarch® HMW DNA Extraction Kit for Tissue Phage protocol

  • Format:Commercial kit
  • Sequencing endpoint: Long-read whole genome
  • Costs: High ($10 per sample)
  • Application notes: Website

Bacteria

See Monarch® HMW DNA Extraction Kit for Tissue Bacteria protocol

  • Format:Commercial kit
  • Sequencing endpoint: Long-read whole genome or PCR amplification/16S
  • Costs: High ($10 per sample)
  • Application notes: Website

Swab collection and DNA preservation

  • Format: Commercial kit
  • Sequencing endpoint: DNA collection and preservation only (no DNA extraction)
  • Costs: Midrange ($7 per sample + kit for DNA extraction)
  • Application notes: Website

Alkaline Method for Bacterial DNA Extraction

  • Format: Homemade
  • Sequencing endpoint: PCR amplification/16S
  • Costs: Low (<$1 sample)
  • Application notes: Protocol

General microbial

Norgen Microbiome DNA Isolation Kit

  • Format: Commercial kit
  • Sequencing endpoint: PCR amplification/16S
  • Costs: Midrange ($6 per sample)
  • Application notes: Website

Soil, environmental

DNeasy PowerSoil Pro Kit

  • Format: Commercial kit
  • Sequencing endpoint: PCR amplification/16S, whole genome
  • Costs: High ($10 per sample)
  • Application notes: Website

Water, environmental

E.Z.N.A.® Water DNA Kit

  • Format: Commercial kit
  • Sequencing endpoint: PCR amplification/16S
  • Costs: Low ($4 per sample)
  • Application notes: Website

Plant

See PacBio Nanobind PanDNA kit formatted for plant nuclei lysis

  • Format: Commercial kit
  • Sequencing endpoint: Long-read whole genome
  • Costs: High ($14 per sample)
  • Application notes: Website

Insect

See Monarch® HMW DNA Extraction Kit for Tissue Insect protocol

  • Format:Commercial kit
  • Sequencing endpoint: Long-read whole genome
  • Costs: High ($10 per sample)
  • Application notes: Website

General

Simple, Robust Invertebrate DNA Barcoding: Chelex-Based DNA Extraction and Optimized COI Amplification

  • Format: Homemade
  • Sequencing endpoint: PCR amplification
  • Costs: Low (<$1 per sample)
  • Application notes: Website

Fire Monkey High Molecular Weight DNA (HMW-DNA) extraction kit

  • Format: Commercial kit
  • Sequencing endpoint: Long-read whole genome
  • Costs: High ($10 per sample)
  • Application notes: Website

Monarch® HMW DNA Extraction Kit for Tissue

  • Format:Commercial kit
  • Sequencing endpoint: Long-read whole genome
  • Costs: High ($10 per sample)
  • Application notes: Website

PacBio Nanobind PanDNA kit

  • Format: Commercial kit
  • Sequencing endpoint: Long-read whole genome
  • Costs: High ($14 per sample)
  • Application notes: Website

New England Biolabs® (NEB) support for ORG.one participants

  • Format: Commercial kit
  • Sequencing endpoint: Long-read whole genome
  • Costs: Varies
  • Application notes: Website

Promega Wizard® HMW DNA Extraction Kit

  • Format: Commercial kit
  • Sequencing endpoint: Long-read whole genome
  • Costs: Midrange ($7 per sample)
  • Application notes: Website

Additional Reading

  1. Oxford Nanopore Extraction protocols page (sign-in required) https://nanoporetech.com/documentation/results?category=prepare&topic=extraction-protocols.
  2. DNA Barcoding 101 — DNA extraction protocols. Website
  3. New England Biolabs® (NEB) support for ORG.one participants. https://nanoporetech.com/oo/extraction-protocols.
  4. Gand, M., Bloemen, B., Vanneste, K. et al. Comparison of 6 DNA extraction methods for isolation of high yield of high molecular weight DNA suitable for shotgun metagenomics Nanopore sequencing to detect bacteria. BMC Genomics 24, 438 (2023). https://doi.org/10.1186/s12864-023-09537-5.

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