Viral Nucleic Acid Purification in a Single Spin

A simple nucleic acid extraction approach quickly purifies genomic viral RNA and DNA while minimizing cross-contamination risks.

The Scientist Creative Services Team and MilliporeSigma
Apr 6, 2022

Disease-causing viruses such as SARS-CoV-2, hepatitis virus, and influenza virus pose serious threats to public health. To combat viral outbreaks, early detection of a virus’s presence in a population is critical for virologists. Analyzing nucleic acids from biological or environmental samples is a preferred virus surveillance method because researchers can precisely characterize different strains and track their evolution in real time. Obtaining pure viral DNA or RNA from samples ensures accurate results in downstream analyses such as PCR, sequencing, and cloning.

A typical nucleic acid purification workflow involves four steps: effective cell disruption, nucleoprotein complex denaturation, nuclease inactivation, and contaminant removal. Although the protocol is straightforward, there is room for error at each step, which can reduce the final product’s quality and quantity. Commercial nucleic acid extraction kits that use solution-based or column-based protocols reduce the number of workflow steps, allowing researchers to isolate viral DNA or RNA from biological samples in a mid- to high-throughput manner. 

Some molecular biologists still use organic solvent-based phenol-chloroform extraction for nucleic acid purification. While this method often generates pure samples, it is laborious, low throughput, and produces hazardous liquid waste. Another key disadvantage of this method is that incomplete phase separation between the liquids reduces the yield, making it difficult to extract genomic material from samples with low viral loads. 

Most commercial kits use solid-phase extraction, where nucleic acids bind to a solid matrix based on affinity or size-exclusion. In affinity-based silica purification, negatively charged nucleic acid molecules bind to positively charged silica particles present in a spin column. These kits involve multiple steps, including cell lysis, nucleic acid adsorption, washing, and elution, before researchers obtain pure DNA or RNA. While kits speed up the extraction process by shortening the time between each protocol step, cross-contamination risk increases with sample number. Also, several steps in the silica column purification workflow require researchers to subject their nucleic acids to centrifugal forces, which decreases product integrity for downstream applications.  

MilliporeSigma Genelute E Kit
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MilliporeSigma

Because of these drawbacks, scientists look for different purification kits that improve extraction speed, yield, purity, and scalability while minimizing cross-contamination. GenElute™-E Single Spin DNA and RNA Purification Kits utilize a size-exclusion chromatography method for nucleic acid purification and offer several advantages over the traditional silica-based purification kits. Exploiting size differences between cellular molecules is at the heart of the GenElute™-E Single Spin Kit. First, a protease mixture in the kit digests proteins, segregating nucleic acids from the protein-DNA complex. When passed through a porous column, smaller biomolecules such as protein fragments, lipids, and sugars get trapped within pores and larger DNA and RNA molecules flow through the column directly into the elution buffer. 

The GenElute™-E Viral DNA/RNA Kit uses the single spin technology, which facilitates faster and more efficient viral nucleic acid purification from a variety of samples, such as nasopharyngeal and genital swabs and stool samples. The kit’s viral lysis buffer utilizes SmartLyseTM protease for viral particle lysis within minutes. After the lysis step, the experimenter separates cellular debris by centrifugation and loads the supernatant with nucleic acid into a single elute column. The column purifies viral DNA or RNA with a single spin, which minimizes the risk of cross-contamination from multiple pipetting steps. By reducing the number of steps in the workflow, the GenElute™-E Viral DNA/RNA Kit has the added benefit of generating less plastic waste. 

The viral purification kit provides high yields of pure nucleic acids without any chemical contaminants from chaotropic salts or organic solvents, which is ideal for downstream applications such as qPCR, multiplex PCR, and next-generation sequencing. An interlaboratory test revealed that commercially-available SARS-CoV-2 PCR kits successfully amplified transcripts from viral RNA isolated with the GenElute™-E Viral DNA/RNA Swab Kit.1 An RNA extraction kit that completes extraction in as little as five minutes with minimal sample handling is therefore attractive to scientists investigating SARS-CoV-2 spread and the emergence of new variants.

References

  1. "SARS-CoV-2 analysis: Swab sample qPCR Cq value differentiation between copy numbers," https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/protein-biology/protein-purification/genelute-e-animation, accessed on March 18, 2022.
  2. S.C. Tan, B.C. Yiap, “DNA, RNA, and protein extraction: The past and the present,” J Biomed Biotechnol, 2009:1-10, 2009.
  3. N. Ali et al., “Current nucleic acid extraction methods and their implications to point-of-care diagnostics” Biomed Res Int, 2017:9306564, 2017. 
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