January 29, 2021

Moving from column to bead-based DNA isolation

By Michael Murphy, Modality Specialist USCAN/Asia, Genomics and Diagnostic Solutions, Cytiva

Magnetic beads are an efficient and easily automatable alternative to column-based methods of nucleic acid isolation. Here are eight key considerations to help you understand how magnetic beads can benefit your workflow.

Successful extraction and purification of nucleic acids is crucial in many molecular biology studies, including those involving next-generation sequencing (NGS). Successful extraction often determines the quality of the overall data. While spin column-based isolation methods have been a staple in labs for many years, they are increasingly being replaced by magnetic bead-based methods.

Why this shift in approach by researchers in academia and industry alike?

Well, magnetic beads (or superparamagnetic beads) are an effective alternative to columns that can simplify the processes of DNA/RNA extraction. They have several advantages over columns when used in sensitive applications, such as NGS library preparation. These advantages include the ability to isolate longer strand nucleic acids from samples, greater flexibility in fragment size selection, and allowing for normalization of libraries.

These eight key considerations might help you decide if magnetic beads can improve your workflows.

1: Simpler, gentler nucleic acid isolation

The first step of sample preparation involves cell lysis, or disruption, usually with a combination of detergent and mechanical force, to release the genetic material.

In column-based methods, you would centrifuge or clarify the lysate, add the supernatant to a silica membrane to bind nucleic acids, wash with buffer via centrifugation or vacuum manifold, then elute the desired nucleic acid in an appropriate volume of buffer.

These steps provide ample opportunity for sample loss and mechanical shearing of the nucleic acids.

Magnetic bead-based methods are gentler and more versatile than columns, require fewer handling steps (and therefore fewer opportunities for shearing), and offer many surface chemistries for different applications.

The result is a simpler workflow that can be easily automated and scaled up to produce more reproducible nucleic acid isolation at greater quantities than spin column-based methods.

Find out more about approaches to DNA/RNA extraction

2: Easier automation with bead-based methods

There has never been a better time to automate your DNA/RNA isolation procedures, with manufacturers now offering benchtop systems for low, medium, and high-throughput automation of nucleic acid isolation for NGS and polymerase chain reaction (PCR) applications.

While column-based isolation methods can be partially automated on generic liquid handling machines, full automation requires systems integrating vacuum manifolds or onboard centrifuges.

Several vendors now offer open platforms that can automate magnetic bead-based reagents from various commercially available kits. Magnetic beads-based kits are well suited for high-throughput applications as, unlike spin column methods, they do not require centrifugation or vacuum processing. As a result, these kits can provide high consistency between experiments and are less prone to sample contamination when automated.

3: Consider the learning curve

In academic research labs, processing many samples for nucleic acids may require a considerable amount of manual pipetting when using column-based methods. This pipetting can lead to frustrating variability in DNA/RNA yield between experiments and individuals. Staff and students may need considerable training and practice to achieve reasonably consistent nucleic acid yields.

Even in automated low or medium-throughput processes using columns, there is likely a requirement for centrifugation and manual handling that affect DNA/RNA integrity.

Washing and eluting DNA/RNA bound to magnetic beads in automated systems is simpler than with columns, requiring little to no additional input from the user, and therefore minimizing the amount of training required.

4: Choose the most appropriate method for your application

Isolation of genomic DNA and total RNA can use silica binding in either the column or magnetic bead format.

However, while columns are well suited for basic applications, such as PCR and electrophoresis to check inserts in vector-based cloning, sample preparation in more sophisticated studies involving methods like quantitative polymerase chain reaction (qPCR) and NGS can be simplified by using magnetic beads.

The simplicity of magnetic bead-based protocols means they can be automated easily, making them an excellent option for procedures that require quick turnaround times, including total viral DNA/RNA extraction for qPCR-based diagnosis of diseases and high-throughput sequencing applications.

Magnetic beads can also support specific diagnostic applications that use samples with low DNA amounts, such as liquid biopsies containing circulating cell-free DNA (cfDNA).

5: Find a surface chemistry for your application

While spin columns are limited to silica, cellulose, or ion exchange resins, magnetic beads provide a range of surface chemistries that extend their use beyond that of standard DNA/RNA extraction and purification techniques.

For example, in exome sequencing, streptavidin-coated magnetic beads enable the capture of specific genes or exon fragments hybridized to biotinylated complementary sequences. These magnetic beads are well suited for use with targeted sequencing panels for cancer, enabling the capture of specific disease-associated sequences for downstream NGS and tumor profiling. Similar techniques are also used for microsatellite isolation in population genetics.

Discover the different coating chemistries used for magnetic beads

6: For unique applications, consider custom bead conjugation

In addition to the standard array of surface chemistries, magnetic beads can be conjugated to enzymes, antibodies, and custom ligands. For example, Protein A/G-conjugated magnetic beads are widely used in immunoprecipitation (IP) and co-IP assays to study protein interactions. As with nucleic acid isolation, protocols with these beads are easily automated, freeing up time and reducing the need for manual handling of samples.

7: Use magbeads for both size selection and clean-up in NGS

NGS sample prep has a series of steps that can influence the quality of the final data: nucleic acid isolation, purification, size selection and clean-up, and library normalization. Wouldn’t it be simpler if you could use the same type of reagent throughout these steps?

While some vendors do offer columns for DNA fragment size selection, these kits are often restricted to removal of fragments below a specific length.

Carboxylate-coated magnetic beads greatly simplify the process of DNA fragment size selection for NGS in both manual and high-throughput automated workflows. Simply altering the ratio of buffer to beads to DNA enables reproducible selection of fragments within a very specific size range.

With magnetic beads, clean-up of libraries for NGS workflows is as simple as eluting the DNA from the beads with a buffer.

Watch our webinar comparing approaches to NGS sample prep

8: The new normal for NGS library normalization

Overlooking the importance of library normalization can lead to wasted capacity and incomplete or unreliable datasets after sequencing. Although columns can be used for some stages of NGS library preparation, unlike magnetic beads, they cannot be used for normalization.

Where your sample is plentiful and time is a factor, magnetic beads remove the need for careful quantitation by qPCR or fluorimetry and provide an equimolar quantity of library fragments across multiple samples.

Find out more about using magnetic beads for NGS library normalization

Magnetic beads are versatile tools for nucleic acid purification, requiring fewer handling steps than columns and offering surface chemistries tailored to different applications. Switching from columns to beads makes automation easier and therefore addresses ongoing high throughput sample demands. For help with making the switch from spin columns to magnetic beads, contact our Scientific Support team.

At Cytiva, we offer technical support and a wide range of products for nucleic acid isolation and library preparation for NGS. For more information about using magnetic beads in your application, visit our Scientist’s guide to magnetic beads.

Request a sample and discuss your magnetic bead applications with a modality specialist