DNA Size Selection: Basics & Development

What’s DNA size selection?

DNA size selection (or DNA sizing) is the targeted capture of DNA fragments of a specific size or a size range. It’s broadly used in sequencing. And it’s a sample preparation step that has the greatest impact on the quality of sequencing results. Ineffective sizing can waste sequencing capacity on low molecular weight material such as adapter-dimers or primer-dimers (see DNA Clean-up) and imprecise sizing can hurt data quality. Precise DNA sizing can boost sequencing efficiency, save money, improve data, and even allow sequencing of low-input samples.

Why is DNA size selection important?

There are two types of sequencers: short-read sequencers (Illumina and Ion Torrent) and long read sequencers (Pacific Biosciences and Oxford Nanopore Technologies). Both benefit from precise sizing, in a slightly different way.

Short-read sequencers operate best when fed DNA libraries that contain fragments of similar sizes, often in a very specific range recommended by the manufacturer. When libraries are not properly size-selected, these sequencers become significantly less efficient. It may take two lanes of sequencing, to accomplish what could be done in a single lane with a well-sized library.

Long read sequencers tend to produce very long read by fully sequencing a large DNA fragment. When fragments are short, the read is limited. So it’s desired to remove smaller fragments using size selection, which allows the sequencers to focus on the DNA fragments most amenable to producing the longest read.

How does DNA size selection work?

MagVigen DNA size selection workflow

Figure 1. DNA size selection workflow.

  1. Mix: Mix MagVigen™ nanoparticles with DNA/RNA sample.
  2. Bind: When mixed with a sample, MagVigen™ nanoparticles bind to the desired DNA/RNA size fragments. This interaction relies on multiple factors between the DNA/RNA molecules and the surfaces of the nanoparticles, such as charge-charge interactions, hydrophilic/hydrophobic interactions, Van der Waal and other intermolecular forces. NVIGEN engineers the surface chemistry of the Magnetic beads and the buffer environment to provide the ideal interaction for desired DNA size fragment selection.
  3. Wash: The beads respond to a magnetic force (by use of a magnet, e.g. magnetic separation rack), allowing bound material to be rapidly and efficiently separated from the rest of the sample. Remove unbound materials by aspiration, what remains is the nanoparticle-bound targets.
  4. Elute: Release the bound target size fragments from the nanoparticle. The selected DNA/RNA sample is ready for downstream applications.

SPRI beads-based protocols among the best methods

SPRI (solid-phase reversible immobilization) beads-based protocols don’t need column or centrifugation. They use magnetic force to aggregate, wash and elute targets. This makes the workflow simple and easy for automation, which leads to higher throughput.(Please see DNA Purification & Isolation Using SPRI and SPRI for the Isolation of PCR Products.)

While some tutorial source (e.g. BiteSizeBio‘s dna-sizing-tutorial) says beads-based protocols should not be used when more precise sizing is needed and they’re most useful in experiments where DNA is abundant, this knowledge is outdated.

The capability and capacity of any DNA size selection methods come from the functional groups assembled to beads, not directly from the beads themselves. Nanobeads, like MagVigen™, manufactured with the state-of-the-art nanotechnologies can be engineered at the molecular level to assemble a variety of functional groups. And nanobeads are in fractional (1/10) size of conventional beads, which creates 10x bigger surface area off the same amount of beads, and new advanced beads dynamics. These new properties have increased the capability and capacity of beads-based protocols to be among the best methods now. 

magvigen-dna-precise-size-selection-by-adjusting-volume-ratio

Figure 2. 1µg Fisher DNA ladder was size-specifically captured by MagVigen™ nanobeads by adjusting the ratios of MagVigen™ volume vs. DNA sample volume. The numbers on top indicate the ratio.

Long read DNA sequencing

Long read DNA sequencing technologies have revolutionized the field by enabling the sequencing of larger fragments (1 kb to 30 kb) compared to traditional short read methods. The advancements offer significant advantages over the short read sequencing, including:

  1. Accurate genome assembly with longer reads.
  2. Improved detection of large-scale genomic variations, including insertions/deletions and structural variants.
  3. Enhanced characterization of complex regions like repetitive sequences and tandem repeats.
  4. Superior transcriptome analysis for diverse RNA forms identification.
  5. Direct epigenetic modification detection, including methylation.
  6. Elimination of PCR amplification and reduction of biases in single molecule sequencing.

Two leading long read sequencing platforms are Pacific Biosciences’ SMRT and Oxford Nanopore Technologies’ Nanopore. PacBio utilizes zero-mode waveguides (ZMWs) for single DNA polymerase enzyme observation, while Nanopore passes a DNA molecule through a tiny pore in a membrane to detect changes in electrical current and identify bases. Both platforms offer real-time sequence data production, which hugely benefits from precise long read DNA Size Selection.

Long read or large fragment DNA Size Selection

DNA fragment purification methods are crucial for long read sequencing applications, depending on the technology used and downstream goals. Common methods include gel electrophoresis and size-selective SPRI magnetic bead-based purification, such as NVIGEN’s MagVigen™ extend DNA size selection kit (Catalog Number: K61001-Extend).

magvigen-dna-precise-size-selection-by-adjusting-volume-ratio

Figure 3. MagVigen™ nanobeads offer unique advantages with extended long read size selection (700bp to 6000bp) and high yield. By adjusting the beads-to-DNA volume ratio, MagVigen™ beads can capture DNA base pairs > 3000 bp with >80% yield. Our MagView™ DNA size selection kit is compatible with various long-read sequencing platforms, ensuring precise size range selection, high yield, and a user-friendly workflow.

Improve your DNA size selection

We cordially invite you to try our state-of-the-art MagVigen™ DNA Size Selection Kits (CAT# K61001), especially our latest extended long bp read (500bp-3000bp) size selection kits (CAT# K61001-Extend) for your DNA purification and size selection needs.

Or interested in sequence specific DNA/RNA capture?
Check our our MagVigen™-Streptavidin products (K61002).

Access this page from this QR code: 2d bar code to dna-size-selection-tutorial