What is DNA clean-up? Why is it important?
DNA clean-up, also known as magnetic beads-enabled cleanup, is the targeted removal of small DNA fragments such as primers, adapters and dimers from a sample mixture for downsteam PCR, DNA ligation/cloning, or DNA library etc.
Sometimes, by DNA clean-up some people may be referring to the removal of other substances, e.g salts, proteins and enzymes etc. that are actually involved in the processes of DNA extraction. We may call it pre-clean-up.
So nowadays by DNA clean-up we sepcifically focus on primers, adapters, and dimers formed from primers or adapters, because they are also formed by DNA oligonucleotides. That’s to say they are also DNA fragments. When these fragments present at high proportions in a sample. They can cause a variety of problems and interfere with proper sequencing on NGS instruments, e.g. they can soak up significant amounts of the sequencing capacity and cause a number of QC metrics to be triggered. And they can lead to NGS alignment errors and increased unaligned reads which hurt the data quality.
Effective DNA clean-up can boost PCR and sequencing efficiency, improve data, even allow sequencing of low-input samples, and save money.
NVIGEN magnetic nanoparticles enable DNA clean-up of DNA products. They can be used for PCR product clean-up, NGS (next generation sequencing) library construction, terminator dye removal, and removal of other contaminants from assay samples, e.g. cell lysate.
What are primers, adapters and dimers?
Primers and adaptors are synthetic DNA oligonucleotides, generally of known sequence. Primers are used in PCR to prime DNA replication reactions. Primers that are used to amplify specific DNA sequences in vitro are called PCR primers, typically 15–30 bases. Note as sequencing includes a step of PCR DNA replication in NGS DNA library construction, primers are also used in sequencing.
Adaptors are any kind of short DNA sequence serving the scope of fishing a (generally unknown) DNA sequence of interest for various purposes. Sequence adapters are used in DNA replication in NGS DNA library construction.
Dimers are 2 pieces of PCR primers or 2 pieces of sequence adapters bound to each other directly without any DNA fragments of interest in between (called inserts). It wastes the raw material and affects results downstream negatively described above.
From illumina, adapter dimers contain full-length adapter sequences (120–170 bp) that are able to bind and cluster on the flow cell and generate sequencing data. And because of their small size, they cluster more efficiently than the intended library fragments. Depending on the quantitative proportion relative to the specific library, they can subtract a significant portion of sequencing reads from the desired library fragments. They can negatively impact sequencing data quality, and may even cause a run to stop prematurely.
In contrast, primer dimers do not contain complete adapter sequences, and are not able to bind or cluster on the flow cell, so are not sequenced.
One reason that primer-dimers and adapter-dimers are formed is that there are excessive primers or adapters present.
Figure 1. PCR annealing, elongation and primer dimers. A. PCR annealing is done, ready for PCR elongation. 2 pieces of PCR primers are bound to each other (the one at the top right corner). B. PCR elongation is complete. A copy of primer dimer is formed (the one on the top).
Figure 2. Prepare Genomic DNA Sample. Randomly fragment genomic DNA and ligate adapters to both ends of the fragments. A. Dna fragments ligated with sequence adapters. B. Adapter dimers formed from excessive adapters.
What causes adapter dimers?
- Insufficient starting input material. The input amount can be measured with a fluorometric-based quantification method. Using an input amount within the range recommended for a sequencing workflow can minimize the chance that adapter dimers present in the final libraries.
- Fragmented or degraded starting input nucleic acid.
- Inefficient beads clean-up
. It is important to follow best practices when handling beads to ensure proper size selection and removal of adapter dimers that may have formed during library preparation.
DNA clean-up methods
BiteSizeBio listed “5 Ways to Clean Up A DNA sample”. To some extent the summary is oudated and misleading. Let’s examine how each works.
- Phenol-Chloroform Extraction to remove proteins. The DNA solution is mixed with phenol and chloroform. The water-soluble DNA partitions into the aqueous phase, while the proteins denature in the presence of organic solvents, thus staying in the organic phase.
- Ethanol Precipitation to desalt. In DNA extraction, salt is used to release the DNA strands by breaking up protein chains that hold nucleic acids together. As DNA is not soluble in Ethanol. In an ethanol solution, the salt makes the DNA less soluble in water while helps to keep the proteins (small organic molecules) dissolved in water. The result is, DNA molecules aggregate and precipitate out of solution.
- Silica Column-Based using a silica gel or silica beads and chaotropic salts. The chaotropic salts disrupt the hydrogen bonding between strands and facilitate binding of the DNA to silica and its separation from the rest of the sample.
- Anion Exchange using positively charged DEAE functionalized resins to bind the negatively charged DNA phosphate backbone.
- Magnetic Beads uses magnetic beads that conditionally bind DNA in a pH-dependent manner, allowing you to separate the DNA from the rest of the sample by simply controlling pH. The magnetic beads are positively charged and bind DNA at low pH, but at high pH they become negatively charged, thus releasing the DNA.
The first two are actually part of the DNA extraction method called Organic Extraction, which includes a lysis step, a phenol chloroform extraction, an ethanol precipitation, and washing steps.
The later 3 are used for DNA clean-up. All includes steps of bind, wash, and elute. Among them the Magnetic Beads based method is most popular becaues there is no need for centrifugation and other time consuming processing steps. It’s Ideal for automation of high throughput processing. As it’s the dominant method now, the cost of beads dropped to half in the past decade.
How to clean up DNA?
Figure 3. DNA clean-up workflow.
- Mix: Mix nanoparticles with DNA sample.
- Bind: When mixed with a sample, nanoparticles bind to the targeted DNA sample.
- Wash: Use a magnetic force (e.g. magnetic separation rack) to pull and aggregate bound material. Remove unbound materials by aspiration, what remains is the nanoparticle-bound target.
- Elute: Release the bound target sample from the nanoparticles. The cleaned DNA sample is ready for downstream applications.
Learn more about the basics & development of DNA Size Selection.
DNA clean up examples
Below are 4 examples in different contamination levels for DNA clean-up.
| Before DNA Clean-up | After DNA Clean-up |
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Figure 4. MagVigen™ Easy DNA Size Selection Kit (K61001-Easy) outperforms conventional brand products for strongly contanminated NGS DNA Library sample (adapter dimer ratio 224%). After clean-up, adapter dimer over library is less than 3% using MagVigen™ (passes quality control (QC)) vs. more than 6% using AmPure XP (fails QC).
| Before DNA Clean-up | After NVIGEN K61001-Easy DNA Clean-up |
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| Tested by Agilent 2100 12000 Assay | Tested by Agilent 2100 High Sensitivity Assay |
Figure 5. MagVigen™ Easy DNA Size Selection Kit (K61001-Easy) outperforms conventional brand products for significantly contanminated NGS DNA Library sample (adapter dimer ratio 186%). After clean-up, adapter dimer over library reduced to 2.3%.
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Figure 6. MagVigen™ Easy DNA Size Selection Kit (K61001-Easy) also works better for medium contanmination NGS DNA Library sample (adapter dimer ratio 68%). After clean-up, adapter dimer over library is 0.45% using MagVigen™ vs. 1.2% using AmPure XP.
| Before DNA Clean-up | After DNA Clean-up |
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Figure 7. MagVigen™ Easy DNA Size Selection Kit (K61001-Easy) still works better for log contanmination NGS DNA Library sample (adapter dimer ratio 20%). After clean-up, adapter dimer undetectable using MagVigen™ vs. 0.6% using AmPure XP.
Example: fast DNA extraction and cleanup using NVIGEN magnetic accessories
Below is a simple tutorial demonstrating fast DNA extraction with NVIGEN MagVigen™ nanoparticles, magnetic rack, magnetic plate and cube magnets.
It shows MagVigen™ allows simple and efficient DNA extraction ideal for automation.
Improve your NGS DNA Library Clean-up
We cordially invite you to try our state-of-the-art MagVigen™ DNA Size Selection Kits (K61001) for your DNA Clean-up or Purification needs.