Next-Generation Sequencing DNA Cleanup and PCR Purification

DNA cleanup

DNA cleanup involves the targeted removal of unwanted fragments and contaminants from a given sample. NGS adapters, primers, primer-dimers, unincorporated dNTPs, and other reaction components are removed prior to use in downstream applications. Cleanup is vital for genomic applications such as next-generation sequencing (NGS) and PCR. Cleanup is made easier through use of solid phase reversible immobilization (SPRI) magnetic bead technology which is widely used throughout NGS library preparation workflows.

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Size selection is a critical step in the next-generation sequencing library preparation workflow for those who are looking to enhance the quality, efficiency, and accuracy of the sequencing process. Size selection involves selecting specific DNA fragments of a desired size range from a pool of prepared DNA fragments before proceeding to the sequencing step.

Solid Phase Reversible Immobilization (SPRI)

Solid Phase Reversible Immobilization (SPRI) is an effective method of purifying nucleic acids from a solution using silica- or carboxyl-coated paramagnetic beads. The beads are engineered to reversibly bind to nucleic acids in the presence of polyethylene glycol and salt. SPRI technology selectively binds nucleic acids by type and size to capture targets and eliminate contaminants within a sample. [1]

Nucleic acid immobilization is achieved when the size selective SPRI beads bind to the target nucleic acids, which are then fixed in place using a magnetic field.

Contaminant removal and wash steps are performed once the select DNA is bound to your paramagnetic beads. A strong magnet is used for particle capture, such as a DynaMag 96 side magnet. Contaminants such as dNTPs, salts, primers, and primer dimers can then be washed thoroughly. Once steps are complete, the sample is removed from the magnetic field, and the purified DNA can be captured.

Nucleic acid elution happens when the beads are removed from the magnetic field and resuspended in water or an elution buffer. The nucleic acids are released from the beads and the mixture is again placed near a magnetic field which separates the beads from the solution.

• Equivalent performance to the gold standard beads for NGS size selection and cleanup

MagMAX Pure Bind is a plug and play replacement for the market leading cleanup beads. It can be seamlessly integrated into existing workflows.

Figure 1. As compared to the leading competitor, MagMAX Pure Bind enables successful PCR cleanup with high recovery of amplicons >90bp (PCR products) and efficient removal of fragments <90bp (primers and primer-dimers). Input of 500 ng of fragmented DNA at a 1.8x bead ratio was used, and post-clean up recovery and removal efficiency was calculated as a percentage of pre-cleanup input amount.
 

• Optimal performance at a fraction of the cost

Achieve up to a 40% cost savings as compared to market leaders without compromising on quality.
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• Compatible with automated workflows

Pair with KingFisher Automated Purification Systems for an efficient, high throughput 30-minute cleanup protocol.
 

Choose your bottle size for NGS clean up depending on throughput needs

MagMAX Pure Bind is available in three bottle sizes to meet throughput needs. Reduce waste with low volume samples with the 5 mL or 50 mL bottle size. Scale up your sample processing with the 250 mL bottle size.

Steps for a complete next generation sequencing workflow

1. Start with a collected sample to begin your NGS workflow
2. Extract nucleic acids manually or with the KingFisher automated purification system
3. Select targets
4. Construct library
5. Fragment size selection and cleanup with MagMAX Pure Bind
6. Prepare template
7. Run sequence
8. Analyze data

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1. DeAngelis MM, Wang DG, Hawkins TL. Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 1995 Nov 25;23(22):4742-3. doi: 10.1093/nar/23.22.4742. PMID: 8524672; PMCID: PMC307455.