- Ideal for extracting DNA from human plasma for cell-free DNA-Seq
- Generates highly concentrated libraries using few PCR cycles
- Automation friendly, mag-bead based format
- Fast, 30 minute protocol with minimal hands-on time
- Ideal for NGS library prep
- No vacuum pumps, vacuum manifolds, or column extenders required
The NextPrep-Mag™ cfDNA Isolation Kit is designed for extracting cell-free DNA (cfDNA) for constructing NGS libraries from human blood plasma or serum, using a fast magnetic bead-based format requiring minimal hands-on time. The isolated cell free DNA is ideal for next generation sequencing and PCR analysis. Suitability of the DNA for use in NGS library construction has been verified using the Bioo Scientific NEXTflex™ Cell Free DNA-Seq Library Prep Kit for Illumina® Sequencing and the NEXTflex Cell Free DNA Library Prep Kit for Ion Torrent Sequencing. This kit is optimized for isolating circulating tumor and cell free circulating fetal DNA from blood plasma.
Cell Free DNA Isolation Protocol
This cell free DNA isolation procedure, which can be completed in approximately 30 minutes, includes an initial lysis and DNA binding step, wash steps, and elution of cfDNA from the magnetic beads. A distinguishing feature of the kit is the very rapid magnetic bead attraction steps.
Cell Free DNA Isolation Challenges
Extraction of DNA from plasma is challenging because the DNA is highly fragmented and present at very low concentration, especially in healthy individuals. Because the expected yields are low, the DNA recovered cannot be reliably detected using standard methods such as NanoDrop spectrophotometer, gel or capillary electrophoresis, or DNA binding dyes.
NextPrep-Mag cfDNA Isolation Kit Outperforms the QIAamp® Circulating Nucleic Acid Kit
The NextPrep-Mag cfDNA Isolation Kit offers a number of advantages over the QIAamp® Circulating Nucleic Acid Kit, including speed of cfDNA isolation and ease of automation, while delivering comparable cfDNA yields and higher concentration NGS libraries.
Bioanalyzer QC Traces for Libraries
DNA libraries constructed with the NEXTflex™ Cell Free DNA-Seq Kit, from cfDNA isolated from plasma of two healthy donors, using either the NextPrep-Mag cfDNA Isolation Kit or the QIAamp Circulating Nucleic Acid Kit. Libraries were amplified for 9 PCR cycles. Input of DNA for each library was 32 μL, representing 52% of the cfDNA extracted from 5 mL of plasma.
The NextPrep-Mag cfDNA Isolation Kit For < 1 mL - 3 mL Plasma Samples contains sufficient reagents for extraction of DNA from 50 samples of 1 mL plasma or less, or 16 samples of 3 mL plasma. The NextPrep-Mag cfDNA Isolation Kit For 3 mL - 5 mL Plasma Samples contains sufficient reagents for extraction of DNA from 50 samples of plasma ranging from 3 mL to 5mL. The kit components are stable for at least 12 months from time of receipt, when properly stored.
cfDNA DNA Proteinase K
cfDNA Binding Solution
cfDNA Magnetic beads
cfDNA Wash Solution 1
cfDNA Wash Solution 2
cfDNA Elution Solution
Required Materials Not Provided
Heat block at 55°C
Microcentrifuge or “picofuge”
2 mL microfuge tubes, nuclease-free. Smaller tubes may be used for the elution step.
Pipettors (P-1000, P-200, P-20 or equivalent) and tips
5 mL disposable pipets
Magnetic stand to hold 2 mL and 15 mL tubes
What is the size distribution of cell free DNA recovered from human plasma?
The cell free DNA isolated from plasma of healthy donors, typically runs as a single small “hump” of ~ 150 bp when analyzed directly on an Agilent 2100 high sensitivity DNA chip. When the cell free DNA is converted into a library using the ligation-based format in the NEXTflex Cell Free DNA-Seq Library Prep Kit, a greater range of sizes of library products is observed, from ~ 300 bp – 5,000 bp. The 5’ and 3’ adapters used to make the library add a total of ~150 bp. This indicates that cell free DNA fragments present at concentrations too low to be visualized directly are detectable after the amplification step used to create cell free DNA libraries.
Do we need special equipment to process large volumes of plasma?
No. The only equipment required is a magnetic stand and a low-speed “picofuge”.
Is it necessary to add carrier nucleic acid or glycogen when processing plasma to recover cell free DNA?
No. Addition of nucleic acid carrier compromises quantification and use of the cell free DNA as input for NGS libraries.
How many PCR cycles are needed to make cell free DNA libraries?
We typically use 9 – 12 PCR cycles for making cell free DNA libraries.