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Improved ccfDNA Enrichment through Utilization of the ProNex® Size-Selective...

Improved ccfDNA Enrichment through Utilization of the ProNex® Size-Selective Purification System
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Abstract

This article describes a magnetic bead-based size selection method that can be used to enrich for the ccfDNA component of a sample, thereby eliminating larger material and effectively decreasing background without affecting levels of the 170bp fragment.

 

Adam Blatter, Samantha Lewis, Charles Cowles and Curtis Knox

 

Promega Corporation
Publication Date: April 2018, tpub_198

Introduction

Circulating cell-free DNA (ccfDNA) is one of the most promising sources of DNA-based information for numerous translational and clinical research applications, such as oncology. Traditionally, obtaining samples for testing has included biopsy or other invasive procedures to physically excise tissue for examination; however, this process is imprecise and can lead to unintended complications. In tumor screening, for example, the heterogeneity of mutations in the clonal evolution of cancer often leads to diverse microenvironments within a tissue that needle biopsies cannot fully capture. Furthermore, the physically invasive nature of tissue biopsy restricts the number of samples that can be collected in both space and time, severely limiting research scope.

Ongoing research into ccfDNA is giving rise to the possibility of “liquid biopsies” that would eliminate the need for invasive tissue collection, potentially providing a more global picture for tumor identification and classification, in real-time. Using easily obtainable blood samples exclusively, scientists now have a powerful tool in liquid biopsies to view their research in an entirely new way.  The detection of clean, enriched ccfDNA is enabling significant breakthroughs in this area.

ccfDNA is DNA that has been released from cells by secretion, necrosis or apoptosis and has been detected in most biological fluids including plasma, urine, cerebrospinal fluid, amniotic fluid, pancreatic juice, saliva, sweat and tears, among others. About 95% of the ccfDNA in circulation has an approximate size of 170bp, representing the molecular size that wraps around a nucleosome, while a lesser component may include dimers (340bp) and trimers (510bp).

The use of ccfDNA as a sample type is not without its challenges. The low concentration and highly fragmented nature of ccfDNA, coupled with the low frequency of biomarkers of interest, present many challenges to the adoption of ccfDNA monitoring. Variations inherent to the sample type, as well as sample handling, can affect the amount of contaminating high molecular weight gDNA. Presence of high levels of contaminating gDNA may result in difficulty detecting low-level mutations present in the minor component ccfDNA.  Magnetic bead-based size selection methods, such as the ProNex® Size-Selective Purification System, can be used to enrich for the ccfDNA component of a sample, thereby eliminating larger material and effectively decreasing background without affecting levels of the 170bp fragment.

Materials and Methods

  • ProNex® Size-Selective Purification System (Cat.# NG2001)
  • Magnetic Tube Stand, i.e. MagneSphere® Technology Magnetic Separation Stand (Cat.# Z5332)
  • 95% Ethanol for Wash Buffer Preparation
  • (optional) LoBind tubes may be used if desired during the size selection protocol and during storage, i.e. Eppendorf® DNA LoBind Tubes (Cat.# 022431021)

ccfDNA Cleanup Protocol

  1. Prepare wash buffer as stated in Section 5 of the ProNex® Size-Selective Purification System Technical Manual #TM508.
  2. Allow the ProNex® Chemistry to equilibrate at room temperature for 30 minutes to 1 hour.
  3. Pipet 50µl of isolated ccfDNA into a tube or well capable of holding up to 250µl.
    Note: For starting volumes other than 50µl of isolated ccfDNA, adjust the volumes of ProNex® Chemistry accordingly to maintain the addition ratios stated in steps 5 and 10.
  4. Ensure that the ProNex® Chemistry bottle cap is tightened securely. Resuspend the resin by vigorous vortexing for 10 seconds.
  5. Mix 55µl of ProNex® Chemistry into the sample (i.e., a 1.1:1 (v:v) ratio of ProNex® Chemistry to 50µl starting sample volume) by pipetting 10 times.
  6. Incubate the sample at room temperature for 10 minutes.
  7. Place the sample on a magnetic stand for 2 minutes.
  8. Carefully transfer the supernatant to a clean tube or well.
    Caution: Do not discard the supernatant. The desired DNA fragments are in the supernatant at this point and the undesired, high molecular weight DNA fragments will be left bound to the resin.
  9. Remove the sample from the magnetic stand.
  10. Mix an additional 95µl of ProNex® Chemistry into the supernatant sample from step 7 (i.e. a 1.9:1 (v:v) ratio of ProNex® Chemistry to 50µl starting sample volume, which results in a final 3:1 (v:v) ratio) by pipetting 10 times.
  11. Incubate the sample at room temperature for 10 minutes.
  12. Place the sample on a magnetic stand for 2 minutes.
  13. Carefully remove and discard the supernatant. The undesired, low molecular weight DNA fragments are in the supernatant. The desired, ccfDNA fragments will be left bound to the resin.
  14. Leaving the sample on the magnetic stand, add 200μl of Wash Buffer to the sample and allow it to incubate for 30–60 seconds. Remove and discard the Wash Buffer. For larger samples, increase the volume of Wash Buffer proportionally to the total volume of sample and ProNex® Chemistry.
  15. Repeat step 15 (for a total of 2 washes).
  16. Allow sample to air-dry for 5 minutes.
    Notes:
    • If working with a multichannel pipette and buffer trough, return the unused Wash Buffer to the Wash Buffer bottle at this point. Tighten the bottle cap to prevent ethanol evaporation.
    • The resin may be allowed to air-dry for longer than 5 minutes. Depending on the sensitivity of downstream applications to ethanol, drying times up to 1 hour can be used. ProNex® Chemistry does not suffer from the loss of high molecular weight DNA upon extended drying.
  17. Remove the sample from the magnetic stand.
  18. Add 50μl of Elution Buffer (or your preferred volume of Elution Buffer, see note below) and resuspend the resin by pipetting and/or shaking on a plate mixer. Incubate the samples at room temperature for 5 minutes to elute the DNA.
    Note: Depending on the downstream application, you can add more or less Elution Buffer to elute the sample. Higher elution volumes do not result in significant yield increases. However, elution volumes <25% of the starting sample volume can be difficult to work with and may result in some yield loss due to the resin void volume.
  19. Return the sample to the magnetic stand for 1 minute, then carefully transfer the eluted DNA to a clean tube or well.
  20. Return the ProNex® Chemistry bottle to storage at 2–10°C.

Results

ccfDNA was isolated from fresh double-spun plasma using the Maxwell® RSC ccfDNA Plasma Kit and spiked with 10ng/µl of HCT116 genomic DNA. Following the DNA spike, half of the samples were processed using the ProNex® Size-Selective Purification System (size selected) while the other half were not (control).

DNA was then sized and visualized on a D5000 ScreenTape using the Agilent TapeStation. ccfDNA bands at 166bp and 332bp were unchanged whether size selection was performed or not; however, a significant reduction in the band above 10,000bp, where gDNA would be expected to run, was seen in selected samples when compared to controls (Figure 1).

Both size selected and control samples were further amplified in a hydrolysis probe-based differential qPCR assay (ProNex® DNA QC Assay) targeting three different fragment sizes: 75bp, 150bp and 300bp. The ratio of 75bp:300bp was used as a ccfDNA purity metric. A low ratio confirmed the presence of spiked gDNA in the control sample while a higher ratio was observed for the size selected sample, indicating effective removal of HMW gDNA (Figure 2).

15065ma-wa
15065ma-wb

Figure 1. Size distribution of 10ng/µl gDNA spiked ccfDNA with or without size selection. Samples were run on a D5000 ScreenTape using the Agilent TapeStation. False gel images are shown on the right.

15066ma-w

Figure 2. Ratio of 75bp to 300bp fragment amplification of gDNA spiked ccfDNA with or without size selection. The ratio of 75bp to 300bp fragment amplification was measured for each sample using the ProNex® DNA QC Assay. N=4 for each condition.

Conclusion

While ccfDNA presents great promise as a source of DNA-based information, the small fraction of circulating molecules of interest compared to gDNA from lysed white blood cells or variations in collection or sample handling can lead to limited sensitivity in downstream assays. We have shown that employing a size selection method after initial purification can improve the enrichment of ccfDNA, thereby increasing the likelihood of detecting relevant low abundance markers of interest in applications such as ddPCR or massively parallel sequencing.

How to Cite This Article

Blatter, A., Lewis, S., Cowles, C. and Knox, C. Improved ccfDNA Enrichment through Utilization of the ProNex® Size-Selective Purification System. [Internet] April 2018, tpub_198. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/tpub-198-improved-ccfdna-enrichment-through-utilization-of-pronex-size-selective-purification-system/

Blatter, A., Lewis, S., Cowles, C. and Knox, C. Improved ccfDNA Enrichment through Utilization of the ProNex® Size-Selective Purification System. Promega Corporation Web site. http://www.promega.com/resources/pubhub/tpub-198-improved-ccfdna-enrichment-through-utilization-of-pronex-size-selective-purification-system/ Updated April 2018, tpub_198. Accessed Month Day, Year.

Promega products referenced in this article are intended for research use only and not for use in diagnostic procedures.
ProNex and Maxwell are registered trademarks of Promega Corporation.
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