ADDRESS FOR CORRESPONDENCE: Stuart Levine, Massachusetts Institute of Technology, 77 Massachusetts Ave. 68-304D, Cambridge, MA 02139; Phone:617-452-2949; E-mail: [email protected]

Disclosure: The authors declare no conflicts of interest.

Summary

Solid-phase reversible immobilization (SPRI) remains a cornerstone of nucleic acid purification and is a key reagent for many protocols. A key aspect of an SPRI-based purification protocol is the ability to select DNAs of certain sizes by changing the properties of the binding buffer. Modification of the standard buffers used in SPRI purification is able to shift the minimum binding length required for SPRI beads to function, allowing them to specifically select DNA of high molecular weight that are required for long read sequencing. A challenge with the modified buffers has been a poorer than expected yield. We have identified that bead saturation is the likely cause of the low yield, and increasing bead fraction improves recovery, particularly for high-molecular-weight fragments.

Results

We have recently shown[1] that adjusting the choice of cations and the cation concentration of the buffers used in SPRI bead–based isolation can increase the length of the size cutoff for binding of DNA to the SPRI beads, allowing size selection of up to 10 kbp. We have observed, however, while sizing is consistent, larger inputs required for long read sequencers may have lower yields, with more than 50% of DNA longer than the cutoff length sometimes remaining in the supernatant.

To address the loss of yield, we hypothesized that SPRI beads may be limiting in buffers with modified cations, unlike typical SPRI reactions in which the beads are always in excess. We tested this under 2 conditions: the addition of 0.1× volumetric of 500 mM MgCl₂ 5%(v/v) polyethylene glycol 8000 (MgPEG, used for 10 kbp+ sizing) and the addition of 0.52× volumetric of 750 mM NaCl, 20% (v/v) polyethylene glycol 8000 (NaPEG, used for 7 kbp+ sizing). In both buffer conditions, we observed that increasing the volume of beads added (while maintaining buffer ratios) increased the amount of DNA recovered off the beads (Figure 1A). Conversely, increasing DNA input to these reactions resulted in a decrease of relative yield with MgPEG, consistent with binding site saturation (Figure 1B, orange). With NaPEG, however, relative yield remained steady with increasing DNA input, indicating dependence on input, in addition to size of SPRI beads pellet (Figure 1B, blue).

While increasing the mass of beads increased the yield of the reaction, the change in bead fraction could impact the efficacy of size selection. To address this, gDNA isolated by high-molecular-weight–selected SPRI and supernatants were analyzed by pulse-field gel-electrophoresis. Using MgPEG, the increased recovery of high-molecular-weight material was accompanied by a modest but noticeable shift to also bind lower molecular weight material, shifting the cutoff threshold from 10 kbp to 6 kbp (Figure 1C and Figure 1E). No reproducible shift was observed with NaPEG (Figure 1D and Figure 1E). Thus, the increase in yield did result in a less stringent sizing, and the experimentalist should carefully consider the trade-offs between yield and sizing in selecting a bead to DNA ratio. Using the larger amount of SPRI beads, consistent recover of ~95% of material is possible (Figure 1F).

Based on our data for high-molecular-weight DNA size selection, SPRI beads are unable to bind several micrograms of DNA per 1 μL of bead suspension using modified buffers (Table 1 and Table 2). This is distinct from applications binding shorter molecules (i.e., [2]). In the current study, we observed the SPRI beads saturate at 6 to 7 ng of high-molecular-weight DNA/μL of beads in the case of MgPEG selection and 7 to 15 ng of high-molecular-weight DNA/μL of beads/μg of input DNA when using NaPEG selection. The results allow for composing a general guide of how to adjust SPRI volume, depending on DNA input, for optimum yield/quality when selecting for >10 kbp and >7 kbp.

Materials

PCRCleanTM-DX beads (C-1003-250; Aline Biosciences), Agencourt AMPureXP beads (A63881; Beckman Coulter), PEG 8000 (HR2-535; Hampton Research), NaCl (AM9759; Thermo Fisher Scientific), CaCl₂ (C5670-100G; MilliporeSigma), nuclease-free water (02-0201-0500; Avantor), TrisHCl (E199; VWR Life Science), 96-well plates (AB-0900 Thermo Fisher Scientific), and pipette tips (17014963 and 17014961, Mettler-Toledo, and 76322-136 and 732-3632, VWR Life Sciences) were used. NaPEG is: 20% (v/v) PEG 8000 and 750 mM NaCl, MgPEG is: 5% (v/v) PEG 8000, 500 mM MgCl2.

Methods

High-molecular-weight DNA for SPRI beads testing was prepared from E. coli MG165s (~10¹¹ bacteria cells), following the procedure described in reference [3], with the following two modifications: (1) after incubation with CTAB/NaCl solution (step 3 of the protocol), E. coli suspension was freeze-thawed 6 times (to ensure gDNA degradation), and (2) instead of CsCl purification (step 16 of the protocol), crude gDNA was cleaned up with 0.6× regular SPRI beads, SPRI pellet washed in 2 × 1 mL 70% ethanol, and eluted in TE (volume equal to initial DNA solution) overnight at room temperature. DNA prepared this way had size distribution with 40% to 50% > 10 kbp and 60% to 70% > 7 kbp.

Required volumes of SPRI beads (Aline or AmpureXP) were conditioned by exchange into nuclease-free water using 2 rounds of magnetic binding and washing (200 μL).

gDNA was conditioned by dilution to 105 μL in 10 mM Tris-HCl pH 8.0, and 100 μL was added to specified volumes of exchanged beads and thoroughly resuspend by pipetting through a large bore tip and briefly spun down. Specified binding buffers were then added to the indicated ratios and mixed by carefully pipetting through a wide bore tip ~12 times and briefly spun down. Reactions were incubated at room temperature for 10’ without shaking or mixing followed by collection on a magnetic stand and both beads and supernatant collected. Beads were washed twice with fresh 70% EtOH and eluted with 10 to 50 μL of 10 mM Tris-HCl pH 8.0.

Supernatant DNA was recovered by mixing with 50 to 100 μL of standard SPRI beads and buffer followed by elution using standard protocols.[1]

Acknowledgments

The authors are thankful for members of the MIT BioMicro Center for their constructive comments and discussions on this work. This work was funded by the National Cancer Institute of the US NIH under award P30-CA14051, by the National Institute of Environmental Health Sciences of the US NIH under award P30- ES002109, and by National Institute of Environmental Health Sciences Superfund Basic Research Program, NIH, P42 ES027707.