How SPRI beads (SPRIselect, AMPure, etc.) work for nucleic acid clean-up & size selection

SPRI (Solid-Phase Reversible Immobilization) beads are used frequently in the preparation of DNA or RNA sequencing libraries. The protocols for library prep involve adding adapters, doing reverse transcription and/or PCR, etc. - steps that leave you with enzymes, primers, adapters, and other things you want to remove. So you need to do a number of purifications along the way to a library that’s ready to sequence. Thankfully, SPRI beads (SPRIselect, AMPure, RNAClean XP, etc.) make this easy(ish).
blog: bit.ly/SPRIbeads
The basic idea is:
take fragmented DNA (or RNA)
add paramagnetic beads (they’ll only act like magnets if you place them on a magnet) in a solution of PEG & NaCI
under those conditions, the DNA/RNA precipitates & binds beads
by modifying bead solution:sample ratio you can selectively bind sizes of interest
use magnet to capture the beads & pipet out other stuff
wash with ethanol while they're bound
add water or aqueous buffer to re-dissolve & elute
How’s it work?
PEG & salt hog water, leaving less for DNA or RNA
PEG causes "crowding" that promotes the DNA or RNA sticking together
salt ions shield charges to prevent the strands repelling one another or the beads
the beads are coated with negatively-charged carboxyl groups that under "normal" conditions will repel DNA or RNA, helping it unstick
The higher the PEG+salt vs. nucleic acid sample solution ratio, the smaller the DNA/RNA will precipitate (& bind beads). Only big stuff binds at a low ratio, but smaller binds too at higher ratios.
You can do a low ratio to remove large (toss beads), followed by a high ratio one on the supernatant to select mid-size ones. This is referred to as a double-sided size selection.
It works because it's harder to precipitate smaller DNA or RNA fragments than bigger ones. So, under milder conditions, you can selectively precipitate & remove big ones.
First get rid of big fragments
start with a low bead (and thus PEG & NaCI) to DNA/RNA solution ratio at which only big DNA pieces bind.
use a magnet to isolate & discard big-DNA/RNA-bound beads
Then wash off small fragments
add supernatant to more beads/PEG/NaCI
here, smaller pieces (but not tiny ones) bind too
wash while bound
Leaving medium-sized ones
add buffer to elute
Sorry I don’t have much text (at least for now)! But I do have resources!
Setting the stage…. You can size-selectively precipitate DNA with PEG/NaCl!
Lis, J. T., & Schleif, R. (1975). Size fractionation of double-stranded DNA by precipitation with polyethylene glycol. Nucleic acids research, 2(3), 383-389. doi.org/10.109...
You can use beads!
DeAngelis, M. M., Wang, D. G., & Hawkins, T. L. (1995). Solid-phase reversible immobilization for the isolation of PCR products. Nucleic acids research, 23(22), 4742-4743. doi.org/10.109...
You can go bigger!
Stortchevoi, A., Kamelamela, N., & Levine, S. S. (2020). SPRI Beads-based Size Selection in the Range of 2-10kb. Journal of biomolecular techniques : JBT, 31(1), 7-10. doi.org/10.717...
The SPRIselect User Guide from Beckman Coulter has good figures, etc. research.fredh...
Here’s a guide to making your own bead mixtures to save money -
Solutions for purifying nucleic acids by solid-phase reversible immobilization (SPRI); Philippe Jolivet and Joseph W. Foley; Ludmer Centre for Neuroinformatics and Mental Health, October 21, 2015 - Based on DeAngelis MM, Wang DG, Hawkins TL, “Solid-phase reversible immobilization for the isolation of PCR products.” Nucleic Acids Res 1995, 23:4742-4743. s3-us-west-2.a...
and even if you go the pre-made route, this guide is still helpful at showing you what’s in it! Their recipes for DNA and RNA are:
DNA mix: 10 mM Tris base, 1 mM EDTA, 2.5 M NaCl, 20% PEG 8000, 0.05% Tween 20, pH 8.0 @ 25 °C
* RNA mix: 1 mM trisodium citrate, 2.5 M NaCl, 20% PEG 8000, 0.05% Tween 20, pH 6.4 @ 25 °C
And they use it with Sera-Mag™ Magnetic SpeedBeads™, carboxylated, 1 μm, 3 EDAC/PA5 (GE Healthcare Life Sciences #65152105050250)
This review talks about a number of considerations and aspects of sequencing library preparation: Quail, M. A., Swerdlow, H., & Turner, D. J. (2009). Improved protocols for the illumina genome analyzer sequencing system. Current protocols in human genetics, Chapter 18, 10.1002/0471142905.hg1802s62. doi.org/10.100...
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