One question we’ve been asked, and one that our NGS providers are frequently asked, is how in principle does PEG precipitate DNA in next generation sequencing library preparation cleanup? We usually hear the question presented as: how do Agencourt’s Ampure XP or SPRIselect beads precipitate DNA? The answer has to do with the chemical properties of DNA, polyethylene glycol (PEG), the beads being used and water. Polystyrene – magnetite beads (Ampure) are coated with a layer of negatively charged carboxyl groups. DNA’s highly charged phosphate backbone makes it polar, allowing it to readily dissolve in water (also polar). When PEG [ H-(O-CH2-CH2)n-OH ] is added to a DNA solution in saturating condition, DNA forms large random coils. Adding this hydrophilic molecule with the right concentration of salt (Na+) causes DNA to aggregate and precipitate out of solution from lack of solvation (1, 2). Too much salt and you’ll have a lot of salty DNA, too little will result in poor recovery. The Na+ ions shield the negative phosphate backbones causing DNA to stick together and on anything else that’s in near vicinity (including carboxylated beads). Once you’re ready to elute your DNA and put it back into solution (after you’ve done your size selection or removal of enzymes, nucleotides, etc.) an aqueous solution is added back (TE or water) fully hydrating the DNA and moving it from an aggregated state back into solution. The negative charge of the carboxyl beads now repel DNA, allowing the user to extract it in the supernatant. Changing the amount of PEG and salt concentration can aid in size selecting DNA (2). This is a common method in NGS library preparation where the user is interested in size selecting a fragment of particular size. It’s often used to replace gel steps in NGS library prep. There is already a wealth of literature out there on conditions to size select DNA, just do a simple google search. The first article we’ve found that describes this selection is referenced below (3).
Since this publication on May 7th, 2014, there are several more commercial, Ampure-like size selection beads on the market:
- MagJet – ThermoFisher
- Mag-Bind – Omega Biotek
- Promega Beads – Promega
- Kapa Pure Beads – Kapa Biosystems
While we haven’t explored each one of these yet, we suspect the chemistry behind precipitation and selection is very similar. If you’d like to share information about these beads, please leave us a comment or send us an email at firstname.lastname@example.org
If you’d like help in constructing your NGS library contact us, and we’d be happy to consult with you on your sequencing project: https://genohub.com/ngs-consultation/
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(1) A Transition to a Compact Form of DNA in Polymer Solutions: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC389314/pdf/pnas00083-0227.pdf
(2) DNA Condensation by Multivalent Cations: https://www.biophysics.org/Portals/1/PDFs/Education/bloomfield.pdf
(3) Size fractionation of double -stranded DNA by precipitation with polyethylene glycol: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC342844/pdf/nar00500-0080.pdf