A Guide to Solid Phase Reversible Immobilization

Scientists today depend heavily on many molecular biology techniques to perform their research. For example, with the advent of next generation sequencing (NGS): scientists are able to look at very minute details, right down to individual genetic sequence variations. However, the increase in experimental complexity means that every extra step becomes more crucial than ever before. In comes solid phase reversible immobilization (SPRI), a technique that is critical in concentrating the amplified PCR product of interest of interest while getting rid of excess nucleotides and reagents. Skipping this step usually causes an increase in noise levels and difficult-to-interpret sequence data (see this webinar on NGS workflow). Therefore, a good SPRI protocol might just be what you need to have a high-resolution NGS sequence data to showcase to your peers

What are SPRI beads?

The SPRI beads technology was originally developed at MIT in 1995 (1). The beads consist of a polystyrene core surrounded by a thin layer of magnetite, which makes it paramagnetic (i.e., the beads will only clump together under a magnetic field). On the surface, the bead is coated by carboxyl molecules, which provide the charge groups for DNA binding. In the presence of polyethylene glycol (PEG) and salt, which work together as “crowding agents,” you can activate the beads to bind to DNA and the binding is reversible. A typical procedure involves the following five simple steps.

Step 1. Binding and mixing. This step is crucial because the ratio between the PEG and the DNA determines the size of the DNA that will bind to the beads.

Step 2. Separation. A powerful magnet pulls the beads down, which are bound to the DNA fragment you want to purify.

Step 3. Wash. Rinse with ethanol (70%)*

Step 4. Elution. The buffer separates the DNA from the beads, which are still bound to the magnet

Step 5. Transfer. Aliquot the DNA to a fresh tube for downstream sequencing work

*A modification called “with-bead” method (2) can also be performed. Instead of eluting the DNA after the wash step, PCR master mix can be directly added to the beads. You can then do all the necessary steps to generate a sequencing library, such as end trimming and addition of poly-A tails for adaptor ligation.

The “magic” ratio determines the size of DNA

Before doing the SPRI pull-down, you should have a clear idea about the size of the DNA fragments. The concentration of PEG is the key factor in determining the size of the DNA fragments that bind to the beads. Just remember, the higher the PEG to DNA ratio, the smaller the DNA fragments will bind to the beads.

SPRI on microfluidic chips

            Another interesting modification of the SPRI technology is the use of microfluidic chips for the purification of dye-labeled DNA sequencing fragments (3). Instead of using the beads format, an immobilization bed that contains UV-sensitive polycarbonate can be activated. The DNA fragments are loaded and bound to the activated surface in the presence of immobilization buffer. The advantage of this method is that it streamlines all the washing and eluting steps by using the microfluidic system, and it can be coupled to a capillary gel electrophoresis system for sequencing.

Examples on current use of the SPRI technology

1. Use of SPRI beads for isolating mitochondrial DNA (4).
2. Use of SPRI beads for extracting Mycobacterial DNA (5).
3. Use of salicylic acid coated magnetic nanoparticles (SAMNPs), a modified form of SPRI technology, to extract mammalian chromatins in its native forms (6).

Reference 

1. DeAngelis MM, Wang DG, Hawkins TL. (1995) Solid-Phase Reversible Immobilization for the Isolation of PCR Products. Nucleic Acids Research 23(22): 4742–43. 

2. Fisher S, Barry A, Abreu J, Minie B, Nolan J, Delorey TM, et al. (2011) A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Genome Biol. 12(1):R1. doi: 10.1186/gb-2011-12-1-r1.

3. Xu Y, Vaidya B, Patel AB, Ford SM, McCarley RL, Soper SA. (2003) Solid-phase reversible immobilization in microfluidic chips for the purification of dye-labeled DNA sequencing fragments. Anal Chem.  75(13):2975-84.
   doi:10.1021/ac030031n.

4. Quispe-Tintaya W, White RR, Popov VN, Vijg J, Maslov AY. Rapid Mitochondrial DNA Isolation Method for Direct Sequencing. In: Weissig V, Edeas M, editors. Methods in Molecular Biology New York: Springer; 2015. pp. 1264

5. Votintseva AA, Pankhurst LJ, Anson LW, Morgan MR, Gascoyne-Binzi D, Walker TM, et al. (2015)Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures. J Clin Microbiol. 53(4):1137-43. doi:
   10.1128/JCM.03073-14.

6. Zhou Z, Irudayaraj J. (2015) A Native Chromatin Extraction Method Based on Salicylic Acid Coated Magnetic Nanoparticles and Characterization of Chromatin. Analyst. 140(3): 938–44. doi:10.1039/C4AN01897D.