Basic strategies and application for DNA fragment sorting

The principle of magnetic bead purification of nucleic acids is based on Solid-Phase Reversible Immobilization technology（SPRI）, of which the mechanism of action is not yet very clear. Salt bridge theory is currently a more scientific explanation, that is, in higher concentrations of alcohol and salt solution seize the water of the hydration layer outside the DNA molecules, resulting in aggregation of DNA molecules precipitation, so that the negatively charged phosphate groups are exposed. The salt ions form a “salt bridge”, also called an “electrical bridge”, with the groups on the surface of the beads, causing the DNA to be adsorbed onto the surface of the beads.

Isopropanol and ethanol precipitation is too mightiness, so fragment sorting is generally not used, but nonionic water-soluble polymers PEG, that is, polyethylene glycol as a precipitant, and because of the viscous PEG so that the magnetic beads in the buffer to obtain a good suspension. In the process of DNA binding, the magnetic beads can be more fully contacted with DNA, and PEG is not easy to cause protein denaturation and non-specific adsorption. しかし, the DNA deposition effect of PEG is easily affected by pH and temperature, and PEG is not easily soluble in water when the temperature is too low, so the magnetic beads are generally required to be equilibrated at room temperature before use, and some low pH stabilizers, such as Tris-HCl, 等, will be added to the magnetic bead buffer.

The longer the DNA, the more negatively charged phosphate groups are exposed on the surface, the stronger the negative charge of the whole molecule is, the easier it is to be adsorbed to the magnetic beads, and only a lower concentration of PEG and NaCl is needed to recover it; the shorter the DNA is, the higher the concentration of PEG and NaCl is needed to destroy the hydration layer on the surface more thoroughly, and to expose enough negatively charged phosphate groups to be adsorbed by the magnetic beads, and thus recovered. and thus recovered back. So if you want to recover shorter DNA fragments, you need to add a larger volume of magnetic beads (shown in Figure 1).

したがって, the general procedure of fragment sorting is to adsorb large fragments with dilute bead buffer in the first round, and then add bead buffer to adsorb the specified fragments in the second round after taking supernatant, to get the desired fragmented DNA. Currently, most of the fragment sorting schemes in the market use carboxylated beads in combination with different concentrations of salt ions and percentages of PEG to recover fragments of different lengths, but of course, there is another scheme, which is Apostle’s method of first adsorbing large fragments with carboxylated magnetic beads (Substrate A shown in Fig. 2) at a certain concentration of salt ions and PEG, and then adsorbing small fragments with silica-hydroxylated magnetic beads (Substrate B shown in Fig. 2) at a certain concentration of salt ions and PEG ^[2].

The magnetic bead method for fragment sorting is an efficient, rapid, and highly specific technique, and fragment sorting is one of the key steps in the NGS library preparation process. Through fragment sorting, it can ensure that the size of DNA fragments in the library meets the requirements of the sequencing platform, thus improving the quality of sequencing data and the accuracy of analysis. In the field of molecular diagnostics, fragment-sorting technology is used to improve the sensitivity and specificity of detection. Fragment sorting magnetic beads can also be used for the purification of PCR products for downstream experiments.

参考文献:

[1] LIU D, LI Q, LUO J, 等. An SPRI beads-based DNA purification strategy for flexibility and cost-effectiveness[J/OL]. BMC Genomics, 2023, 24(1): 125. DOI:10.1186/s12864-023-09211-w.

[2] ZHANG B, ZHAO S, WAN H, 等. High-resolution DNA size enrichment using a magnetic nano-platform and application in non-invasive prenatal testing[J/OL]. The Analyst, 2020, 145(17): 5733-5739. DOI:10.1039/D0AN00813C.

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