Fractionation of nucleic acids into single-stranded and double-stranded forms

We describe a rapid and efficient procedure for the fractionation of mixtures of nucleic acids (NA) into double-stranded (ds) and single-stranded (ss) forms regardless of the nature of the nucleic acid (DNA or RNA). The procedure is based on the differential binding of dsand ss-NA forms to silica particles in different lysis/binding buffers which have in common that they contain a high concentration of the chaotropic agent guanidinium thiocyanate (GuSCN). Previously we reported on a procedure (protocol Y) for the routine purification of total NA from clinical specimens (1). The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent GuSCN together with the NA-binding properties of silica particles (or diatoms) in the presence of this agent. Comparison of different GuSCN-containing lysis/binding buffers with respect to the binding of different NA-types to silica particles revealed that only ds-forms were bound when using lysis/binding buffer L11 (see below) whereas both dsand ss-forms were bound in lysis/binding buffer L6 (1). This observation formed the basis for the development of a procedure (protocol R) for the fractionation of mixtures of ssand ds-NA. The procedure is summarised in Figure 1. A 50 μl specimen (containing a mixture of NA-types in TE buffer) was added to a mixture of 900 μl lysis/binding buffer L11 and 40 μl size-fractionated silica particles (SC) in an Eppendorf tube and subsequently homogenized by vortexing. After a 10 min binding step at room temperature, the tube was centrifuged (2 min at ∼12 000 g) which resulted in a silica/ds-NA pellet (‘initial silica pellet’) and a supernatant containing ss-NA. To recover ss-NA forms (protocol R-sup) 900 μl of the supernatant were added to a mixture of 400 μl binding buffer L10 (see below) and 40 μl SC. Thereafter the ss-NA was bound during a 10 min binding step at room temperature. The tube was subsequently centrifuged (15 s at ∼12 000 g), and the supernatant discarded (by suction). The resulting pellet was subsequently washed twice with 1 ml of washing buffer L2 (1), twice with 1 ml ethanol 70% (vol/vol) and once with 1 ml acetone. The silica pellet was dried (10 min at 56 C with open lid in an Eppendorf heating block) and eluted (10 min at 56 C; closed lid) in 50 μl TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). After centrifugation (2 min at ∼12 000 g) the supernatant contained the ss-NA fraction. To recover ds-NA forms (protocol R-pellet) from the initial silica-pellet, the remaining supernatant was discarded, and the silica pellet was washed twice with 1 ml lysis/binding buffer L11 to remove unbound ss-NA. The resulting silica pellet Figure 1. Outline of protocol R. Recovery of ds-NA takes place from the initial pellet (R-pellet), recovery of ss-NA takes place from the initial supernatant (R-sup). L11, L10, L6 and L2 are GuSCN containing buffers; SC is silica particle suspension. For details see text.