Fractionation of nucleic acids into single-stranded and double-stranded forms
Fractionation of nucleic acids into single-stranded and double-stranded forms
Marcel
Beld
,
Cees
Sol
,
Jaap
Goudsmit
and
René
Boom
*
Department of Human Retrovirology, Academic Medical Center, University of
Amsterdam, Meibergdreef 15,
1105 AZ
Amsterdam
,
The Netherlands
Received March 11, 1996;
Revised and Accepted May 15, 1996
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 ds- and 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 ds- and 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 ss- and ds-NA. The procedure is summarised in Figure
1
. A 50 [mu]l specimen (containing a mixture of NA-types in TE buffer) was added to a mixture of 900 [mu]l lysis/binding buffer L11 and 40 [mu]l 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 [mu]l of the supernatant were added to a mixture of 400 [mu]l binding buffer L10 (see below) and 40 [mu]l 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 56oC with open lid in an Eppendorf heating block) and eluted (10 min at 56oC; closed lid) in 50 [mu]l 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 was subsequently washed twice with washing
buffer L2, twice with ethanol 70%, once with acetone, dried and eluted as described for protocol
R-sup. After centrifugation (2 min at ~12 000
g
) the supernatant contained the ds-NA fraction.
REFERENCES
1 Boom,R., Sol,C.J.A., Salimans,M.M.M., Wertheim van Dillen,P.M.E. and van der Noordaa,J. (1990) J. Clin. Microbiol., 28,495-503.
2 Holmes,I.H. (1988) In Lenette,E.H, Halonen,P. and Murphy,F.A., Laboratory Diagnosis of Infectious Diseases, vol. II. Springer Verlag, New York Inc., pp. 384-413.
3 Aaij,C. and Borst,P. (1972) Biochim. Biophys. Acta., 269,192-200.
