Isolation of nucleic acids
Methods of isolating nucleic acids comprising DNA from biological material are disclosed. The methods comprise a lysis step using an aqueous composition which comprises lithium at a concentration of 0.05 to 1.0M, a chelating agent, and a surfactant to produce a lysed composition. The lysed composition is treated with a solid support that is capable of immobilising DNA in the presence of a dissolved chaotropic agent at a concentration of 0.05 to 2M and 25% to 60% by volume of a C 1-3 alkanol. The support is then washed with a first wash solution containing lithium dissolved in a C 1-3 alkanol. Subsequently, the support is washed with a liquid comprising at least 80% by volume of a C 1-3 alkanol. The nucleic acid comprising DNA is then eluted from the support.
1. A method of isolating nucleic acids comprising DNA from biological material, the method comprising the steps of:
(i) effecting a lysing of the biological material with an aqueous solution which comprises lithium at a concentration of 0.6 to 0.8 M, a chelating agent, and a surfactant to produce an aqueous lysed composition,
(ii) treating the lysed composition with a solid support in the presence of a dissolved chaotropic agent at a concentration of 0.05 to 2 M and 25% to 60% by volume in the liquid of a dissolved C 1-3 alkanol, said solid support being one capable of immobilising DNA,
(iii) separating the solid support from liquid, wherein said separation is effected in a spin-tub in which said solid support is a filter,
(iv) treating the solid support with a first wash solution containing lithium at a concentration of 0.05 to 1 M, said first wash solution containing 20% to 90% by volume of a dissolved C 1-3 alkanol,
(v) separating the solid support from the first wash liquid,
(vi) treating the solid support with a second wash liquid which comprises at least 80% by volume of a C 1-3 alkanol, the balance if any being water,
(vii) separating the solid support from the second wash solution, and
(viii) eluting DNA from the solid support, wherein steps (iii), (v), (vii) and (viii) are effected with centrifugation.
2. A method as claimed in claim 1 wherein the lysis solution consists essentially of water, lithium chloride at a concentration of 0.7 to 0.8 M, EDTA or a sodium salt thereof at a concentration of 5 to 15 mM and 0.9 to 1.1% by weight SDS.
3. A method as claimed in claim 2 wherein in step (ii) the chaotropic agent is guanidinium chloride at a concentration of 0.6 to 0.7 M and the alkanol is ethanol present in an amount of 35-40% by volume of the liquid.
4. A method as claimed in claim 1 wherein the first wash liquid consists essentially of water, lithium chloride at a concentration of 0.3 to 0.6 M, and 45% to 55% by volume ethanol.
5. A method as claimed in claim 1 wherein the first wash liquid consists essentially of water, lithium chloride at a concentration of 0.6 to 0.8 M, and 45% to 55% by volume ethanol.
6. A method as claimed in claim 1 wherein the second wash liquid consists essentially of 80% to 95% by volume of ethanol and water.
7. A method as claimed in claim 1 wherein the solid support comprises silica.
8. A method as claimed in claim 1 wherein the filter comprises borosilicate glass fibres.
9. A method as claimed in claim 8 wherein a plurality of said filters are provided in the spin tube.
10. A method as claimed in claim 1 wherein the biological material from which the nucleic acids comprising DNA is to be isolated comprises cells.
11. A method as claimed in claim 10 wherein the cells are in the form of a pellet.
12. A kit for extracting nucleic acids comprising DNA from cells, the kit comprising in combination:
(i) an aqueous solution which comprises, consists essentially of, or consists of water, lithium chloride at a concentration of 0.6 to 0.8 moles per litre, EDTA or a sodium salt thereof at a concentration of 5 to 15 mM, and 0.9 to 1.1% by weight SDS,
(ii) an aqueous solution of a guanidinium salt,
(iii) ethanol,
(iv) a first wash liquid containing lithium chloride in a concentration of 0.3 to 0.6 M, said first liquid containing ethanol in an amount of 45% to 55% by volume,
(v) a second wash liquid which comprises at least 80% by volume ethanol, the balance if any being water,
(vi) an elution buffer, and
(vii) a spin column having a filter for immobilising DNA.
13. A kit as claimed in claim 12 wherein the filter comprises at least one sheet of borosilicate fibres.
14. A kit as claimed in claim 13 wherein the filter comprises four sheets of borosilicate fibres.
15. A lysis composition which comprises water, lithium chloride at a concentration of 0.7 to 0.8 M, EDTA or a sodium salt thereof at a concentration of 5 to 15 mM and 0.9 to 1.1% by weight SDS.
16. A method of lysing biological material comprising treating the biological material with an aqueous lysis composition as claimed in claim 15 .
17. A method as claimed in claim 16 wherein the biological material comprises cells.