Methods for obtaining liquid from a solid phase

Abstract

A method for obtaining a liquid from a porous solid phase is described. The method comprises forming a liquid seal at a first end of a porous solid phase to which a liquid is bound, wherein liquid of the liquid seal is immiscible with the liquid bound to the solid phase, and applying a pressure differential across the porous solid phase to cause the immiscible liquid to move through the porous solid phase towards a second end of the porous solid phase, thereby displacing the liquid bound to the porous solid phase towards the second end and releasing this liquid from the second end. Recovery of liquid from the solid phase using such methods is increased compared with corresponding methods in which no liquid seal is formed. In preferred embodiments, the liquid used to form the liquid seal is a mineral oil. The methods have particular application in nucleic acid extractions which utilize capture of nucleic acid to a solid phase. Kits and apparatus for performing the methods are also described.

Claims

1. A method for obtaining a liquid from a porous solid phase, comprising: forming a liquid seal at a first end of a porous solid phase to which a liquid is releasably bound, wherein the liquid seal comprises a layer of an immiscible liquid that is immiscible with the liquid that is releasably bound to the solid phase; and applying a pressure differential across the porous solid phase to cause the layer of immiscible liquid to move through the porous solid phase towards a second end of the porous solid phase, thereby displacing the liquid that is releasably bound to the porous solid phase towards the second end and releasing said releasably bound liquid from the second end.

2. The method of claim 1, wherein the immiscible liquid is selected from (i) an immiscible liquid that is less dense than the liquid that is releasably bound to the solid phase, (ii) an immiscible liquid that comprises a hydrophobic liquid, and (iii) a hydrophobic liquid that is a mineral oil.

3. The method of claim 1, wherein the porous solid phase is within a column.

4. The method of claim 1, wherein the liquid that is releasably bound to the porous solid phase comprises a component of a biological sample, which it is desired to release the liquid that is releasably bound from the solid phase.

5. The method of claim 4, wherein the component comprises a nucleic acid.

6. The method of claim 5, wherein the porous solid phase comprises a material that is selected from (i) a material to which the nucleic acid binds at a lower pH and from which nucleic acid is eluted at a higher pH, (ii) an inorganic oxide, and (iii) silica.

7. The method of claim 4, wherein either or both of (i) the component comprises a protein, and (ii) the porous solid phase comprises an ion-exchange material for protein purification.

8. The method of claim 1, wherein the liquid that is releasably bound to the solid phase comprises an elution buffer.

9. The method of claim 1, wherein the pressure differential is applied for a sufficient time that at least some of the immiscible liquid is released from the second end of the porous solid phase.

10. The method of claim 9, wherein the released releasably bound liquid and the immiscible liquid that is released from the solid phase are collected in an uncapped collection tube.

11. The method of claim 1, which further comprises heating the porous solid phase.

12. A method for isolation of a biological component, comprising: (a) contacting (i) a liquid that comprises a biological component, with (ii) a porous solid phase, under conditions and for a time sufficient to releasably bind the biological component to the porous solid phase; (b) releasing the releasably bound biological component from the porous solid phase into a liquid that is releasably bound to the porous solid phase; (c) subsequently forming a liquid seal at a first end of the porous solid phase to which the liquid is releasably bound, wherein the liquid seal comprises a layer of an immiscible liquid that is immiscible with the liquid that is releasably bound to the solid phase; and (d) applying a pressure differential across the porous solid phase to cause the layer of immiscible liquid to move through the porous solid phase towards a second end of the porous solid phase, thereby displacing the liquid that is releasably bound to the porous solid phase towards the second end and releasing said releasably bound liquid from the second end, and thereby isolating the biological component.

13. The method of claim 1, which is an automated method.

Description

(1) Preferred embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

(2) FIG. 1 shows the volume of liquid remaining in a porous solid phase after different volumes of air were pumped through the solid phase using a syringe in the absence of a liquid seal;

(3) FIG. 2 shows the volume of liquid collected from a porous solid phase by pumping air through the solid phase with and without a liquid seal (a layer of mineral oil) at an upper end of the porous solid phase;

(4) FIG. 3 shows the strength of the assay signal obtained following amplification and detection of HIV-1 RNA eluted from a porous solid phase with and without a liquid seal (a layer of mineral oil) at an upper end of the porous solid phase;

(5) FIG. 4 shows photographs of the lower end of columns containing a silica-based solid phase through which liquid containing a red coloured dye has been passed (a) without, and (b) with a liquid seal (a layer of mineral oil) at an upper end of the solid phase;

(6) FIG. 5 shows photographs of the lower end of columns containing a silica-based solid phase with a buffer containing a red coloured dye bound to the solid phase. In figure (a) there is no layer of mineral oil over the buffer bound to the solid phase. In figure (b) there is a layer of mineral over the buffer bound to the solid phase. A semi-spherical meniscus can be seen at the interface between the mineral oil and the buffer;

(7) FIG. 6 shows a comparison of the results from detection of amplified HIV-1 RNA eluted from porous solid phases in a capped column with no mineral oil, an uncapped column with no mineral oil, and an uncapped column with a layer of mineral oil over the solid phase; and

(8) FIG. 7 shows the yield of nucleic acid eluted from a porous solid phase using a method of the invention compared with a conventional method in which liquid bound to the column was removed by centrifugation. The black horizontal bars show the average yield in each case.

EXAMPLE 1

Use of Mineral Oil Increases the Recovery of Liquid from a Solid Phase

(9) A silica-based solid phase was loaded with 0.5 ml lysis buffer (0.2M sodium citrate buffer pH 4.3, 0.3 M ammonium sulphate, 0.4% Triton-X 100), and then 30 ml, 100 ml, 200 ml, 300 ml, or 400 ml of air was passed through it using a syringe. The volume of liquid left in the solid phase is recorded in FIG. 1. The data shows that there is always a volume of residual buffer retained in the solid phase, even after 400 ml of air has been pumped through it.

(10) The recovery of liquid from the solid phase was increased by use of mineral oil according to the invention. A silica-based solid phase was loaded with 150 μl elution buffer (10 mM Tris-HCl pH 8.5). Mineral oil was then applied to form a liquid seal at the upper end of the solid phase, prior to pumping air through the solid phase with a syringe. The volume of liquid collected from the solid phase was recorded, and compared with the volume of liquid collected without application of mineral oil. The results are shown in FIG. 2. The data points represent six individual measurements of liquid collected with and without mineral oil. The black bar indicates the average volume collected, and “CV” is the coefficient of variation. The results show that mineral oil increases the recovery of liquid from the solid phase and reduces the variation in the amount of liquid recovered.

EXAMPLE 2

Use of Mineral Oil Increases the Yield of Nucleic Acid from a Solid Phase

(11) RNA was extracted from human plasma spiked with HIV-1 RNA using a silica-based solid phase in a column. RNA was eluted from the solid phase with and without a liquid seal formed by a layer of mineral oil at the upper end of the solid phase. HIV-1 RNA in the eluate was amplified, and specifically detected by dipstick assay using a method as described in Dineva et al (Journal of Clinical Microbiology, 2005, Vol. 43(8): 4015-4021). The assay signals were scored from 0.5 to 5 using a scorecard, with 5 being strongest and 0.5 weakest. The results, shown in FIG. 3, demonstrate that the assay signal was increased by over 50% when mineral oil was used. It is concluded that the yield of nucleic acid from the solid phase is increased by use of mineral oil.

EXAMPLE 3

Use of Mineral Oil to Obtain Liquid Trapped at Interfaces

(12) Liquid containing a red coloured dye was applied to a column containing a porous solid phase, and then removed from the solid phase by pumping air through the column using a syringe. The liquid leaves the column through an nozzle at the lower end of the column. A photograph of the lower end of the column with the nozzle is shown in FIG. 4(a). Liquid remains trapped in at an interface between the solid phase and the column, and in the nozzle. FIG. 4(b) shows the effect of applying a layer of mineral oil over the solid phase before air is pumped through the column. No liquid is trapped at the interface of the solid phase with the column, or in the nozzle.

(13) It is concluded that use of mineral oil also increases the amount of liquid that can be obtained from the solid phase by removing liquid from the interface of the solid phase with the solid phase support.

EXAMPLE 4

Formation of a Semi-Spherical Meniscus at the Interface Between Mineral Oil and Aqueous Liquid Bound to the Solid Phase

(14) FIG. 5 shows that when mineral oil is layered at an upper end of a porous solid phase to which an aqueous liquid is bound, a semi-spherical meniscus is formed at the interface between the mineral oil and the aqueous liquid. When a pressure differential is applied across the solid phase, the force at the semi-spherical meniscus is directed towards the centre. It is believed that this reduces the pressure differential required to displace the liquid bound to the solid phase towards the second end of the solid phase.

EXAMPLE 5

A Procedure for Isolation of Nucleic Acid from a Plasma Sample Using a Method of the Invention

(15) In this example, a detailed procedure for isolation of nucleic acid from a plasma sample using a method of the invention is described.

(16) A plasma sample is lysed, digested with a proteinase, and then applied to a porous solid phase contained within a column. Nucleic acid in the lysed plasma sample binds to the solid phase, and is then washed with a wash buffer. Next, elution buffer is applied to the column to release the nucleic acid from the solid phase. Elution buffer containing the released nucleic acid is then obtained from the solid phase using a method of the invention.

(17) Buffers:

(18) Lysis buffer: comprises a kosmotropic salt, and a non-ionic detergent at acidic pH;

(19) Wash buffer: comprises Tris-HCl at acidic pH;

(20) Elution buffer: comprises Tris-HCl at alkaline pH.

(21) Procedure:

(22) ##STR00001##

EXAMPLE 6

Nucleic Acid Yield Using a Method of the Invention is Improved in Uncapped Columns

(23) In this example, RNA was extracted from human plasma spiked with HIV-1 RNA by carrying out the procedure described in Example 5 using an uncapped column. For comparison, extractions were also performed with: (i) a capped column; or (ii) an uncapped column; without adding mineral oil after the addition of elution buffer.

(24) HIV-1 RNA in the eluate was amplified, and specifically detected by dipstick assay using a method as described in Dineva et al (Journal of Clinical Microbiology, 2005, Vol. 43(8): 4015-4021). The assay signals were scored from 0.5 to 5 using a scorecard, with 5 being strongest and 0.5 weakest. The results, shown in FIG. 6, demonstrate that the best dipstick signal was obtained when mineral oil was used, indicating that the yield of RNA was greatest from the uncapped column with mineral oil.

EXAMPLE 7

Nucleic Acid Yield Using a Method of the Invention is Comparable with a Centrifugation Method

(25) Plasma was spiked with HIV-1 at 5000 copies/ml. RNA was extracted from the plasma and bound to a porous solid phase in two separate columns. Elution buffer was added to each column, followed by addition of a layer of mineral oil to form a liquid seal at the upper end of the solid phase of one of the columns. RNA was then eluted from the solid phase with the mineral oil by applying air pressure to the top of the solid phase. RNA was eluted from the other column by centrifugation. HIV-1 RNA in the eluted samples was quantified by reverse transcription-polymerase chain reaction (RT-PCR). The results are shown in FIG. 7. The black horizontal bars indicate average extraction yield. It is concluded that the yield of RNA using a method of the invention was comparable with the centrifugation method.