Nucleic Acid Preparation Method

Abstract

A method for processing a nucleic acid, in which the nucleic acid is exposed to an aqueous medium which includes a polyol in sufficient proportion for at least a portion of the nucleic acid to enter or remain in an extra-solution phase. Thus, a polyol may be used to bind a nucleic acid which is in solution to a solid support or to wash a nucleic acid on a solid support whilst maintaining it on the support. The polyol may for example be a C 2 -C 10 alkanediol.

1 . Use of a polyol or a liquid comprising a polyol to induce or facilitate nucleic acid to enter or, as the case may be, to stay in an extra-solution phase. 
     
     
         2 . The use according to  claim 1 , wherein said polyol is not subjected to a specific removal step prior to a nucleic acid amplification. 
     
     
         3 . The use of  claim 1  or  claim 2 , wherein the nucleic acid in an extra-solution phase is bound to a solid support. 
     
     
         4 . The use of any one of  claims 1  to  3 , wherein said liquid is an aqueous composition comprising at least about 10% v/v of the polyol, for example at least about 25% v/v of the polyol, preferably at least 40% v/v of the polyol, more preferably at least 50% v/v of the polyol, e.g. at least 60% v/v of the polyol. 
     
     
         5 . The use of any one of  claims 1  to  4 , wherein said liquid is an aqueous composition comprising not more than about 70% v/v of the polyol, preferably not more than 75%, more preferably not more than about 90% v/v of the polyol. 
     
     
         6 . The use of any one of  claims 1  to  5 , wherein the polyol is present in the liquid at a concentration of at least about 30% v/v and not more than about 80% v/v. 
     
     
         7 . The use of any one of the preceding claims, wherein the viscosity of the polyol or the liquid is preferably below 50 Centipoises or mPa s at 4° C. and atmospheric pressure, more preferably below 25 Centipoises at 4° C., most preferably between 1 and 20 Centipoises, 1 and 10 Centipoises, 1 and 5 Centipoises or 1 and 3 Centipoises. 
     
     
         8 . The use according to any one of the preceding claims, wherein said polyol is a C 2 -C 10  polyol which is an alkane or alkene substituted by at least two hydroxy groups and optionally interrupted by 1, 2 or 3 ether linkages. 
     
     
         9 . The use of any one of the preceding claims, wherein the polyol has a flashpoint of at least 50° C., preferably at least 70° C. 
     
     
         10 . The use of any one of the preceding claims, wherein the polyol is a polyhydric alkanol. 
     
     
         11 . The use of any one of the preceding claims, wherein the polyol is a polyhydric alkanol ether comprising 1, 2 or 3 ether linkages. 
     
     
         12 . The use of any one of the preceding claims, wherein the polyol is a branched chain polyol. 
     
     
         13 . The use of any one of the preceding claims, wherein the polyol is a straight chain polyol. 
     
     
         14 . The use of any one of the preceding claims, wherein the polyol is a diol. 
     
     
         15 . The use of any one of the preceding claims, wherein the polyol is a triol. 
     
     
         16 . The use of any one of the preceding claims, wherein the polyol has from 3 to 9 carbon atoms, optionally 3 to 7 carbon atoms, further optionally 3, 4 or 5 carbon atoms. 
     
     
         17 . The use of any one of the preceding claims, wherein the polyol has 4 carbon atoms. 
     
     
         18 . The use of any one of the preceding claims, wherein the polyol is selected from 2-methyl-1,3-propanediol, 2,3-butanediol, 1,2-propanediol and tripropyleneglycol. 
     
     
         19 . The use of any one of the preceding claims, wherein the nucleic acid is DNA. 
     
     
         20 . The use of any one of the preceding claims, wherein the nucleic acid is RNA. 
     
     
         21 . The use of any one of the preceding claims, wherein the extra-solution phase is a solid support-bound phase and the polyol is used to induce or facilitate nucleic acid to bind to a solid support. 
     
     
         22 . The use of  claim 21  wherein an aqueous medium having nucleic acid dissolved therein is combined with the polyol and the solid support. 
     
     
         23 . The use of  claim 22 , wherein the polyol is in the form of an aqueous solution thereof prior to the combining. 
     
     
         24 . The use of any one of  claims 1  to  20 , wherein the extra-solution phase is a solid support-bound phase and the polyol is used to wash nucleic acid bound to a solid support. 
     
     
         25 . The use of  claim 24 , wherein the support-bound nucleic acid is contacted with an aqueous solution of the polyol. 
     
     
         26 . The use of any of  claims 21  to  23  which is followed by separating the solid support from liquid and then by the use of  claim 24  or  claim 25 . 
     
     
         27 . The use of any of  claims 24  to  26 , which is followed by elution of the nucleic acid from the solid support. 
     
     
         28 . The use of any of  claims 21  to  27 , wherein the solid support is beads comprising a silica surface. 
     
     
         29 . The use of  claim 28 , wherein the beads are magnetic. 
     
     
         30 . The use of any one of the preceding claims, wherein the nucleic acid is after the use further subjected to one or more additional processes, optionally selected from detection, cloning, analysis, sequencing, amplification, study, transfection, hybridisation, cDNA synthesis, studying nucleic acid structure and/or composition (e.g. the methylation pattern of DNA), size separation and chromatography. 
     
     
         31 . The use of  claim 30  wherein the amplification comprises qPCR. 
     
     
         32 . A method for processing a nucleic acid, in which method the nucleic acid is exposed to an aqueous medium which includes a polyol in sufficient proportion to induce or facilitate at least a portion of the nucleic acid to adopt an extra-solution phase. 
     
     
         33 . The method of  claim 32 , wherein the extra-solution phase is a solid support-bound phase. 
     
     
         34 . The method of  claim 33 , wherein the solid support comprises beads capable of binding nucleic acid. 
     
     
         35 . The method of  claim 33  or  claim 34 , which is a method for binding a nucleic acid to a solid support, wherein the method comprises combining an aqueous medium having nucleic acid dissolved therein with the polyol and the solid support to form a composition comprising support-bound nucleic and an aqueous liquid. 
     
     
         36 . The method of  claim 35 , which further comprises separating the solid phase and its bound nucleic acid from the aqueous liquid. 
     
     
         37 . The method of  claim 33  or  claim 34 , which is a method for washing solid support-bound nucleic acid, wherein the method comprises contacting the solid support and its bound nucleic acid with a liquid comprising the polyol. 
     
     
         38 . The method of  claim 37 , which further comprises recovering the solid support and its bound nucleic acid from the liquid. 
     
     
         39 . The method of any of  claims 32  to  38 , wherein the liquid comprises an aqueous medium comprising the polyol in an amount of at least 40% v/v. 
     
     
         40 . The method of any of  claims 32  to  39 , wherein the polyol is as defined by any of  claims 7  to  18 . 
     
     
         41 . A method of washing a composition comprising a nucleic acid bound to a solid support, the method comprising:
 a. contacting the solid support with a liquid comprising a polyol optionally as defined in any one, or a permitted combination, of  claims 7  to  18 ; and   b. separating the liquid and the solid support.   
     
     
         42 . The method of  claim 41 , further comprising preparing the composition comprising the nucleic acid bound to the solid support by:
 a′. contacting a biological sample with the solid support, whereby the nucleic acid binds to the solid support;   b′. separating the sample and the solid support; and   c′. optionally washing the solid support with an alcohol free wash buffer, preferably lysis/binding buffer employed for said lysis and contacting.   
     
     
         43 . The method of  claim 41  or  claim 42 , wherein the sample comprises or is a biological fluid, cell lysate, physiological buffer or transport medium. 
     
     
         44 . The method of  claim 43 , wherein the sample is serum or plasma. 
     
     
         45 . The method of any one of  claims 42  to  44  further comprising:
 c. after the separating of the liquid and the solid support, contacting the solid support with an elution buffer, whereby the nucleic acid is separated from the solid support; and 
 d. separating the elution buffer and the solid support. 
 
     
     
         46 . The method of any of  claims 42  to  45 , wherein the solid support is as defined in  claim 28  or  claim 29 . 
     
     
         47 . A kit comprising a polyol, optionally as defined in any of  claims 7  to  18 , and one or both of a lysis/binding buffer and a solid support. 
     
     
         48 . The kit of  claim 47 , wherein the solid support is as defined in  claim 28  or  claim 29 . 
     
     
         49 . The kit of any of  claim 47  or  48 , further comprising an elution buffer to elute bound nucleic acid from the solid support. 
     
     
         50 . A method for preparing nucleic acid from a sample for nucleic acid amplification, characterised in that said nucleic acid is not contacted with ethanol or isopropanol and is subjected to least one washing step employing a liquid comprising a polyol to remove contaminants without requiring a step to specifically remove said polyol prior to amplification. 
     
     
         51 . The method of  claim 50 , wherein the nucleic acid is RNA. 
     
     
         52 . The method of  claim 50  or  51 , wherein the polyol is as defined in any one, or a permitted combination, of  claims 7  to  18  and the liquid is an aqueous medium comprising, or consisting of, water and the polyol wherein the liquid is defined according to any one of  claims 4  to  7 . 
     
     
         53 . The method of  claims 50  to  52 , wherein said sample is a biological sample and prior to said at least one washing step said nucleic acid is extracted from cells, organelles or viral particles of said sample by a lysis step, optionally in the presence of Proteinase K, and captured on a solid support, for example beads, in the presence of a monohydric alcohol-free lysis/binding buffer, followed by a separation step. 
     
     
         54 . The method of  claim 53 , wherein said solid support is silica-coated beads, preferably magnetic silica-coated beads. 
     
     
         55 . The method of  claim 53  or  claim 54 , wherein said separation step is followed by an initial washing with the lysis/binding buffer. 
     
     
         56 . The method of  claims 50  to  55 , wherein after said at least one washing step employing a liquid comprising a polyol the nucleic acid is eluted from the solid support. 
     
     
         57 . The method of  claims 50  to  56 , which further comprises amplifying the washed nucleic acid, optionally wherein the nucleic acid amplification is qPCR. 
     
     
         58 . Use of a solid support for nucleic acid capture in a method according to any one of  claims 53  to  57 . 
     
     
         59 . Use of an alcohol-free buffer, which can release nucleic acid from cells, organelles or viral particles in a biological sample in a method according to any one of  claims 53  to  57 , wherein said buffer is employed as the lysis/binding buffer. 
     
     
         60 . A composition of matter comprising (i) a liquid which comprises water and a polyol, and (ii) a nucleic acid not in solution in the liquid. 
     
     
         61 . The composition of  claim 60 , wherein the nucleic acid is bound to a solid support, e.g. a bead. 
     
     
         62 . The composition of  claim 60  or  61 , wherein the nucleic acid is RNA. 
     
     
         63 . The composition of any one of  claims 60  to  62 , wherein the polyol is as defined in any one of, or a permitted combination of,  claims 7  to  18 . 
     
     
         64 . A kit for carrying out a method according to any one of  claims 50  to  57  which comprises said washing liquid comprising a polyol as defined in any of  claims 7  to  18  and one or both of said solid support and said lysis/binding buffer.