(19) TZZ¥Z_ ¥_T (11) EP 3 061 823 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 31.08.2016 Bulletin 2016/35 C12N 15/10 (2006.01) (21) Application number: 15156548.8 (22) Date of filing: 25.02.2015 (84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • Hucklenbroich, Jörg GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 42929 Wermelskirchen (DE) PL PT RO RS SE SI SK SM TR • Singer, Thorsten Designated Extension States: 42699 Solingen (DE) BA ME (74) Representative: f & e patent (71) Applicant: QIAGEN GmbH Fleischer, Engels & Partner mbB, Patentanwälte 40724 Hilden (DE) Braunsberger Feld 29 51429 Bergisch Gladbach (DE) (54) Method for extracting nucleic acids from an agarose matrix (57) The present invention provides a method for extracting one or more nucleic acids from a sample comprising gelled agarose (an agarose matrix) as well as kit for carrying out this method. EP 3 061 823 A1 Printed by Jouve, 75001 PARIS (FR) 1 EP 3 061 823 A1 2 Description nidinium thiocyanate, guanidinium isothiocyanate, or so- dium iodide. The nucleic acids are then selectively bound [0001] The present invention relates to a method for to silica particles, so-called "glass milk", diatomaceous extracting one or more nucleic acids from a sample com- earth, or further commercially available binding matri- prising gelled agarose (agarose matrix) and at least one 5 ces/resins in the presence of a high concentration (usu- nucleic acid, preferably from agarose gels, by using a ally > 2 M) of one or more of the aforementioned chao- solid phase, which selectively binds nucleic acids in the tropic substances. Based on this general principle, vari- presence of a binding mediating agent without the need ous kits for DNA extraction from agarose gels are com- for chaotropic substances, as well as to a kit for carrying mercially available, for example the GENECLEAN kit out said method. 10 from Bio101 (Davidson, MC, USA), the agarose gel ex- [0002] For the separation of nucleic acids, including traction kit from Jena Bioscience (Jena, Germany) or the ribonucleic acids (RNA) and desoxyribonucleic acids QIAquick and QIAex kits, both from Qiagen (Hilden, Ger- (DNA), on the basis of their size, fractionated agarose many). gel electrophoresis is the method of choice. Nucleic acids [0007] One drawback of these chaotrope-mediated isolated from agarose gels are frequently used as tem- 15 methods is the fact that the used chaotropic salts are plates for polymerase chain reaction (PCR) or reverse toxic and/or corrosive and moreover usually are required transcript-PCR (RT-PCR) as well as for hybridization, se- ina concentration of morethan 2 M in the binding solution. quencing, ligation, and various other recombinant mo- Furthermore, using these methods, recovery of nucleic lecular techniques. acid fragments having a length of less than 150 base [0003] All these downstream analyses depend upon 20 pairs (bp) often is poor. the quality and purity of the nucleic acids isolated from [0008] Accordingly, the object of the present invention the agarose matrix, i.e. the method employed for extract- was to provide a method for extracting one or more nu- ing the nucleic acids from the matrix. In attempt to recover cleic acids from a sample comprising gelled agarose (an nucleic acids, mainly DNA, from agarose gels in an intact agarose matrix) which avoids the use of the chaotropic form and free of contaminants, several methods have 25 substances, in particular of guanidine hydrochloride, been developed during the past 30 years. These meth- guanidinium (iso)thiocyanate, or sodium iodide. ods include for example processes for physical gel de- [0009] This object is met by the method of the present struction, like the so-called "freeze and squeeze" proce- invention. The present invention provides a method for dure (R.W.J. Thuring, J.N.P. Anders, P. Borst Anal. Bi- extracting one or more nucleic acids from a sample com- ochem. 1975, 66, 213 ) or the so-called "crush and soak" 30 prising gelled agarose (agarose matrix) and at least one method. nucleic acid, preferably from agarose gels of nucleic ac- [0004] Physical extrusion of DNA molecules from a gel ids, comprising the steps of: at least partly liquefying the (or a part thereof) includes e.g. DNA extraction by electro- agarose matrix and separating the nucleic acid from the elution inside a dialysis bag into an electrophoresis buff- sample using a solid phase, which selectively binds the er,from which theDNA can berecovered by precipitation. 35 nucleic acid in presence of a binding mediating agent, According to another procedure, a small trough is cut in wherein neither the binding mediating agent nor any oth- the agarose gel just in front of the DNA band to be re- er compound added to the sample during the procedure covered. The DNA then is electrophoretically eluted onto comprises a chaotropic substance, selected from the a diethylaminoethyl (DEAE)-cellulose paper strip, an af- group comprising sodium perchlorate, sodium iodide, po- finity membrane, a gel insert comprising a mixture of so- 40 tassium iodide, guanidinium (iso)thiocyanate, and gua- dium acetate and sucrose, or a dialysis tubing (P.N. Hen- nidine hydrochloride. Preferably neither the binding me- gen Trends in Biochemical Sciences 1994, 19 (9), 388). diating agent nor any other compound added to the sam- [0005] The gel may also be dissolved chemically or ple during the whole procedure nor the sample itself com- digested enzymatically to release the nucleic acids into prise more than 0.5 M of any chaotropic substance. Pref- solution. For example, low melting temperature (LMT) 45 erably any of the added components comprises less than agarose can be digested using GELase (Epicentre Bio- 0.1 M, more preferably less than 0.05 M of any chaotropic technologies, Madison, WI, USA), a DNase-free combi- substance. nation of enzymes comprising beta-agarase, which di- [0010] It has surprisingly been found, that a nucleic gests the agarose into multimeric subunits. However, di- acid can be extracted from agarose matrices, in particular gestion takes several hours to overnight and in general 50 excised agarose gel bands of DNA or RNA electrophore- additional steps of phenol extraction and/or precipitation sis gels by an extraction procedure which preferably are required for purifying the nucleic acid. avoids the use of any chaotopic substances, but never- [0006] Following the fundamental procedure de- theless makes use of the solid phase-based separating scribed by Vogelstein and Gillespie (Proc. Nat. Acad. Sci. step typically employed in these chaotrope-based meth- USA 1979, 76 (2), 615) several methods have been de- 55 ods. Thus, the method of the present invention combines veloped, wherein at first the agarose gel matrix is ther- the known advantages of solid phase-based separation mally and/or chemically dissolved in the presence of a technology and the further advantage that no toxic and/or chaotropic salt such as guanidine hydrochloride, gua- corrosive chaotropic substances have to be employed 2 3 EP 3 061 823 A1 4 during the whole extracting procedure. This is possible, any chaotropic substance at all. Alternatively and/or ad- since it has surprisingly been found that a non-chaotropic ditionally, the agarose matrix may also be liquefied by binding mediating agent is able to selectively mediate melting, as described below. the binding between the solid phase and the nucleic ac- [0015] In terms of the present invention a binding me- ids, even in the presence of residual agarose and/or sub- 5 diating agent is a non-chaotropic substance which me- stances used to dissolve and/or remove the agarose ma- diates selective binding between the solid phase and the trix, for example precipitating agents used for removing nucleicacids. By "mediating selective binding" it is meant, the agarose matrix. that in the absence of the binding mediating agent the [0011] By using the method of the present invention nucleic acids do not bind to the solid phase or at least nucleic acids can be extracted from any sample compris- 10 only in a limited extent (e.g. less than 15 %, preferably ing gelled agarose / an agarose matrix. Preferably aga- less than 10 %, more preferably less than 5%, even more rose gels from gel electrophoresis (or parts thereof) are preferably less than 2 % and most preferably less than used as a sample in the present invention. Preferably, 0.5 %, based on the total amount of nucleic acids bound parts of an agarose gel, i.e. parts including particular to the solid phase in the presence of the optimum amount bands of interest excised from an agarose gel using for 15 of binding agent), while in the presence of said binding example a razor blade or a scalpel may be used. In the mediating agent the nucleic acids present in a sample following the term "agarose gel" comprises both, a com- can be attached to the solid phase, for example via ionic plete gel as well as excised parts of such a gel, e.g. in- or electrostatic interactions, without being limited to cluding nucleic acid fragments. these. Preferably all kind of nucleic acids present in the [0012] Agarose is a commercial available linear poly- 20 sample may be bound to the solid phase in the presence mer, from which agarose gels are prepared by melting a of said binding mediating agent or at least all ribonucleic defined amount of said polymer in an appropriate buffer acids and/or desoxyribonucleic acids present in the sam- untila clear solution isobtained. Upon cooling, the melted ple, irrespective of their particular substitution pattern.