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WO1997030152A1 - Separation d'un acide nucleique a un brin d'un acide nucleique a deux brins - Google Patents

Separation d'un acide nucleique a un brin d'un acide nucleique a deux brins Download PDF

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Publication number
WO1997030152A1
WO1997030152A1 PCT/NL1997/000062 NL9700062W WO9730152A1 WO 1997030152 A1 WO1997030152 A1 WO 1997030152A1 NL 9700062 W NL9700062 W NL 9700062W WO 9730152 A1 WO9730152 A1 WO 9730152A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
solid phase
single stranded
stranded nucleic
acid material
Prior art date
Application number
PCT/NL1997/000062
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English (en)
Inventor
Jaap Goudsmit
Cornelis Johannes Andreas Sol
Marcellinus Gualbertus Hubertus Maria Beld
Willem René BOOM
Original Assignee
Amsterdam Support Diagnostics B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amsterdam Support Diagnostics B.V. filed Critical Amsterdam Support Diagnostics B.V.
Priority to AU16765/97A priority Critical patent/AU1676597A/en
Publication of WO1997030152A1 publication Critical patent/WO1997030152A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • the invention relates to the field of purification and separation of nucleic acids from nucleic acid containing starting materials, especially from biological materials such as urine, faeces, sperm, saliva, whole blood, serum or other body fluids, fractions of such fluids such as leucocyte fractions (buffy coats), cell cultures and the like, but also samples from the environment such as soil, water and the like.
  • biological materials such as urine, faeces, sperm, saliva, whole blood, serum or other body fluids, fractions of such fluids such as leucocyte fractions (buffy coats), cell cultures and the like, but also samples from the environment such as soil, water and the like.
  • the nature of the target nucleic acid may not be known beforehand, or there may be many different targets necessary to be analyzed. In these cases the rapid but rather crude method described above may not be sophisticated enough and further separations of the crude material may be wanted. Fractionation of mixtures of double- (ds) and single-stranded (ss) nucleic acids (NA) into single- and double-stranded forms is frequently needed e.g. in the separation of labelled ss-NA probes from ds-hybrids, in the separation of in vitro transcripts from ds-DNA templates, and in the separation of genomic DNA from mRNA.
  • ds double-
  • ss single-stranded nucleic acids
  • Electrophoresis can be used to fractionate different forms of nucleic acids, because of differences in size and shape (1-3). Centrifugation takes advantage of differences in density (4), and more recently the technology of high-performence liquid chromatography (HPLC) has been applied to separate and purify single- and double-stranded DNA and RNA molecules (5-8).
  • HPLC high-performence liquid chromatography
  • RNA purified from eukaryotic cells by the currently most widely used procedure (9) appears to contain significant amounts of genomic DNA, an adaptation which reduces genomic DNA contamination of the ss-RNA fraction has recently been described (10).
  • the present invention therefor provides a method for separating single stranded nucleic acid material from double stranded nucleic acid material comprising contacting a mixture of the both with a liquid comprising a chaotropic agent and a nucleic acid binding solid phase, whereby the liquid has a composition such that double stranded nucleic acid binds to the solid phase and a substantial amount of single stranded nucleic acid does not and separating the solid phase from the liquid. Suitable circumstances to arrive at such a separation can be determined by the person skilled in the art.
  • Circumstances under which double stranded material binds to the solid material and single stranded material will vary, however important parameters to obtain such differential binding are the concentration of the chaotropic agent, which should roughly be between 1-10 M, preferably between 3-6 and particularly about 5 M; the concentration of chelating agent, which in the case that EDTA is applied should be equal to or greater than 10 mM and preferably not higher than 1 M; the pH of the aqueous solution in which the separation is carried out should be above 2 when a thiocyanate is used as chaotropic agent and it should be below 10 because otherwise there is a risk that the ds material will become ss.
  • the temperature at which the process is carried out seems to be non-critical, however, it is probably best to keep it between 4°C and 60°C.
  • An important aspect of the process is of course that the ds material remains double stranded during the separation. Under the circumstances as disclosed above this will normally be the case if the ds nucleic acid is at least 50 bp long at 40% GC basepairs . The skilled artisan knows how this length may vary with lower or higher GC content. In Van Ness et al (17) and/or Thompson et al (18) it is shown that the whole process depends on intricate interactions between a.o. the factors mentioned above. Using this disclosure and the cited references the skilled artisan will be able to adjust the circumstances to his or her particular process.
  • Chaotropic agents are a very important feature of the present invention. They are defined as any substance that can alter the secondary, tertiary and/or quaternary structure of nucleic acids . They should have no substantial effect on the primary structure of the nucleic acid. If nucleic acids are present associated with other molecules, such as proteins, these associations can also be altered by the same or different chaotropic agents. Many chaotropic agents are suitable for use in the present invention, such as sodium iodide, potassium iodide, sodium (iso)thiocyanate, urea or guanidinium salts, or combinations thereof. A preferred class of chaotropic agents according to the invention are guanidinium salts, of which guanidinium thiocyanate is most preferre .
  • the solid phase to be used is less critical . Important is that it should bind nucleic acids reversibly. Many such materials are known, of which a number are silicium based, such as aluminium silicate and the like, preferably silica. Silica is meant to include Si ⁇ 2 crystals and other forms of silicon oxide, such as diatom skeletons, glass powder and/or particles and amorphous silicon oxide.
  • the solid phase may be present in any form, it may even be the vessel which contains the nucleic acid mixtures or a part of such a vessel. It may also be a filter or any other suitable structure. Apart from silicium based materials other materials will also be suitable, such as nitrocellulose (filters), latex particles and other polymeric substances.
  • a preferred form of the solid phase is a particulate form, which allows for easy separation of bound and free material, for instance by centrifugation.
  • the particle size of the solid phase is not critical. Suitable average particle sizes range from about 0.05 to 500 ⁇ m. Preferably the range is chosen such that at least 80, preferably 90 % of the particles have a size between the values just mentioned. The same holds true for the preferred ranges of which the average particle sizes are between 0.1 and 200 ⁇ m, preferably between 1 and 200 ⁇ m.
  • the binding capacity of a given weight of the particles increases with decreasing size, however the lower limit of the size is when particles cannot easily be redispersed after separation through for instance centrifugation.
  • a further embodiment of the present invention is a method for isolating single stranded nucleic acid material from a mixture of nucleic acid material, comprising the steps of subjecting the mixture to a method as described hereinabove and treating the supernatant containing the single stranded nucleic acid material with a second liquid comprising a chaotropic agent and a second nucleic acid binding solid phase, whereby the second liquid has a composition such that the resulting mixture of supernatant and second liquid allow for binding of the single stranded nucleic acid material to the second solid phase.
  • the double stranded nucleic acid material is removed from the crude mixture and the single stranded nucleic acid is purified from the remaining still crude mixture in another single step.
  • Both the double stranded material and the single stranded material are reversibly bound to the respective solid phases, so that they may be easily eluted from said solid phases to undergo further analysis or other treatments.
  • a very useful further treatment is the amplification of the (double or single stranded) nucleic acid material.
  • Phage MS-2 ss-RNA 3569 nt
  • E. coli rRNA 16 and 23S; l,7kb and 3,5kb respectively
  • phage M13 ss-DNA 7599 nt
  • Hindlll digested phage lambda ds-DNA were purchased from
  • Rotavirus ds-RNA was purified from feces of an infected individual by protocol Y/SC (11) .
  • Plasmid DNA was purified from E. coli HB101 as described by Ish-Horowicz and Burke (13) followed by column chromotography with Sepharose CL2B (Pharmacia, Inc. Uppsala, Sweden).
  • Total NA was purified from E.coli by protocol Y/D (11). Chemicals .
  • Guanidiniu thiocyanate was obtained from Fluka (Buchs, Switzerland) .
  • EDTA Tetriplex
  • MgCl2.6H20 were obtained from Merck (Darmstadt, Germany) .
  • TRIS was obtained from Boehringer (Mannheim, Germany) .
  • the preparation of size-fractionated silica particles (silica coarse, SC) and diatom suspension has been described (11).
  • Triton X-100 was from Packard (Packard Instrument Co., Inc., Downers Grove, 111).
  • 0.2M EDTA (pH 8.0) was made by dissolving 37.2 g EDTA (Merck, Germany) and 4.4 g NaOH (Merck, Germany) in aqua in a total volume of 500 ml.
  • Binding buffer L10 was prepared by dissolving 120 g GuSCN in 100 ml 0.35M TRIS.HCI (pH 6.4); subsequently 22 ml 0.2M EDTA (pH 8.0) and 9.1 g Triton X-100 were added and the solution was homogenized; finally 11 g of solid MgCl 2 -6H 2 ⁇ was added. The final concentration of MgCl 2 in L10 is about 0.25M. L10 is stable for at least 1 month when stored at ambient temperature in the dark.
  • ss-NA forms protocol R-sup
  • 900 ⁇ l of the supernatant were added to a mixture of 400 ⁇ l L10 and 40 ⁇ l SC and ss-NA was bound during a 10 min. incubation at room temperature.
  • the tube was subsequently centrifuged (15 sec. at approx. 10.000 x g), and the supernatant was discarded (by suction) .
  • the resulting pellet was subsequently washed twice with 1 ml of L2, twice with 1 ml ethanol 70% (vol/vol) and once with 1 ml acetone.
  • the silica pellet was dried (10 min. at 56°C with open lid in an Eppendorf heating block) and eluted in 50 ⁇ l TE buffer (10 min.
  • the supernatant contains the ss-NA fraction.
  • the remaining supernatant was discarded, and the silica pellet was washed twice with Lll to remove unbound ss-NA.
  • the resulting silica pellet was subsequently washed twice with L2, twice with ethanol 70%, once with acetone, dried and eluted as described above.
  • the supernatant contains the ds-NA fraction.
  • protocol R Due to trapping of ss-NA into high-molecular-weight genomic DNA, protocol R as described above gives only low yields of ss-NA. This can be circumvented by first isolating total NA by protocol Y/D (11), which causes some shearing of the high-molecular-weight genomic DNA, sufficient enough to prevent trapping of the ss-NA. Total NA thus purified can subsequently be used as input for protocol R.
  • NA was electrophoresed (8 to 10 V/cm) through neutral agarose slab gels containing ethidiumbromide (l ⁇ g/ml) in the buffer system (40mM TRIS-20 mM sodium acetate- 2mM EDTA adjusted to pH 7.7 with acetic acid; ethidium bromide was added to a concentration of l ⁇ g/ml of buffer) described by Aaij and Borst (14) .
  • DNA fragments were transferred to nitrocellulose filters by the procedure of Southern (15) and hybridized with [alpha- 32 P]dCTP labelled pHC624 (16) prepared by random labeling (Boehringer, Germany) . Hybridization conditions were as described previously (12).
  • Double-stranded and single-stranded forms can subsequently be purified by washing and eluting the silica-NA complexes (protocol R) . Double ⁇ stranded nucleic acid is recovered from the initial silica- pellet (protocol R-pellet) , whereas single-stranded forms are recovered from the initial supernatant (protocol R-sup) .
  • protocol R For optimization of protocol R we performed reconstruction experiments in which previously purified or commercially available, nucleic acids were mixed and subsequently fractionated by protocol R. Fractionation of a mixture of double-stranded DNA and single-stranded DNA.
  • Figure 3 shows the fractionation of a mixture of ds-RNA (human Rotavirus genome segments 1-11; for review see 14) and ss-RNA (phage MS2 RNA) into double stranded- and single stranded forms.
  • the estimated recovery of ds-RNA and ss-RNA was at least 80% .
  • fractionation into ds- and ss-forms was complete.

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Abstract

L'invention concerne un procédé simple pour séparer un acide nucléique à un brin d'un acide nucléique à deux brins, dans un échantillon contenant les deux. En choisissant correctement au moins un agent chaotrope, de préférence un sel de guanidine, en l'utilisant à une concentration choisie et en opérant dans des conditions appropriées, par exemple en présence d'agents chélatants, en ajustant le pH et similaire, il est possible de fixer l'acide nucléique à deux brins à une phase solide, par exemple à des particules de silice, alors que dans ces circonstances, l'acide nucléique à un brin ne se fixe pas. En séparant les particules de silice de l'échantillon, on peut facilement séparer l'acide nucléique à deux brins. Il peut ensuite être facilement élué des particules de silice. Dans une seconde étape, l'acide nucléique à un brin peut être fixé à une phase solide, par sélection de conditions appropriées. Ici encore, les particules peuvent être séparées de l'échantillon et l'acide nucléique à un brin peut ensuite être élué. Pour des séparations très poussées, le procédé peut être répété.
PCT/NL1997/000062 1996-02-14 1997-02-14 Separation d'un acide nucleique a un brin d'un acide nucleique a deux brins WO1997030152A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU16765/97A AU1676597A (en) 1996-02-14 1997-02-14 Separation of single stranded and double stranded nucleic acid materials

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EP96200355 1996-02-14
EP96200355.4 1996-02-14

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WO1997030152A1 true WO1997030152A1 (fr) 1997-08-21

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1479769A1 (fr) * 2003-05-19 2004-11-24 Hitachi High-Technologies Corporation Procédé d'isolement séparé d'ADN et d'ARN et kit pour l'isolement d'acides nucléiques
WO2008003776A2 (fr) * 2006-07-06 2008-01-10 Aj Innuscreen Gmbh Procédé permettant d'isoler en parallèle des acides nucléiques à simple brin et à double brin et de retirer de façon sélective des acides nucléiques à double brin d'un mélange d'acides nucléiques à simple brin et à double brin
EP1963526A2 (fr) * 2005-12-09 2008-09-03 Promega Corporation Purification d'acide nucleique avec une matrice de liaison
US20110046361A1 (en) * 2008-04-30 2011-02-24 Ge Healthcare Bio-Sciences Corp. Method for separation of double-stranded and single-stranded nucleic acids
US8241475B2 (en) * 2004-09-02 2012-08-14 Lifeind Ehf. Two-dimensional strandness-and length-dependent separation of nucleic acid fragments

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5075430A (en) * 1988-12-12 1991-12-24 Bio-Rad Laboratories, Inc. Process for the purification of DNA on diatomaceous earth
US5155018A (en) * 1991-07-10 1992-10-13 Hahnemann University Process and kit for isolating and purifying RNA from biological sources
WO1995021849A1 (fr) * 1994-02-11 1995-08-17 Qiagen Gmbh Procede de separation de structures d'acides nucleiques a deux brins et a un brin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5075430A (en) * 1988-12-12 1991-12-24 Bio-Rad Laboratories, Inc. Process for the purification of DNA on diatomaceous earth
US5155018A (en) * 1991-07-10 1992-10-13 Hahnemann University Process and kit for isolating and purifying RNA from biological sources
WO1995021849A1 (fr) * 1994-02-11 1995-08-17 Qiagen Gmbh Procede de separation de structures d'acides nucleiques a deux brins et a un brin

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1479769A1 (fr) * 2003-05-19 2004-11-24 Hitachi High-Technologies Corporation Procédé d'isolement séparé d'ADN et d'ARN et kit pour l'isolement d'acides nucléiques
US7737268B2 (en) 2003-05-19 2010-06-15 Hitachi High-Technologies Corporation Method of recovering nucleic acids and kit for recovering nucleic acids
US8241475B2 (en) * 2004-09-02 2012-08-14 Lifeind Ehf. Two-dimensional strandness-and length-dependent separation of nucleic acid fragments
EP1963526A2 (fr) * 2005-12-09 2008-09-03 Promega Corporation Purification d'acide nucleique avec une matrice de liaison
EP1963526A4 (fr) * 2005-12-09 2009-11-18 Promega Corp Purification d'acide nucleique avec une matrice de liaison
WO2008003776A2 (fr) * 2006-07-06 2008-01-10 Aj Innuscreen Gmbh Procédé permettant d'isoler en parallèle des acides nucléiques à simple brin et à double brin et de retirer de façon sélective des acides nucléiques à double brin d'un mélange d'acides nucléiques à simple brin et à double brin
WO2008003776A3 (fr) * 2006-07-06 2008-02-21 Aj Innuscreen Gmbh Procédé permettant d'isoler en parallèle des acides nucléiques à simple brin et à double brin et de retirer de façon sélective des acides nucléiques à double brin d'un mélange d'acides nucléiques à simple brin et à double brin
US9222084B2 (en) 2006-07-06 2015-12-29 Aj Innuscreen Gmbh Method for isolating parallel double and single-stranded nucleic acids and for selectively removing double-stranded nucleic acids from a mixture of double and single-stranded nucleic acids
US20110046361A1 (en) * 2008-04-30 2011-02-24 Ge Healthcare Bio-Sciences Corp. Method for separation of double-stranded and single-stranded nucleic acids

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