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WO1997037040A2 - Isolation et/ou amplification d'acides nucleiques du virus de l'hepatite c (hcv) a partir d'echantillons susceptibles de contenir ce virus - Google Patents

Isolation et/ou amplification d'acides nucleiques du virus de l'hepatite c (hcv) a partir d'echantillons susceptibles de contenir ce virus Download PDF

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Publication number
WO1997037040A2
WO1997037040A2 PCT/NL1997/000167 NL9700167W WO9737040A2 WO 1997037040 A2 WO1997037040 A2 WO 1997037040A2 NL 9700167 W NL9700167 W NL 9700167W WO 9737040 A2 WO9737040 A2 WO 9737040A2
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Prior art keywords
nucleic acid
hcv
solid phase
single stranded
hepatitis
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PCT/NL1997/000167
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English (en)
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WO1997037040A3 (fr
Inventor
Jaap Goudsmit
Marcellinus Gualbertus Hubertus Maria Beld
Cornelis Johannes Andreas Sol
Willem René BOOM
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Akzo Nobel N.V.
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Priority to AU21809/97A priority Critical patent/AU2180997A/en
Publication of WO1997037040A2 publication Critical patent/WO1997037040A2/fr
Publication of WO1997037040A3 publication Critical patent/WO1997037040A3/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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D

Definitions

  • the invention relates to the field of purification and amplification 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.
  • 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
  • an infectious agent such as HCV in a sample containing other (ds) nucleic acids.
  • electrophoresis can be used to fractionate different forms of nucleic acids , because of differences in size and shape. Centrifugation takes advantage of differences in density, 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.
  • HPLC high-performence liquid chromatography
  • RNA purified from eukaryotic cells by the currently most widely used procedure (1) appears to contain
  • causative agent or in any combination.
  • HVC Hepatitis C Virus
  • HCV Hepatitis delta virus
  • ALT alanine aminotransferase
  • the present invention therefore provides a method for separating causative agents of Hepatitis from each other and from host material by applying differential binding
  • the invention provides a method for separating HCV RNA from a sample suspected to contain said RNA comprising contacting said sample with a first liquid comprising a chaotropic agent and a nucleic acid binding solid phase, whereby the first 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
  • the single stranded HCV nucleic acid material will be present in the supernatant where it can be directly detected or further purified or amplified.
  • Suitable circumstances to arrive at a separation of double stranded nucleic acids from the single stranded HCV material can be determined by the person skilled in the art.
  • the concentration of the chaotropic agent which should roughly be between 1-10 M, preferably between 3-6 M 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.
  • 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
  • nucleic acids 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 preferred.
  • the solid phase to be used is less critical. Important is that it should bind nucleic acids reversibly.
  • silicium based such as aluminium silicate and the like, preferably silica.
  • Silica is meant to include SiO 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.
  • other materials will also be suitable, such as
  • nitrocellulose filters
  • latex particles latex particles
  • 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.
  • the range is chosen such that at least 80, preferably 90 % of the particles have a size between the values just mentioned.
  • 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. This will be the case in starting material rich in nucleic acids containing many nucleic acids of a higher molecular weight. The particles and the nucleic acids may form aggregates in these cases.
  • the person skilled in the art will be able to choose the right particle size for the particular application
  • a further embodiment of the present invention is a method as disclosed above further comprising treating the supernatant containing the single stranded HCV 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 compositon such that the resulting mixture of supernatant and second liquid allow for binding of the single stranded HCV nucleic acid material to the second solid phase.
  • the double stranded nucleic acid material is removed from the crude mixture in the first step 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.
  • Both types can be amplified, or both types may be converted into one another so that they can be amplified.
  • the present invention provides in yet another embodiment a method for amplifying single stranded nucleic acid material comprising the steps of hybridizing the single stranded nucleic acid with primers and elongating the probes using an enzyme which adds nucleotides to the primer sequence using the hybridized single strand material as a template, whereby at least one primer comprises a random hybridizing sequence and an amplification motif.
  • the criteria for amplification are well known in the art.
  • the length of suitable primers, suitable buffers, suitable melting temperatures for separating strands, suitable hybridization conditions can all be determined using standard handbooks in the field.
  • primers will be at least 10 bases long and not much longer than 100 bases.
  • amplification embodiments of the invention are exemplified using PCR (polymerase chain reaction). Other amplification methods are of course equally suitable.
  • the exemplified label (or tag) on the primers is DIG (digoxygenin).
  • DIG digoxygenin
  • other labels are available and well known in the art. The invention will now be explained in further detail in the following detailed description.
  • Serum samples were obtained from patients
  • ALT alanine aminotransferase
  • Guanidiniumthiocyanate (GuSCN) was obtained from Fluka (Buchs, Switzerland).
  • Triton X-100 was from Packard (Packard Instrument Co., Inc., Downers Grove, I11).
  • the lysis/binding buffer L6, washing buffer L2, and TE (10mM Tris.HCl, 1 mM EDTA; pH 8.0) have been described (27).
  • Binding buffer L10 was prepared by dissolving 120 g GuSCN in 100 ml 0.35M TRIS.HCl (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 O 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.
  • silica pellet was washed twice with L11 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 (27), 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 (1 ⁇ 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 1 ⁇ g/ml of buffer) described by Aaij and Borst (25).
  • DNA fragments were transferred to nitrocellulose filters by the procedure of Southern and hybridized with [alpha- 32 P]dCTP labelled pHC624 prepared by random labeling (Boehringer, Germany). Hybridization conditions were as described previously (29).
  • 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).
  • Figure 3 shows the fractionation of a mixture of ds-RNA (human Rotavirus genome segments 1-11) 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. Fractionation of a mixture of double-stranded DNA and single-stranded RNA.
  • HCV RNA, HDV RNA and HBV DNA were purified from
  • the nucleic acid in the specimens was allowed to complex with the silica particles.
  • the tubes were subsequently vortexed again and centrifuged for 15 s at 10,000 rpm (24 - hole Hettich KG centrifuge), the supernatant was discarded and the silica-nucleic acid complexes were washed twice with 1 ml of buffer L2 (see herein), twice with 1 ml of 70% (vol/vol) ethanol and once with 1 ml of acetone and were dried at 56° C (10 min); the nucleic acids were then eluted at 56° C (10 min) in either 50 1 of 10 mM Tris.HCl 0.1 mM EDTA pH
  • lysis buffer L6 was made by dissolving 120 g of GuSCN in 100 ml of 0.1 M Tris. HCl (pH 6.4) and, subsequently, 22 ml of 0.2 M EDTA (pH 8.0) and 2.6 g of Triton X-100 were added.
  • Washing buffer L2 was made by dissolving 120 g of GuSCN in 100 ml of 0.1 M Tris.HCl (pH 6.4).
  • EDTA, KCl, MgCl 2 .6H 2 O, NaCl and tri-Sodium citrate dihydrate were obtained from Merck (Darmstadt, Germany).
  • TRIS and BSA were obtained from Boehringer (Mannheim, Germany).
  • Triton X-100 was obtained from Packard (Packard Instruments Co., Inc., Downers, I11, USA).
  • Sodium Dodecylsulfate (SDS) was obtained from Serva (Heidelberg, Germany).
  • the dNTP's and Dextran Sulphate were obtained from Pharmacia (Uppsala, Sweden).
  • Reverse transcriptase Superscript II was purchased from Life Technologies (Gaithersburg, Maryland, USA). DNA polymerase Sequenase 2 was obtained from Amersham (United
  • RNAse H was obtained from Boehringer (Mannheim, Germany). Salmon sperm DNA was obtained from Sigma (St. Louis, USA).
  • protocol R The preparation of the buffers used in protocol R have been described herein, except that the lysis buffer and washing buffers (L10, L11, and L2) used in protocol R for the isolation of nucleic acids were filtered through a column packed with Diatoms (27) in order to remove any endogenous nucleic acids in the lysis buffer and washing buffers.
  • lysis buffer and washing buffers L10, L11, and L2
  • the 10 x reverse transcription buffer (CMB1) consists of 100 mM Tris.HCl (pH 8.5), 500 mM KCl and 1% Triton X-100.
  • the 10 x PCR buffer consists of 500 mM Tris.HCl (pH 8.3), 200 mM KCl and 1 mg/ml BSA.
  • the elution buffer Tris/EDTA (TE, pH 8.0) consists of 10 mM Tris.HCl (pH 8.0) and 1 mM EDTA (pH 8.0). Primers and probes
  • the primer used for reverse transcription of HCV RNA was HCV-6: 5'ACC.TCC 3' (nt 319-324, nt numbering according to (23).
  • the anti sense PCR primer for HCV was RB-6B: 5' ACT .CGC.MG.CAC.CCT.ATC.AGG 3'(nt 292-312) and the sense PCR primer was RB-6A:5 ' GTG . AGG . AAC . TAC . TGT . CTT . CAC . G 3 '(nt 47- 68).
  • the oligonucleotide RB-6P 5 ' TTG.GGT.CGC.GM.AGG.CCT.TGT.
  • GGT.ACT.G 3'(nt 264291) was labelled at the 5-end with digoxiginine and was used as a probe in hybridization experiments to determine the specificity of PCR products.
  • the HCV oligos were specific for the 5' untranslated region of the HCV genome.
  • the primer used for reverse transcription of HDV RNA was E21: 5 'CCT.CGA.GM.CM.GM.GM.GC 3' (nt
  • the primers used for HDV PCR were E21, the primer also used for reverse transcription, and E22: 5 ' CGG.CTG.GGC.MC.ATT.CCG.AG 3 (nt 718-737).
  • the plasmid pG4Z(D3) was a generous gift of John Taylor (Fox Chase Cancer Center, Philadelphia) and it contains 3 complete cDNA copies of the HDV genome in tandem, cloned in the Eco RI site of pGem4Z (Promega).
  • pG4Z(D3) Through digestion of pG4Z(D3) with Bgl II , isolation of the 4.4 kb fragment from an agarose gel, ligation and transfection into E.coli C 600, plasmid pG4Z(D1) was obtained containing a single cDNA copy of the HDV genome.
  • pG4Z(Dl) was labelled with digoxigenine-dUTP by random primer DNA synthesis according to the manufacturer's protocol (Boehringer
  • the primers used for HBV PCR were LBL: 5
  • the plasmid PCP10 contains in tandem two complete copies of the HBV genome inserted at the Eco RI site of pBR 322 and was a generous gift of R. Heytink
  • pHBt-III was derived from PCP10 by Eco RI digestion, isolation of the HBV Eco RI linear from an agarose gel and ligating it into the Eco RI cleaved high copy plasmid pHC 624 (28). pHBtlll was labelled with digoxigenine-dUTP by random primer DNA synthesis according to the manufacturer s protocol (Boehringer
  • Reverse transcription was performed in a 25 I reaction volume containing 20 U of RNase inhibitor (Promega Biotec, Madison, Wis.), 67 mM Tris.HCl pH 8.8, 17 mM Ammonium
  • deoxynucleoside Triphosphates 11.5 ⁇ l of the 50 ⁇ l eluate from the nucleic acid purification (see above), and 200 U of superscript reverse transcriptase I (GIBCO-BRL,
  • the PCR was performed in a 50 ⁇ l volume containing, 2.5 U of Taq polymerase (Perkin Elmer Cetus), 50 mM Tris.HCl pH 8.3, 20 mM KCI, 1,2 mM MgCl 2 and 1 mg/ml BSA), 12.5 ⁇ l of the RT reaction mix, 200 uM of each deoxynucleoside
  • Samples were denaturated at 95°C for 5 min and subjected to 35 rounds of thermal cycling in a DNA thermal cycler (type 480; Perkin Elmer Cetus).
  • a cycle consisted of denaturation for 1 min at 95°C, annealing for 1 min at 55°C, and
  • HDV RNA and HBV DNA are co-purified by the method described above.
  • HBV DNA is poorly eluted from the silica particles in TE at 56°C presumably because of co-purification of the protein which is covalently bound to the HBV DNA genome.
  • 7 ⁇ l of the eluate (25 ⁇ l) was taken from the tube for HDV RT-PCR.
  • 18 ⁇ l TE containing 200 ng proteinase-K/ml was added and the tube was incubated for 10 min at 56°C. After heating at 95°C for 10 min to inactivate the proteinase and centrifugation (1 min at 12,000 x g), 20 ⁇ l of the elate was used for HBV PCR.
  • Annealing of the primer with template HDV RNA was performed in a mixture containing 7 ⁇ l eluate, 1 ng of antisense primer E21, 50 mM Tris.HCl pH 8,3, 40 mM KCI, 6 mM MgCI 2 , 10 mM dithiothreitol, and 150 ⁇ M (each)
  • deoxynucleoside triphosphates dNTPs
  • AMV reverse transcriptase Boehringer Mannheim GmbH
  • RT was performed for 1 hr at 42°C in a total volume of 10 ⁇ l.
  • Reverse transcriptase was denatured by incubation for 5 min at 95°C (27).
  • PCR was performed in a reaction mixture of 100 ⁇ l containing 200 ng primer E21, 200 ng primer E22, 50 mM Tris.HCl pH 8.3, 20 mM KCI, 1 mM MgCI2, 0.1 mg BSA, 1.5 U Taq polymerase, 200 uM of each deoxynucleoside
  • the HBV PCR was performed in a 100 ⁇ l reaction mixture containing 200 ng primer LBL, 200 ng primer RAL, 50 mM Tris.HCl pH 8.3, 20 mM KCI, 1 mM MgCI2, 0.1 mg BSA, 1.5 U Taq polymerase, 200 uM of each deoxynucleoside triphosphate and 20 ⁇ l of the eluate. After incubation for 5 min at 95°C the sample was subjected to 35 cycles of amplification. The same cycling program was used as for HCV and HDV.
  • HBV and HDV containing plasmids were labelled with digoxigenine-dUTP by random priming (Boehringer Mannheim GmbH). Nitrocelluse filters were pretreated as described previously (26). Hybridization and post hybridization washings were as previously described. Detection of
  • digoxigenine was done according to the protocol provided by the manufacturer of the kit (Boehringer Mannheim GmbH).
  • HCV sequences from nt 47 to 1032 were cloned after RT PCR into the pSP 64 (poly A) vector (Promega, Madison, Wis). The presence of the right insert was confirmed by DNA sequence analysis. The construct was named pMOZ.1.HCV.
  • HCV template RNA was transcribed in vitro from
  • RNAsin ribonucloside Triphosphate
  • 10 mM dithiothreitol 10 mM dithiothreitol
  • 40 mM Tris-Hcl PH 7.5
  • 6 mM Mgcl2 6 mM spermidine
  • 10 mM NaCl 10 mM NaCl in a total reaction volume of 100 ul.
  • the DNA template was degraded by two rounds of digestion with RNase free DNase (Boehringer) for 30 min at 37°c with 10 U of enzyme. Upon completion of the digestion, 2 rounds of extraction using phenol-chlorophorm-isopropyl alcohol, followed by ethanol precipitation were done.
  • the HCV RNA transcripts which contained a poly (A) Tail, were further purified on an oligo dT cellulose column.
  • the RNAsin 10 mM dithiothreitol
  • 40 mM Tris-Hcl PH 7.5
  • HDV RNA was synthesized and except for the oligo-dT cellulose selection, purified in a similar way as described above for HCV RNA.
  • pG4B (D1) linearized with Hind III was the substrate for SP6 RNA polymerase.
  • Enzyme-Linked Immunosorbent Assay (ELISA) kits were, provided by Abbott Laboratories (Chicago, Illinois), or Sorin Biomedica (Sallugia, Italy) were used for the

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Abstract

Procédés permettant de séparer aisément une substance composée d'acides nucléiques monocaténaires (HCV) d'une substance composée d'acides nucléiques bicaténaires, ces substances étant contenues dans un échantillon. Par le choix judicieux d'au moins un agent chaotropique, de préférence un sel de guanidine, à une concentration sélectionnée, et d'autres conditions appropriées, relatives à la présence d'agents chélateurs, à la valeur du pH, etc., il est possible de lier une substance bicaténaire à une phase solide, telle que des particules de silice, alors que, dans ces mêmes conditions, une substance monocaténaire ne se lie pas. En séparant les particules de silice de l'échantillon, on en extrait en même temps la substance composée d'acides nucléiques bicaténaires, qui peut être aisément séparée par élution des particules de silice. Au cours d'une deuxième étape, on peut lier la substance composée d'HCV monocaténaire à une phase solide en sélectionnant un ensemble de conditions différentes. De nouveau, on peut séparer les particules de l'échantillon, et la substance monocaténaire peut alors être extraite par élution. On peut répéter ce processus pour améliorer l'efficacité de séparation. Après séparation, chaque type d'acide nucléique peut être amplifié. On décrit des procédés d'amplification ne nécessitant pas de données de séquençage de la substance à amplifier. Selon ces procédés, une amorce sera pourvue d'un motif d'amplification et d'un motif d'hybridation aléatoire.
PCT/NL1997/000167 1996-04-03 1997-04-03 Isolation et/ou amplification d'acides nucleiques du virus de l'hepatite c (hcv) a partir d'echantillons susceptibles de contenir ce virus WO1997037040A2 (fr)

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AU21809/97A AU2180997A (en) 1996-04-03 1997-04-03 Isolation and/or amplification of hepatitis c virus (hcv) nucleic acids from samples suspected to contain hcv

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NL1002781A NL1002781C1 (nl) 1996-04-03 1996-04-03 Isolatie en/of amplificatie van hepatitis-C-virus-(HCV) -nucleïnezuren uit monsters waarvan vermoed wordt dat zij HCV bevatten.
NL1002781 1996-04-03

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006031764A1 (de) * 2006-07-06 2008-01-10 Aj Innuscreen Gmbh Verfahren zur parallelen Isolierung doppel- und einzelsträngiger Nukleinsäuren sowie zur selektiven Entfernung doppelsträngiger Nukleinsäuren aus einem Gemisch von doppel- und einzelsträngigen Nukleinsäuren
EP2663649A1 (fr) * 2011-01-10 2013-11-20 American University In Cairo Détection directe de l'arn non amplifié du virus de l'hépatite c à l'aide de nanoparticules d'or non modifiées

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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
WO1995034569A1 (fr) * 1994-06-14 1995-12-21 Invitek Gmbh Procede universel d'isolement et de purification d'acides nucleiques a partir de quantites extremement reduites de differents materiaux de depart fortement contamines
WO1996003528A2 (fr) * 1994-07-27 1996-02-08 Cambridge University Technical Services Limited Oligonucleotides et leur utilisation

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EP0389063A2 (fr) * 1989-03-23 1990-09-26 Akzo Nobel N.V. Procédé de purification d'acides nucléiques
EP0488243A1 (fr) * 1990-11-30 1992-06-03 Sanwa Kagaku Kenkyusho Co., Ltd. Procédé d'extraction du génome du virus d'un échantillon derivé d'un corps vivant infecté par le virus et procédé de détection du génome
US5155018A (en) * 1991-07-10 1992-10-13 Hahnemann University Process and kit for isolating and purifying RNA from biological sources
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DE102006031764A1 (de) * 2006-07-06 2008-01-10 Aj Innuscreen Gmbh Verfahren zur parallelen Isolierung doppel- und einzelsträngiger Nukleinsäuren sowie zur selektiven Entfernung doppelsträngiger Nukleinsäuren aus einem Gemisch von doppel- und einzelsträngigen Nukleinsäuren
DE102006031764B4 (de) * 2006-07-06 2009-10-01 Aj Innuscreen Gmbh Verfahren zur parallelen Isolierung doppel- und einzelsträngiger Nukleinsäuren sowie zur selektiven Entfernung doppelsträngiger Nukleinsäuren aus einem Gemisch von doppel- und einzelsträngigen Nukleinsäuren
EP2663649A1 (fr) * 2011-01-10 2013-11-20 American University In Cairo Détection directe de l'arn non amplifié du virus de l'hépatite c à l'aide de nanoparticules d'or non modifiées
EP2663649A4 (fr) * 2011-01-10 2014-08-27 American University In Cairo Détection directe de l'arn non amplifié du virus de l'hépatite c à l'aide de nanoparticules d'or non modifiées

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