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WO1992007957A1 - Procedes et compositions servant a reduire au minimum l'inhibition de l'amplification d'acides nucleiques - Google Patents

Procedes et compositions servant a reduire au minimum l'inhibition de l'amplification d'acides nucleiques Download PDF

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Publication number
WO1992007957A1
WO1992007957A1 PCT/US1991/007895 US9107895W WO9207957A1 WO 1992007957 A1 WO1992007957 A1 WO 1992007957A1 US 9107895 W US9107895 W US 9107895W WO 9207957 A1 WO9207957 A1 WO 9207957A1
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nucleic acid
compound
amplification
composition
reaction mixture
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George D. Cimino
Stephen T. Isaacs
John J. Sninsky
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    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the present invention relates to nucleic acid amplification techniques. More particularly, the invention relates to methods and compositions for use with agents employed to prevent further amplification of nucleic acids.
  • nucleic acid technology has made possible the manipulation, selection and characterization of a large number of eukaryotic, pro aryotic and viral genes.
  • the application of nucleic acid amplification techniques has provided access to greater volumes of nucleic acid within relatively short periods of time, thereby speeding each of these processes.
  • PCR poly erase chain reaction
  • the basic PCR technique for increasing the concentration of a target sequence (amplification) in a nucleic acid mixture is described in U.S. Patent Nos. 4,683,195 and 4,683,202.
  • This amplification technique consists of introducing a large excess of two oligonucleotide primers to the nucleic acid mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a nucleic acid polymerase, usually Thermus a ⁇ uaticus (Tag) polymerase.
  • the primers are complementary to their respective strands of region surrounding the double-stranded target sequence.
  • the mixture is denatured and then the primers are allowed to anneal to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form complementary strands.
  • Each of these new products then forms a new target sequence capable of serving as a template for the primer pair.
  • the steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e. denaturation, annealing and extension constitute one "cycle;” there can be numerous “cycles") to obtain a high concentration of an amplified segment of the desired target sequence. Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR-amplified".
  • nucleic acid sequence carryover can occur either through aerosols containing nucleic acid sequences or through direct introduction.
  • activation compounds particularly photoreactive compounds
  • Photoactivation compounds are activated by activating wavelengths of electromagnetic radiation.
  • the amplification reaction is first performed in the presence of photoactivation compounds but in the absence of the activating wavelength. Once the reaction has been completed, an appropriate wavelength is used to activate the photoactivation compounds, thereby sterilizing the large quantity of amplified nucleic acid. Sterilization is believed to be achieved in part because,-- upon activation, these photoreactive compounds form covalently-bound photoadducts with nucleic acids. In certain PCR amplification systems it is necessary to operate with high concentrations of sterilizing compounds in order to ensure that carryover is avoided.
  • compositions useful in nucleic acid amplification techniques which increase the amplification of the desired segment of a target sequence in the presence of an effective amount of a sterilizing compound used to avoid carryover.
  • nucleic acid amplification reaction mixture composition comprising a nucleic acid sample containing at least one nucleic acid sequence of interest, all standard reagents necessary to amplify at least a portion of said nucleic acid sequence, at least one amplification enzyme, at least one sterilizing compound, and a compound selected from the group consisting of DMSO and glycerol.
  • Another aspect of the invention provides a method for nucleic acid amplification comprising the steps of a) providing, in any order, a nucleic acid sample containing at least one amplifiable nucleic acid sequence, all standard reagents necessary to amplify at least a portion of said nucleic acid sequence, at least one amplification enzyme capable of initiating an amplification reaction resulting in a nucleic acid product, at least one sterilizing compound, and a compound selected from the group consisting of DMSO and glycerol; b) adding, in any order, the nucleic acid sample, the amplification reagents, the sterilizing compound and the selected compound, to form a reaction mixture; and c) adding the amplification enzyme to initiate the amplification reaction. It is desired that the selected compound is added in an amount sufficient to increase the amount of nucleic acid product obtained from said nucleic acid sample by the amplification reaction performed in the presence of said sterilizing compound.
  • Another aspect of the invention provides a method for nucleic acid sterilization comprising the steps of a) providing, in any order, a nucleic acid sample containing at least one amplifiable nucleic acid sequence, all standard reagents necessary to amplify at least a portion of said nucleic acid sequence, at least one amplification enzyme capable of initiating an amplification reaction resulting in a nucleic acid product, at least one sterilizing compound, and a compound selected from the group consisting of DMSO and glycerol; b) adding, in any order, the nucleic acid sample, the amplification reagents, the sterilizing compound and the selected compound, to form a reaction mixture; c) adding amplification enzyme to the reaction mixture so that the amplifiable nucleic acid sequence is amplified; and d) treating the reaction mixture so that the sterilizing compound renders the amplified nucleic acid substantially unamplifiable.
  • the sterilizing compound is a photoreactive compound and the treating of step (d) comprises photoactivating the photoreactive compound.
  • the nucleic acid is DNA or RNA
  • the standard amplification reagents and the amplification enzyme are those appropriate to the particular amplification system.
  • the amplification enzyme is Tag polymerase.
  • Figure l is a photograph of an autoradiograph of electrophoresed, amplified nucleic acid generated in the presence or absence of unirradiated AMDMIP (200 ⁇ g/ml) using standard PCR conditions in the presence of 0%, 1%, 5% or 10% DMSO.
  • Figure 2 is a graph of photochemical binding results of isopsoralens to DNA in the presence of DMSO, showing that 3 H-AMDMIP binding decreased as the percentage of DMSO was increased.
  • Figure 3 is a photograph of an autoradiograph of electrophoresed, amplified nucleic acid generated in the presence of unirradiated or irradiated AMDMIP (100 ⁇ g/ml) using standard PCR conditions in the presence of DMSO.
  • Figure 4 is a photograph of an autoradiograph of electrophoresed, amplified nucleic acid generated in the presence of unirradiated or irradiated AMDMIP (200 ⁇ g/ml) using standard PCR conditions in the presence of DMSO.
  • Figure 5 is a photograph of an autoradiograph of electrophoresed, amplified nucleic acid generated in the presence or absence of unirradiated AMDMIP using standard PCR conditions in the presence of glycerol.
  • the amount of a sterilizing compound capable of rendering amplified nucleic acids subsequently unamplifiable can also inhibit the amplification of the nucleic acid sequence of interest during the amplification reaction.
  • the present invention is directed to an improvement in the above discussed sterilization technique whereby the impact of the unactivated, activation compound on the amplification reaction is minimized.
  • high concentrations of sterilizing compounds may function to stabilize amplified product (particularly long products or products which are exceptionally GC rich) , such that less of the double-stranded product will denature during each amplification cycle. This reduced availability of single-stranded product for subsequent priming and extension would reduce the product yield in each cycle. This reduced efficiency over many cycles would result in drastic reduction in the yield of PCR product.
  • one method to overcome excessive stabilization of amplified product due to the use of sterilizing compounds is to modify the amplification conditions such that denaturation is carried out at higher (above 95°C) temperatures. In so doing, more of the double-stranded product is denatured each cycle, thereby providing more single-stranded target for subsequent priming and extension. While it might be expected that the net result of the modified conditions is a higher yield of product, such a modification has the disadvantage of concomitant inactivation of the amplification enzyme; even Tag polymerase would be inactivated at such temperatures. Thus the modification would require the addition of fresh Tag polymerase after each inactivation. This is both a costly proposition and one that risks further carryover problems.
  • the present invention involves a more desirable solution to the problem.
  • the present invention involves adding a compound selected from the group consisting of dimethylsulfoxide (DMSO) and glycerol to the amplification reaction mixture.
  • DMSO dimethylsulfoxide
  • one aspect of the present invention provides a nucleic acid amplification reaction mixture composition comprising a nucleic acid sample containing at least one nucleic acid sequence of interest, all standard reagents necessary to amplify at least a portion of said nucleic acid sequence, at least one amplification enzyme, at least one sterilizing compound, and a compound selected from the group consisting of DMSO and glycerol.
  • PCR is a method for amplifying target nucleic acid sequences as disclosed in U.S. Patent Nos. 4,683,195 and 4,683,202, the entire contents of which are hereby incorporated by this reference.
  • the standard reagents necessary to amplify a portion of a nucleic acid sequence (“amplification reagents") are defined as those reagents (primers, deoxyribonucleoside triphosphates, etc.) needed for amplification, except for a nucleic acid sequence template and the amplification enzyme. Standard reagents will vary, depending on the amplification reaction employed.
  • Standard PCR reagents will include all reagents necessary to carry out amplification except polymerase and nucleic acid template.
  • Standard PCR reagents normally include the deoxyribonucleoside triphosphates (dCTP, dTTP, etc.) and the nucleic acid primers in an appropriate buffered solvent (usually aqueous) (See, e*»g*», PCR Technology, supra) .
  • nucleic acid sample which comprises nucleic acid sequences which may contain a target sequence of interest.
  • a target sequence will include a nucleic acid template for the amplification reaction.
  • nucleic acid sample Any source of nucleic acid, in purified or non-purified form, can be utilized as the source for the nucleic acid sample.
  • the practice of the invention may employ any nucleic acid, for example DNA or RNA, including messenger RNA, which may be single-stranded or double-stranded.
  • a DNA-RNA hybrid which contains one strand of each distinct nucleic acid may be utilized. It is also possible to utilize a mixture of any one or more of such nucleic acids.
  • the nucleic acid sample does not need to be provided in pure form; it may be a fraction of a more complex mixture, e.g., it may constitute only a minor fraction of a particular sample of biological origin.
  • the present invention utilizes "sterilizing compounds” and methods for using “sterilizing compounds.”
  • “Sterilizing compounds” are defined such that, when used to treat nucleic acid according to the sterilization method of the present invention, the nucleic acid is rendered substantially unamplifiable, i.e. substantially sterilized.
  • the preferred sterilizing compounds of the present invention are activation compounds.
  • photoreactive compounds are members of the activation compound family that undergo chemical change in response to electromagnetic radiation. Generally, such compounds are capable of forming covalent bonds with nucleic acids. Such photoreactive compounds include those identified in Table 1.
  • furocoumarins A presently preferred genus of photoreactive compounds is commonly referred to as the furocoumarins.
  • the furocoumarins belong to two main categories:
  • psoralens [7H-furo(3,2-g)-(l)-benzopyran-7-one, or 5-lactones of 6-hydroxy-5-benzofuranacrylic acid] , which are linear and in which two oxygen residues appended to the central aromatic moiety have a 1, 3 orientation, and further in which the furan ring moiety is linked to the 6 position of the two ring coumarin system, and
  • isopsoralens [2H-furo(2,3-h)-(l)-benzopyran-2-one, or ⁇ S-lactones of 4-hydroxy-5-benzofuranacrylic acid], which are angular and in which the two oxygen residues appended to the central aromatic moiety have a 1, 3 orientation, and further in which the furan ring moiety is linked to the 8 position of the two ring coumarin system.
  • Psoralen derivatives are obtained by substitution of the linear furocoumarin at the 3, 4, 5, 8, 4', or 5' positions
  • isopsoralen derivatives are obtained by substitution of the angular furocoumarin at the 3, 4, 5, 6, 4 1 , or 5' positions.
  • Presently preferred photoreactive compounds are isopsoralens.
  • the isopsoralen(s) is selected from the group consisting of 5- methylisopsoralen (MIP) , 5-aminomethylisopsoralen (AMIP) , and their radiolabelled derivatives.
  • the isopsoralen is selected from the group consisting of 4,5*- dimethylisopsoralen (DMIP) , 4'-aminomethyl-4,5•- dimethylisopsoralen (AMDMIP) , and their radiolabelled derivatives.
  • DMIP 4,5*- dimethylisopsoralen
  • AMDMIP 4'-aminomethyl-4,5•- dimethylisopsoralen
  • a mixture of isopsoralens will be used. While the preferred sterilizing compound for controlling carryover according to the method of the present invention is an isopsoralen, the present invention contemplates amplification reaction mixtures with psoralens as well.
  • the linear furocoumarin 4'-aminomethyl-4,5• , 8-trimethylpsoralen (AMT) is used as a post-amplification agent to render the nucleic acid substantially refractory to further amplification.
  • compositions of the invention utilize compounds selected from the group consisting of glycerol and dimethylsulfoxide (DMSO) .
  • DMSO dimethylsulfoxide
  • these compounds have been used in particular nucleic acid techniques. See Nucleic Acid Hybridization: A Practical Approach. Hames and Higgins, Ed. (1985) .
  • these compounds have been used in protocols for the PCR.
  • DMSO has been reported to improve DNA sequencing reactions using the PCR with Sequenase*TM (Winship, P.R., "An Improved Method For Directly Sequencing PCR Amplified Material Using Dimethyl Sulfoxide," Nucl. Acids Res.
  • Standard amplification reagents are defined as those reagents (primers, deoxyribonucleoside triphosphates, etc.) needed for nucleic acid amplification, except for nucleic acid sequence template and the amplification enzyme.
  • Such reagents are specified in detail in publications known to those in the art (See, e.g. Saiki, R.K., "The Design and Optimization of the PCR," in PCR Technology, supra and Chamberlain, J.S. et al. in PCR Protocols. A Guide To Methods and Applications, supra) .
  • the composition of the present invention will generally be held in a standard reaction vessel such as a test tube or microwell.
  • the sterilizing compound used in the composition of the invention is preferably an isopsoralen.
  • a preferred isopsoralen is 4'- aminomethyl-4,5•-dimethylisopsoralen (AMDMIP).
  • AMDMIP is a known compound, the synthesis of which is described in U.S. Patent No. 4,312,883 to Baccichetti et al., the contents of which are hereby incorporated by reference.
  • M millimolar
  • mM millimolar
  • ⁇ M micromolar
  • all quantities are given as moles (mol), millimoles (mmol) ,.micromoles ( ⁇ mol) or nanomoles (nmol) and all volumes are given as liters (1) or milliliters (ml) , unless otherwise indicated.
  • MW molecular weight
  • OD optical density
  • EDTA ethylenediaminetetraacetic acid
  • TE buffer buffer: lOmM Tris/1 mM EDTA, pH 7.5
  • TBE buffer Tris-Borate-EDTA
  • Tris-Acetate-EDTA Tris-Acetate-EDTA
  • Taq buffer 50mM KC1, 2.5 MgCl 2 , lOmM Tris, pH 8.5, 200 ⁇ g/ml gelatin
  • PAGE polyacrylamide gel electrophoresis
  • V voltage
  • W watts
  • mA milliamps
  • bp base pair
  • kb kilobase pairs
  • CPM counts per minute
  • PCR was carried out using 175-200 ⁇ M dNTPs (deoxyribonucleoside 5'-triphosphates) and 0.5 to 1.0 ⁇ M primers. 5 Units/ 100 ⁇ l of Taq polymerase was used. PCR reactions were overlaid with 30-100 ⁇ l light mineral oil.
  • a typical PCR cycle for HIV amplification using a Perkin-Elmer Cetus DNA Thermal Cycler (Part No. N8010150) was: denaturation at 93°C for 30 seconds; annealing at 55°C for 30 seconds; and extension at 72°C for 1 minute. PCR cycles were normally carried out in this manner for 30 cycles followed by 7 minutes at 72°C.
  • amplification of HLA Class II genes was performed using primer pair RS-134/RS-135 and human placental DNA to produce a 242- mer product.
  • the sequences of these primers are: RS-134 5'-GTGCTGCAGGTGTAAACTTGTACCAG-3' RS-135 5•-CACGGATCCGGTAGCAGCGGTAGAGTTG-3• These primers and other primers are described in PCR Protocols: A Guide To Methods and Applications f Innis, M.A. et al. eds., pp. 261-271 (1990)).
  • the sequence of the 242-mer product is:
  • amplification of HIV sequences was performed using primer pair SK-145/SK-431 and plasmid pBKBHIOS, to produce a 142-mer product.
  • the sequences of these primers are:
  • AGAACCAAGGGGAAGTGACATAGCA-3 The plasmid is described in AIDS Research and Ref rence Reagent Program Catalog. NIH Publication No. 90-1536 (1990) , and was contributed by Dr. John Rossi to the repository established by the National Institute of
  • Photoactivation was performed using a device ("HRI-100") sold commercially by HRI Research Inc. (Berkeley, California, USA) and ULTRA-LUM, INC. (Carson, California, USA) .
  • RS-134/RS-135 is inhibited at AMDMIP concentrations above lOO ⁇ g/ml (3.5 x 10 ⁇ 4 M) , which is believed to result from stabilization of the 242-mer amplicon. If concentrations higher than lOO ⁇ g/ml are required for effective use of AMDMIP as a sterilizing compound, one possibility is to use DMSO during PCR.
  • Nucleic acid samples were prepared which contained l ⁇ g of human placental DNA, either with or without (unirradiated) AMDMIP (200 ⁇ g/ml) .
  • the samples were amplified for 30 cycles under standard PCR conditions in the presence of 0%, 1%, 5% or 10% DMSO.
  • the reaction mix contained ⁇ - 32 P-dCTP.
  • the samples were analyzed by PAGE. The results ( Figure 1) indicated that, while
  • nucleic acid samples were prepared containing lO ⁇ g/ml calf thymus DNA and 50 ⁇ g/ml 3 H-AMDMIP in 1XTE buffer with
  • Amplification of the HLA locus using primer pair RS-134/RS-135 is inhibited by AMDMIP concentrations greater than lOO ⁇ g/ml (3.5 x 10 ⁇ 4 M) .
  • AMDMIP concentrations greater than lOO ⁇ g/ml (3.5 x 10 ⁇ 4 M) .
  • 3% or 5% DMSO allows amplification to proceed in the presence of 200 ⁇ g/ml AMDMIP.
  • the ability of AMDMIP to sterilize the 242-mer amplicon with DMSO present is shown below.
  • Amplification of the HLA locus with primer pair SK-145/SK-431 is inhibited at AMDMIP concentrations above lOO ⁇ g/ml (3.5 x l ⁇ "4 M) .
  • the inhibitory effect due to isopsoralen i.e., low amplification efficiency
  • isopsoralen i.e., low amplification efficiency
  • concentrations higher than lOO ⁇ g/ml are required for effective use of AMDMIP as a sterilizing compound, one possibility is to use glycerol during PCR.
  • Nucleic acid samples were prepared which contained
  • the DMSO or glycerol effect is quite broad, and will reestablish amplification of a low copy sample (e.g., 10 copies) in the presence of a high concentration of isopsoralen. While the precise mechanism is not clear, these results may be understood by considering that the presence of positively charged isopsoralens act to stabilize the double-stranded amplicon and that these effects will be more pronounced as the length of the amplicon increases.
  • the inhibitory effect may be concealed because the reaction still achieves plateau after 30 cycles. When this occurs, the amplification efficiency appears to be the same. As the starting copy number decreases, plateau is no longer reached and differences in PCR efficiency become apparent.
  • the interaction between the isopsoralen and the DNA prior to photoreaction is weakened, perhaps reducing the enhanced stability of the amplicon duplex. While a concomitant reduction in isopsoralen photoaddition to DNA in the presence of effective concentrations of DMSO or glycerol is found, the observed reduction in photoreactivity is so great as to impact sterilization significantly. It should be emphasized that careful selection of the sterilizing compound concentration is important. For example, where the sterilizing compound is an isopsoralen, if the initial copy number is high (e.g., genomic DNA) , a relatively high concentration of isopsoralen is preferred.

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Abstract

L'invention se rapporte à des procédés et à des compositions, qui servent à accroître la quantité de produits obtenus par des réactions d'amplification d'acides nucléiques et dont le mélange de réaction contient des agents qui rendent les acides nucléiques, après leur amplification, réfractaires à toute autre amplification et qui inhibent la réaction d'amplification.
PCT/US1991/007895 1990-10-31 1991-10-24 Procedes et compositions servant a reduire au minimum l'inhibition de l'amplification d'acides nucleiques Ceased WO1992007957A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
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WO1993025706A1 (fr) * 1992-06-05 1993-12-23 Buchardt, Dorthe Utilisations d'analogues d'acide nucleique pour inhiber les procedures d'amplification de l'acide nucleique
WO1995014790A1 (fr) * 1993-11-23 1995-06-01 Ciba Corning Diagnostics Corp. Utilisation d'oligomeres antisens dans un procede destine a limiter la contamination dans les reactions d'amplification de l'acide nucleique
EP0587298A3 (en) * 1992-08-04 1995-08-30 Gen Probe Inc Nucleic acid sequence amplification method
US5766849A (en) * 1989-07-11 1998-06-16 Gen-Probe Incorporated Methods of amplifying nucleic acids using promoter-containing primer sequence
US5846489A (en) * 1994-04-09 1998-12-08 Boehringer Mannheim Gmbh System for opening closures of vessels and for the contamination-free operation of reaction sequences
US6187572B1 (en) 1990-04-16 2001-02-13 Baxter International Inc. Method of inactivation of viral and bacterial blood contaminants
WO2002014548A1 (fr) * 2000-08-10 2002-02-21 Applied Gene Technologies, Inc. Compositions et procedes de traitement et d'amplification d'echantillons d'acides nucleiques
US6448047B2 (en) 1999-07-30 2002-09-10 Applied Gene Technologies, Inc. Sample processing to release nucleic acids for direct detection
US6686162B2 (en) * 2001-12-04 2004-02-03 Quest Diagnostics Investments, Incorporated Oligonucleotides and methods for detecting Borrelia burgdorferi
US7009041B1 (en) 1989-07-11 2006-03-07 Gen-Probe Incorporated Oligonucleotides for nucleic acid amplification and for the detection of Mycobacterium tuberculosis

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WO1990001563A1 (fr) * 1988-08-01 1990-02-22 Cimino George D Identification de sequences d'acide nucleique specifiques d'alleles par hybridation au moyen de sondes d'oligonucleotides reticulables
EP0362042A1 (fr) * 1988-09-26 1990-04-04 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procédé d'analyse d'une séquence spécifique d'ADN ou d'ARN, réactifs et nécessaires pour sa mise en oeuvre
EP0407291A1 (fr) * 1989-07-03 1991-01-09 Institut Pasteur Perfectionnements apportés aux techniques d'amplification d'acide nucléique
WO1991006665A1 (fr) * 1989-10-26 1991-05-16 Isaacs Stephen T Composes d'activation et procedes de sterilisation de l'acide nucleique

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Title
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NUCLEIC ACIDS RESEARCH vol. 18, no. 22, November 1990, ARLINGTON, VIRGINA page 6739; JINNO,Y. ET AL.: 'USE OF PSORALEN AS EXTINGUISHER OF CONTAMINATED DNA IN PCR' *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5766849A (en) * 1989-07-11 1998-06-16 Gen-Probe Incorporated Methods of amplifying nucleic acids using promoter-containing primer sequence
US5908744A (en) * 1989-07-11 1999-06-01 Gen-Probe Incorporated Detecting Mycobacterium tuberculosis by nucleic acid sequence amplification
US7009041B1 (en) 1989-07-11 2006-03-07 Gen-Probe Incorporated Oligonucleotides for nucleic acid amplification and for the detection of Mycobacterium tuberculosis
US6187572B1 (en) 1990-04-16 2001-02-13 Baxter International Inc. Method of inactivation of viral and bacterial blood contaminants
WO1993025706A1 (fr) * 1992-06-05 1993-12-23 Buchardt, Dorthe Utilisations d'analogues d'acide nucleique pour inhiber les procedures d'amplification de l'acide nucleique
EP0587298A3 (en) * 1992-08-04 1995-08-30 Gen Probe Inc Nucleic acid sequence amplification method
WO1995014790A1 (fr) * 1993-11-23 1995-06-01 Ciba Corning Diagnostics Corp. Utilisation d'oligomeres antisens dans un procede destine a limiter la contamination dans les reactions d'amplification de l'acide nucleique
US5763186A (en) * 1993-11-23 1998-06-09 Chiron Diagnostics Corporation Use of antisense oligomers in a process for controlling contamination in nucleic acid amplification reactions
US5846489A (en) * 1994-04-09 1998-12-08 Boehringer Mannheim Gmbh System for opening closures of vessels and for the contamination-free operation of reaction sequences
US6448047B2 (en) 1999-07-30 2002-09-10 Applied Gene Technologies, Inc. Sample processing to release nucleic acids for direct detection
WO2002014548A1 (fr) * 2000-08-10 2002-02-21 Applied Gene Technologies, Inc. Compositions et procedes de traitement et d'amplification d'echantillons d'acides nucleiques
US6686162B2 (en) * 2001-12-04 2004-02-03 Quest Diagnostics Investments, Incorporated Oligonucleotides and methods for detecting Borrelia burgdorferi

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