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WO1992013963A1 - Procede de preparation d'adn circulaire ferme - Google Patents

Procede de preparation d'adn circulaire ferme Download PDF

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Publication number
WO1992013963A1
WO1992013963A1 PCT/US1992/000540 US9200540W WO9213963A1 WO 1992013963 A1 WO1992013963 A1 WO 1992013963A1 US 9200540 W US9200540 W US 9200540W WO 9213963 A1 WO9213963 A1 WO 9213963A1
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nuclease
dna
exonuclease
circular dna
enzymes
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PCT/US1992/000540
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English (en)
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Edward D. Hyman
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Hyman Edward D
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Publication of WO1992013963A1 publication Critical patent/WO1992013963A1/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
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Definitions

  • This application relates to a new method for preparing plasmid and other closed circular DNA which is readily automated.
  • Plasmids are small circular pieces of DNA found naturally and as a result of human intervention in many bacteria, particularly E. coli. Because many copies of an individual plasmid are freguently present within the host organism, and because plasmids are both reproduced and in many cases expressed by the host organism, plasmids have come to play a major role in many biotechnological processes. Indeed, the preparation of plasmid DNA containing a nucleic acid fragment of interest is a routine procedural precursor to such other procedures as DNA seguencing, restriction digestion, cloning, probing, amplification (e.g., PCR) , hybridization, in vitro transcription or mutagenesis.
  • the boiling method for preparing plasmids uses lysozyme in a boiling water bath to lyse the cells.
  • Chromosomal DNA and cellular debris are removed by centrifugation and the supernatant containing the plasmid DNA is treated with sodium acetate and isopropanol to precipitate the plasmids. This precipitate is recovered by centrifugation, and treated with RNase.
  • One commercially available instrument for preparing plasmid DNA contains a built in centrifuge and isolates plasmid DNA using the alkaline lysis method.
  • the throughput of this machine is limited to about 40 plasmid preps per day, the eguivalent of what a single technician can achieve by hand in one day.
  • the human genome project and other efforts which could involve the need for hundreds of thousands of plasmid preparations before completion make it clear that this throughput level is unacceptable. There exists, therefore, a real need for a preparation technique which can be performed rapidly and simply by automated equipment. This invention satisfies that need.
  • closed circular DNA can be recovered from cells or subcellular organelles containing closed circular DNA by a method comprising, in sequence, the steps of:
  • the method is particularly applicable to recovery and purification of plasmid DNA from E. coli.
  • Fig. 1 shows an automated instrument for performing the method of the invention
  • Fig. 2 shows a modified microtiter sample holder useful in automating the present invention.
  • Figs. 3 and 4 show cross sections of a single sample well.
  • the present invention is applicable generally to the isolation of closed circular DNA, both single and double stranded, from other DNA.
  • the invention is applicable both to the preparation of plasmid samples and to preparation of circular phage DNA (e.g. phage M13 which is frequently used as a cloning vector) and DNA isolated from organelles such as chloroplasts and mitochondria.
  • the method can also be employed in modified form to isolate viral DNA from host nucleic acids.
  • the first step of the method for preparing purified circular DNA in accordance with the invention is lysis of the host microorganism.
  • lysis can be accomplished by a combination of enzymatic treatment with lysozyme followed by heat treatment at a ti e and temperature sufficient to accomplish the result of making DNA available for subsequent enzymatic attack.
  • Other methods for cell lysis can also be employed provided that the lysis method does not nick the plasmid DNA. For example, exposure to alkaline conditions (pH 13-14) after lysozyme treatment or species specific-lytic agents such as lysostaphin for staphylococci may be employed.
  • Lyticase or zymolase can be used to lyse yeast cells, while detergent alone should be sufficient in the case of mammalian cells, insect cells (e.g. for baculovirus isolation), chloroplasts or mitochondria.
  • Cellulase may be a suitable lytic enzyme for isolation from plant cells. Following cell lysis, the cell preparation is enzymatically treated with a protease enzyme to destroy the cell structure and free nucleic acids from proteins (e.g., histones) which might interfere with subsequent enzymatic degradation steps.
  • a suitable enzyme for this purpose is Proteinase K, a commercially available protease secreted extracellularly by Tritirachium album (Sigma Chemical Co., St. Louis, MO).
  • Proteinase K is a serine endoproteinase of the bacterial subtilisin family that rapidly and non-specifically hydrolyzes native proteins. Conveniently, proteinase K remains active in the presence of urea and SDS and other detergents such that denaturants may be added to facilitate the proteolytic digestion and is active at relatively high temperatures which allows accelerated proteolysis. Detergents, such as TRITON X-100, may also be added to facilitate the proteolytic digestion. After incubating the lysed bacteria in the protease enzyme for a period of time sufficient to substantially clarify the preparation, the protease enzyme is inactivated to prevent degradation of subsequently added enzymes.
  • This inactivation is conveniently done by heating the preparation, which not only inactivates the protease but also denatures substantially all of the bacterial DNA other than supercoiled and relaxed closed plasmid DNA into single stranded form.
  • Other inactivators may be used (e.g., metal chelators, phenylmethylsulfonyl fluoride, iodoacetate or a change in pH (increasing or decreasing from optimum as appropriate for the enzyme used) or buffer conditions) , however if heat inactivation is ineffective for a given protease, provided that these inactivators do not interfere with subsequent enzymatic steps (e.g., nuclease activity) or can be removed.
  • chemical means for denaturing non-circular DNA may be employed, including the enzyme helicase, exposure to alkali (pH 13-14) , acid (pH 1-4) , urea, dimethylsulfoxide or dimethylformamide or other denaturing conditions.
  • the chromosomal DNA Upon cooling the solution, the chromosomal DNA is likely to remain in single stranded form as a result of the low incidence of highly repetitive sequences in bacterial, e.g. E. coli. genomes.
  • the chromosomal DNA, along with the RNA is susceptible to enzymatic digestion by a variety of nucleases, including single stranded exo- and endonucleases and double stranded exonucleases.
  • Suitable nuclease enzymes for this purpose include Mung Bean Nuclease, SI Nuclease (US Biochemical Corp., “USB”) , PI Nuclease (Bethesda Research Labs, “BRL”) , T7 exonuclease (USB) , Bal 31 Nuclease (USB) , Exonuclease I (USB) , Exonuclease III (USB) , Exonuclease VII (BRL) and Lambda Exonuclease (BRL) .
  • RNA from the microorganism is digested either before or concurrently with the DNA, depending on the conditions needed for enzymatic activity, using an RNase enzyme such as pancreatic ribonuclease (USB) or Ribonuclease T ! (Pharmacia) .
  • an RNase enzyme such as pancreatic ribonuclease (USB) or Ribonuclease T ! (Pharmacia) .
  • URB pancreatic ribonuclease
  • Ribonuclease T ! Pharmacia
  • Bal 31 nuclease will completely hydrolyze supercoiled plasmid DNA due to the fact that supercoiled plasmid exists transiently in single stranded form, which is a substrate for Bal 31. Legerski et al., J. Biol. Chem 252. 8740 (1977). To avoid unwanted digestion of plasmid DNA, enzymes of this type should be used after treatment with an enzyme such as topoisomerase I (BRL) which relaxes supercoiled plasmid DNA. Other enzymes which would achieve this same result would be a combination of DNase I (nickase) , DNA ligase and ATP/NAD; or T4 DNA ligase and AMP.
  • BRL topoisomerase I
  • Both of these combinations would nick supercoiled DNA to yield a nicked, relaxed plasmid, and then repair the nick via the ligase. Relaxation of supercoiled DNA can also be achieved non-enzymatically with intercalating agents such as ethidium bromide.
  • T7 exonuclease degrades the 5' end strand of double.stranded DNA attacking only chromosomal DNA which exists as linear double strands. Ausubel et al., eds. Current Protocols in Molecular Biology, p. 3.12.1. T7 exonuclease will not degrade either supercoiled or relaxed plasmid. In fact, the resistance of plasmids to degradation serves as a quality control test for T7 exonuclease purity used by the manufacturer (USB) .
  • DNA polymerase enzymes which under certain conditions (absence of dNTP's and excess pyrophosphate, i.e., about lOOmM) catalyze the reverse polymerization of double stranded nucleic acids. This activity requires a free 3'-OH group and thus is specific for linear (non-plasmid) DNA. These enzymes also have exonuclease activity for either double or single stranded DNA. Kornberg, DNA Replication. W.H. Freeman & Co., pp 127-130 (1980). Suitable enzymes include Klenow fragment, T7 DNA polymerase (USB) , Taq polymerase and T4 DNA polymerase.
  • Additional enzymes might be used in conjunction with a reverse polymerization system to enhance the rate of degradation. These enzymes include T4 polynucleotidekinase to remove the 3'-terminal phosphate and to yield a 3'-OH group, enzymes selected to degrade the dNTP product, such as hexokinase and nucleoside diphosphate kinase together with appropriate reagents (ADP and glucose) .
  • T4 polynucleotidekinase to remove the 3'-terminal phosphate and to yield a 3'-OH group
  • enzymes selected to degrade the dNTP product such as hexokinase and nucleoside diphosphate kinase together with appropriate reagents (ADP and glucose) .
  • Terminal deoxynucleotidyl transferase could also be theoretically used to achieve selective degradation of single stranded chromosomal DNA in the presence of pyrophosphate.
  • this enzyme requires a free 3'-OH group, but it acts on single stranded as opposed to double stranded nucleic acids.
  • the dNTP's released could be further hydrolyzed with the aid of the same enzyme systems discussed above. This system is not a preferred means for selective single stranded DNA removal, however, as the reaction rate is very slow. Restriction enzymes are potentially useful in the present invention provided the plasmid to be isolated does not have the restriction site for which the enzyme is specific.
  • a simple test can be used to assess the presence or absence of a given restriction site in a plasmid by exposing the plasmid preparation to the restriction enzyme prior to heat treatment and digestion of chromosomal DNA and running a gel to see if plasmid DNA remains. If plasmid DNA is observed, the restriction site is not present in the plasmid. If restriction enzymes are used it is appropriate to maintain chromosomal DNA in a double stranded state until restriction digestion has occurred. This may require lowering the temperature used to denature the protease.
  • nucleases with single-strand endonuclease activity cannot be used as these enzymes will degrade the desired product.
  • a combination of nucleases is selected from among single-strand exonucleases and double-strand exonucleases.
  • Double- strand endonucleases can also be employed provided that the circular DNA does not self anneal to produce double stranded regions or if conditions (temperature/buffer) are maintained to prevent formation of double stranded regions.
  • the final step in the nucleic acid preparation of the present invention is the recovery of plasmid DNA from the solution.
  • Example 1 A 1.5 ml E. coli culture containing the cloning vector pUCll ⁇ was grown overnight to saturation. The E. coli were pelleted and resuspended in 300 ⁇ l of 100 mM Tris-Cl (tris(hydroxymethyl)aminomethane hydrochloride) , 2.5 mM MgCl 2 , 0.5% Triton X-100, pH 8.0. Lysozyme solution, 30 ⁇ l at 10 ⁇ g/ ⁇ l, was added and incubated at 37°C for 30 minutes. The lysozyme alone did not create microscopically visible ruptures in the E. coli cells. The E ⁇ _ coli were heated at 95 °C for 5 minutes, and then cooled to 40°C.
  • Tris-Cl tris(hydroxymethyl)aminomethane hydrochloride
  • Proteinase K solution 10 ⁇ l at 10 ⁇ g/ ⁇ l, was added and the E ⁇ coli were incubated at 50°C for 30 minutes. This enzymatic step resulted in the complete clearing of the solution. Microscopic examination confirmed that no E. coli or fragments were present. Apparently, the lysozyme and heating step had introduced holes in the bacteria allowing the protease to enter and dissolve the cells completely. This lysed solution was again heated at 95°C for 5 minutes, then cooled to 25°C, to inactivate the proteinase K.
  • the contents of the tube were precipitated by adding 35 ⁇ l of 3.0 M NaOAc (pH 5.2) + 800 ⁇ l ethanol, pelleted at 12,000g for 5 minutes, and resuspended in 50 ⁇ l of TE buffer with ribonuclease (10 mM Tris-Cl, 1 mM EDTA, 20 ⁇ g/ml pancreatic ribonuclease, pH 8.0).
  • ribonuclease 10 mM Tris-Cl, 1 mM EDTA, 20 ⁇ g/ml pancreatic ribonuclease, pH 8.0.
  • the preparation at this point was analyzed by gel electrophoresis (0.6% agarose in 40mM TrisOAc, 2 mM EDTA, pH 8.3). Following ethidium bromide staining, evaluation of the gel indicated that the lysate consisted largely of fragmented chromosomal DNA, fragmented RNA, and intact
  • DTT dithiothreitol
  • USB Exonuclease III
  • the DNA was precipitated from the sample using 3.0 M NaOAc and ethanol and stored overnight at -20°C.
  • the DNA was collected by centrifugation at 15,000 g for 5 minutes, washed with 0.5 ml 70% ethanol and dissolved in 40 ⁇ l Exonuclease I buffer (67 mM glycine, 6.7 mM MgCl 2 , 10 mM mercaptoethanol, pH 9.5).
  • Exonuclease I incubation resulted in only a slight improvement in the purity of plasmid DNA, although more improvement might be achieved if more enzyme were used. No degradation of either the supercoiled or relaxed plasmid DNA is observed (compared to pre-exonuclease I control) due to the specificity of Exonuclease I for the ends of single-stranded DNA.
  • the solution was next heated at 95°C for 5 minutes and cooled to 25°C.
  • the DNA was precipitated using 3.0 M NaOAc and ethanol and redissolved in 21 ⁇ l of 30 mM NaOAc, 200 mM NaCl, 1 mM ZnCl 2 , pH 4.6.
  • Example 2 E. coli containing the plasmid pUCll ⁇ was grown by inoculating 500 ⁇ l of TYGPN both (2.0 g tryptone, 1.0 g yeast extract, 800 ⁇ l glycerol 0.5 g sodium phosphate and 1.0 g potassium nitrate in 100 ml of deionized water, pH 7.0.) containing 50 ⁇ g/ml ampicillin.
  • the E. coli was incubated in a sealed 0.5 ml Eppendorf tube at 37°C without agitation or aeration for 10 hours.
  • the cell density before harvesting was about 1.6 X 10 9 cells/ml.
  • the cells were harvested by centrifugation at 12,000g (1 min, 4°C) and resuspended in 300 ⁇ l of 100 mM Tris-Cl, 2.5 mM MgCl 2 , 0.5% TRITON X-100, pH 8.0. 30 ⁇ l of lysozyme (10 mg/ml) was added to the resuspended cells and the mixture was incubated at 37°C for 30 minutes. The mixture was then heated to 95°C for 5 minutes and then cooled to 50°C.
  • Total DNA was precipitated by addition of 3.0 M NaOAc (pH 5.2) +800 ⁇ l ethanol and incubating at -20°C for 30 minutes. The precipitated DNA was recovered by centrifugation (12,000g) for 5 minutes at 4°C. The pellet was recovered, washed with 70% ethanol and dried at room temperature.
  • the pellet was then redissolved in 50 ⁇ l of 50 mM Tris-Cl, 50 mM KCl, 10 mM MgCl 2 , 0.1 mM EDTA, l.'O mM DTT, 20 ⁇ g/ml RNase (USB), pH 7.5. 30 units of Topoisomerase I (BRL) were then added and the mixture was incubated at 37°C for six hours to degrade RNA and relax supercoiled plasmid DNA. At the end of the incubation time, the mixture was heated to 95°C for
  • Total DNA was again recovered by precipitation using 4.5 ⁇ l of 3.0 M NaOAc (pH 5.2) and 110 ⁇ l of ethanol. After 30 minutes of incubation time at -20°C, the precipitated DNA was collected by centrifugation (12000 g, 5 min. 4°C), washed with 70 % ethanol and dried at room temperature. The resulting material, containing both plasmid and chromosomal DNA was dissolved in 40 ⁇ l of 30 mM NaOAc, 200 mM NaCl, 1 mM ZnCl 2 , pH 4.6. 118 units of SI nuclease (USB) were added and the mixture was incubated at 37°C for 60 minutes. The resulting solution was analyzed by agarose gel electrophoresis in 40 mM TrisOAc, 1 mM EDTA, pH 8.3 buffer. The gels showed plasmid DNA of approximately 95% purity.
  • Example 3 The experiment of example 2 was repeated except that T4 DNA ligase and AMP was used in place of topoisomerase to relax the plasmid DNA.
  • the DNA pellet was dissolved in 50 ⁇ l of 50mM Tris-Cl, 10 mM MgCl 2 , ImM DTE (dithioerythritol) , 20 ⁇ g/ml RNase (USB), pH 7.6.
  • Example 4 Ml3 DNA was prepared from a culture of E. coli transformed with M13mpl9 replicative form DNA that had been grown in LB broth overnight with agitation. The culture had achieved saturation density.
  • the dried nucleic acids were dissolved in 75 ⁇ l of 50 mM Tris-Cl (pH 8.0), 10 mM MgCl 2 , 50 mM NaCl. 20 units of Mbo I (BRL) , a restriction enzyme, were added. Following incubation at 37°C for 60 minutes, the mixture was heated to 95°C for 5 minutes and then cooled to 37°C. 20 units of T4 polynucleotide kinase (BRL), 2.5 ⁇ g RNase and 5 ⁇ l of 100 mM DTT were then added and the mixture was incubated at 37°C for 60 minutes.
  • Example 5 A simple modification of the protocols discussed above makes it possible to use the present invention to isolate RNA from a sample. In this case, RNases are omitted and a mixture of DNases is used to degrade all DNA. Selective membrane filtration of the sample using a 25,000 dalton cut-off membrane isolates total RNA including messenger RNA, transfer RNA and ribosomal RNA.
  • Example 6 The method of the invention can be used to isolate viral particles. Lytic viruses produce viral particles within a host cell which are protected by viral coat proteins. Host DNA, on the other hand is exposed to the surrounding environment upon lysis of the cell. Viral DNA/RNA can therefore be recovered, separate from host DNA, by the steps of enzymatic lysis of the cell walls or membranes, addition of nucleases to the lysed cells to degrade host nucleic acids, addition of nuclease inhibitors, proteases or heat to inactivate the nucleases and then treatment with a proteolytic enzyme to destroy the nucleases and remove the viral coat protein.
  • Fig. 1 shows a schematic of a basic instrument for accomplishing this purpose.
  • sample containers 2 are held in a temperature control block 1 and supplied with reagents via an automated pipettor 4 controlled by a computer 5.
  • the temperature of the block can be controlled by introducing a circulating liquid (e.g. water) via line 9 to flow through the block and exit via line 10.
  • Liquid flow can be controlled via value 7 whose operation is controlled to maintain the sample in the holders 2 at the necessary temperatures.
  • Feedstock supplies 6 may conveniently be included within the apparatus housing 3, as may a pump 8 to provide PEG or vacuum to the sample holder as discussed further below.
  • the sample containers may advantageously be microtiter plates having a plurality of wells, the bottom of each of which is in contact through a filtration membrane with a circulating solution of polyethylene glycol
  • Fig. 2 shows schematically a microtiter plate 21 adapted for use in the present invention.
  • Each well 22 of the plate 21 is in contact through an ultrafiltration membrane 24 with a stream of PEG flowing through tube 23.
  • PEG is supplied from vessel 25 into tube 23 and flows through the microtiter plate
  • Fig. 3 shows a cross section of a sample well in the microtiter plate 21 and the adjoining tube 23.
  • PEG solution 31 circulating within tube 23 past ultrafiltration membrane 24 osmotically draws water and materials of molecular weight below the filter's cut- off out of the sample well 22.
  • the height of the impermeable wall 34 defines the final volume of the sample if buffer is not continuously added.
  • Fig. 4 shows an alternative sample well arrangement in which vacuum rather than osmotic pressure is employed.
  • each sample well

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Abstract

On peut récupérer de l'ADN circulaire fermé à partir de cellules ou d'organites sous-cellulaires contenant de l'ADN circulaire fermé selon un procédé comprenant, en séquence, les étapes consistant: (a) à lyser les cellules ou organites afin de libérer l'ADN, par exemple avec une combinaison de lysozyme et de chaleur ou de détergents, (b) à traiter de manière enzymatique l'ADN plasmidique libéré avec une enzyme protéolytique telle que la protéinase K, (c) à traiter de manière enzymatique l'ADN plasmidique traité par enzyme protéolytique avec de la RNase et de la topoisomérase I, (d) à digérer sélectivement de manière enzymatique les acides nucléiques non circulaires provenant du microorganisme, et (e) à récupérer l'ADN circulaire fermé. Le procédé est notamment applicable à la récupération et à la purification d'ADN plasmidique à partir de E. coli.
PCT/US1992/000540 1991-01-30 1992-01-22 Procede de preparation d'adn circulaire ferme WO1992013963A1 (fr)

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US647,789 1991-01-30

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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0517515A3 (en) * 1991-06-04 1993-06-16 Tosoh Corporation Method of purifying dna
WO1995021250A3 (fr) * 1994-02-01 1996-02-15 Vical Inc Production d'adn plasmidique d'une qualite permettant son utilisation dans l'industrie pharmaceutique
WO1996018744A3 (fr) * 1994-12-16 1996-08-29 Rhone Poulenc Rorer Sa Purification d'adn par formation de triple helice avec un oligonucleotide immobilise
EP0987327A1 (fr) * 1998-09-14 2000-03-22 QIAGEN GmbH Methode pour la purification d'ADN circulaire fermé
EP0967274A3 (fr) * 1998-06-15 2000-04-12 Mologen GmbH Procédé pour la purification des molecules d'ADN fermés covalents
EP0992583A1 (fr) * 1998-09-14 2000-04-12 QIAGEN GmbH Methode pour la purification d'ADN circulaire fermé
WO2000049179A1 (fr) * 1999-02-18 2000-08-24 Promega Corporation Procedes de determination de la presence de sequences cibles d'acides nucleiques et leurs applications
WO2000066723A1 (fr) * 1999-05-04 2000-11-09 Millipore Corporation Procede d'ultrafiltration
WO2001029209A1 (fr) * 1999-10-21 2001-04-26 Cobra Therapeutics Ltd Preparation de composants cellulaires sensiblement depourvus d'adn chromosomique
US6235480B1 (en) 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
US6268146B1 (en) 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection
US6270973B1 (en) 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6312902B1 (en) 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6335162B1 (en) 1998-03-13 2002-01-01 Promega Corporation Nucleic acid detection
US6391551B1 (en) 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
US6498240B1 (en) 1999-09-17 2002-12-24 Millipore Corporation Method for sequencing reaction cleanup by constant pressure diffential ultrafiltration
EP1388588A1 (fr) * 2002-07-29 2004-02-11 JSR Corporation Méthode de séparation d'acide nucléiques et solutions pour les extraire
US6703211B1 (en) 1998-03-13 2004-03-09 Promega Corporation Cellular detection by providing high energy phosphate donor other than ADP to produce ATP
US6759233B2 (en) 2000-04-13 2004-07-06 Millipore Corporation Method of plasmid recovery and apparatus for doing so
US7038026B2 (en) 2000-05-26 2006-05-02 Centelion Purification of a triple heli formation with an immobilized oligonucleotide
US7090975B2 (en) 1998-03-13 2006-08-15 Promega Corporation Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection
WO2014071178A1 (fr) * 2012-11-05 2014-05-08 Icahn School Of Medicine At Mount Sinai Procédé d'isolement d'adn mitochondrial pur
US8748168B2 (en) 2004-08-16 2014-06-10 Nature Technology Corp. Strains of E. coli for plasmid DNA production
US10612032B2 (en) 2016-03-24 2020-04-07 The Board Of Trustees Of The Leland Stanford Junior University Inducible production-phase promoters for coordinated heterologous expression in yeast

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Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0517515A3 (en) * 1991-06-04 1993-06-16 Tosoh Corporation Method of purifying dna
WO1995021250A3 (fr) * 1994-02-01 1996-02-15 Vical Inc Production d'adn plasmidique d'une qualite permettant son utilisation dans l'industrie pharmaceutique
US5561064A (en) * 1994-02-01 1996-10-01 Vical Incorporated Production of pharmaceutical-grade plasmid DNA
US6287762B1 (en) 1994-12-16 2001-09-11 Rhone-Poulenc Rorer S.A. Purification of a triple helix formation with an immobilized oligonucleotide
WO1996018744A3 (fr) * 1994-12-16 1996-08-29 Rhone Poulenc Rorer Sa Purification d'adn par formation de triple helice avec un oligonucleotide immobilise
US8399636B2 (en) 1994-12-16 2013-03-19 Centelion Purification of a triple helix formation with an immobilized obligonucleotide
US8017744B2 (en) 1994-12-16 2011-09-13 Centelion Purification of a triple helix formation with an immobilized oligonucleotide
US6319672B1 (en) 1994-12-16 2001-11-20 Aventis Pharma S.A. Purification of a triple helix formation with an immobilized oligonucleotide
US7622566B2 (en) 1995-11-08 2009-11-24 Centelion Purification of a triple helix formation with an immobilized oligonucleotide
US6730479B2 (en) 1998-03-13 2004-05-04 Promega Corporation Detection of nucleic acid hybrids
US6312902B1 (en) 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6703211B1 (en) 1998-03-13 2004-03-09 Promega Corporation Cellular detection by providing high energy phosphate donor other than ADP to produce ATP
US6268146B1 (en) 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection
US6270973B1 (en) 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6270974B1 (en) 1998-03-13 2001-08-07 Promega Corporation Exogenous nucleic acid detection
US6235480B1 (en) 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
US6653078B2 (en) 1998-03-13 2003-11-25 Promega Corporation Multiplex method for nucleic acid detection
US7090975B2 (en) 1998-03-13 2006-08-15 Promega Corporation Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection
US6335162B1 (en) 1998-03-13 2002-01-01 Promega Corporation Nucleic acid detection
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US6391551B1 (en) 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
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