WO2001097868A9 - The inactivation of nucleic acids using broad-spectrum pulsed light - Google Patents
The inactivation of nucleic acids using broad-spectrum pulsed lightInfo
- Publication number
- WO2001097868A9 WO2001097868A9 PCT/US2001/018571 US0118571W WO0197868A9 WO 2001097868 A9 WO2001097868 A9 WO 2001097868A9 US 0118571 W US0118571 W US 0118571W WO 0197868 A9 WO0197868 A9 WO 0197868A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- inactivated
- bspl
- treatment
- nucleic acids
- Prior art date
Links
Classifications
-
- 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3681—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/04—Liquids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/04—Liquids
- A61M2202/0413—Blood
- A61M2202/0427—Platelets; Thrombocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/20—Pathogenic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/30—Wastewater or sewage treatment systems using renewable energies
- Y02W10/37—Wastewater or sewage treatment systems using renewable energies using solar energy
Definitions
- the present invention is directed to a method of inactivating nucleic acids. More particularly, the method of the present invention utilizes broad-spectrum pulsed light (BSPL) to inactivate nucleic acid strands in biologically derived compositions, reagents and devices, waste products and in air.
- BSPL broad-spectrum pulsed light
- compositions The use of biologically derived compositions in scientific research and in the manufacture of therapeutic substances and vaccines has become very common. Various human and animal sera are routinely used as a source of protein agents for treatment of various diseases and disorders. Animal and plant cell tissue culture methods are now commonly used in the production of numerous pharmaceutical/therapeutic agents, vaccines and related compositions, such as recombinant DNA and/or recombinant protein (from genetically engineered animal and plant cell lines) , virus vectors (from animals and plants) , amino acids, peptides, insulin and monoclonal antibodies. Because these compositions are derived from living organisms they can be contaminated with exogenous and/or endogenous nucleic acids or animal or plant viruses. Further, any materials that come in contact with humans during manufacturing processes can potentially be contaminated with nucleic acids .
- Biologically derived compositions used as biotechnological and/or biological products are required to meet certain criteria prescribed by the U.S. Food and Drug Administration (FDA) in terms of purity. Sections of the FDA code require minimization of molecular entities, such nucleic acids, in biologically derived compositions . In an attempt to comply with regulations that require minimization of nucleic acid levels, manufacturing processes and methodologies have been developed that are designed to minimize, inactivate or remove nucleic acids from biologically derived compositions.
- FDA U.S. Food and Drug Administration
- 3,941,670 describes a method of sterilizing materials, including foodstuffs, by exposing the materials to laser illumination to inactivate microorganisms.
- materials including foodstuffs
- laser illumination to inactivate microorganisms.
- such methods have various deficiencies, such as limited throughput capacity, adverse effects on the product itself, inefficient energy conversion and economic disadvantages .
- PCR polymerase chain reaction
- any contaminating nucleic acids having a sequence greater than 100 bp could potentially cause interference with the assay.
- contamination of reactions is an often encountered problem with PCR.
- Contaminating nucleic acids often result in false positives in the PCR assay.
- contamination problems are avoided by using dedicated reagents, Gilsons, and plugged tips. These reagents and devices are typically treated with various sterilization techniques, including UV light (see U.S. Patent No. 5,993,749) to inactivate or denature any nucleic acids that may be present .
- Waste Treatment Manufacturing facilities, hospitals, and laboratories generate both solid and liquid wastes that may contain nucleic acids and which are often classified as biohazardous waste. Many countries, such as Germany, now have regulations that require that waste which contains nucleic acids be sterilized or denatured.
- a waste stream that includes a sterilized or denatured nucleic acid is subsequently treated in a clarification plant, measures must be carried out to insure that no substance is introduced into the clarification plant that would kill off the microorganisms of the activated sludge.
- care must be taken that the substances used for sterilization or denaturing the nucleic acids in the waste product are not toxic or, before introduction into the clarification plant, are so broken down that they can cause no harm.
- airborne nucleic acids may be of a concern, especially in terms of contaminating other devices or products.
- Various clean room facilities, manufacturing equipment and hospital operating rooms require decontamination prior to their use and require systems to insure that they remain decontaminated during use.
- an air treatment approach In order to be effective, an air treatment approach must be able to first provide an uncontaminated area and then process flowing air as it passes from a contaminated space into an uncontaminated or sterile space. Methods employed to provide an initial uncontaminated area have included various chemical methods, UV light irradiation, and combinations of the two.
- the most common method of air treatment has been to employ micro filters, such as HEPA filters, in a duct in order to physically remove particulate contaminants from the flowing air.
- micro filters such as HEPA filters
- micro filters due to the relatively high resistance to air flow posed by such micro filters, leakage of air around the filters becomes a significant factor in their design. Such filters also are subject to releasing trapped contaminants into the duct when they are removed for replacement and such released contaminants can be subsequently carried by the duct into areas sought to be protected by the filter. Furthermore, some microfilters may be unable to remove particularly small contaminant particles, including exogenous or endogenous nucleic acids.
- the present invention provides a fast, reliable and efficient method for the inactivation of nucleic acid and/or nucleic acid strands.
- the method of the present invention utilizes a broad- spectrum pulsed light treatment apparatus to illuminate a sample and inactivate any nucleic acid and nucleic acid strands present in or on the sample.
- the method is effective for use on a wide variety of sample types including biologically derived compositions, reagents and devices, waste products, and air. These samples may be aqueous, solid or semi- solid in form.
- the method of the invention can economically be incorporated into a number of different research and industrial scale processes which can provide a further assurance that nucleic acids are inactivated.
- the method of the invention includes illuminating a sample with pulsed light having an intensity of at least about 0.01 J/cm 2 , preferably about 0.02 J/cm 2 to about 50 J/cm 2 , most preferably about 0.05 J/cm 2 to about 1.0 J/cm 2 , wherein the energy intensity is measured at the surface of the composition being illuminated.
- the light pulses are preferably, and advantageously, of very short duration. In particular, pulse durations of less than about 100 ms are preferred, for example durations of between about 10 ns and 100 ms, such as about 0.3 ms are most preferable.
- the pulsed light being of high intensity and short duration, it is characterized in that it is incoherent, polychromatic light in a broad spectrum.
- the light includes wavelengths from about 170 nm to about 2600 nm (i.e. frequencies of about 1.8 x 10 15 Hz to about 1.8 x 10 1£ Hz). Due to the short time between pulses, less than about 100 ms, a single, multi- pulse inactivation treatment can be completed in less than a minute for each treatment. This is in stark contrast to heretofore known methods for inactivating nucleic acids, which can require up to hours to complete. In another important aspect of the invention, the method of the invention is versatile enough to be used for a wide variety of process applications and sample types.
- the nucleic acid to be inactivated may be in a biologically derived composition which is either in a liquid or semi-solid state or which is a suspension, in a reagent or on the surface of a device or various target objects, in a waste material, or present as an airborne particle.
- the method of the present invention is effective for inactivating nucleic acids which may be exogenous or endogenous as part of a mammalian cell, a eukaryotic cell, plant cell, any biological tissue, a tumor cell, chloroplasts, cellular organelles such as mitochondria and ribosomes, virus, bacteria, fungi, phage, transposon, spore, vaccine or antigen purified therefrom, prion and/or vector.
- the method of the present invention can be easily, quickly and economically administered at various points during the processing of the sample to be treated, thereby providing further assurance of complete or near complete inactivation of nucleic acids.
- the invention provides a method for inactivating a nucleic acid strand in a biologically derived composition.
- Biologically derived compositions include pharmaceutical compositions and compositions such as vaccines, plasma, monoclonal antibodies, protein from genetically engineered mammalian cell lines, gene therapy products, human and/or animal blood derived products, plant derived products, biological pharmaceutics such as heparin and/or collagen, bovine serum, sheep blood, peptones/amino acids and/or bovine insulin/transferrin, fermentation broths and mixtures thereof.
- a biological composition is exposed to a broad-spectrum pulsed light treatment as described above either in a batch process or continuous process.
- nucleic acid strands Exposure of the biologically derived composition results in an inactivation of nucleic acid strands as compared to biological compositions that have not been exposed to BSPL.
- nucleic acid strands are inactivated to a level where they are no longer a concern for regulatory purposes or interfere with various types of assays such as PCR.
- the invention provides a method for inactivating nucleic acid strands present in reagents or on the surface of a device or various target objects which may be used in connection with the production of biologically derived compositions or for various assays.
- reagents or target objects are exposed to BSPL treatment either in a batch process or in a continuous mode type of process . Exposure of the reagents or target objects to BSPL 5 results in an inactivation of nucleic acid strands. When these reagents or target objects are used in conjunction with the production of biologically derived compositions, no active nucleic acids are added to the biologically derived composition. Further exposure of the reagents
- BSPL 10 and target objects to BSPL provides reagents and target objects that do not add active nucleic acids to the biologically derived composition and no nucleic acids are added to an assay such that they would interfere with the assay.
- the present invention provides a method for inactivating nucleic acid strands in waste products and/or waste product streams.
- waste products or waste product streams are exposed to BSPL
- Exposure of the waste products or waste product streams to BSPL is effective for inactivation of nucleic acid strands to levels which permit disposal of these wastes,
- the present invention provides a method for inactivating nucleic acid strands in air.
- air is flowed into a treatment region and exposed to BSPL in an amount effective for the inactivation of nucleic acid
- the nucleic acid strand may be exogenous or on the surface of an airborne particle.
- the present invention provides a method for randomly producing nucleic acid fragments .
- the present invention provides a method for randomly producing nucleic acid fragments .
- a sample containing nucleic acid strands is illuminated with at least one high- intensity, short duration pulse of incoherent polychromatic light in a broad spectrum.
- the method is effective for forming nucleic acid fragments of different length.
- Fig. 1 shows a gel electrophoresis of DNA ladder treated with increasing levels of BSPL as more fully described in Example 1.
- Fig. 2 shows a gel electrophoresis of E. coli DNA treated with increasing levels of BSPL as more fully described in Example 2.
- Fig. 2a is a density scan of the gel electrophoresis shown in Fig . 2.
- the graph shows the % of degraded Eumble coli DNA with increasing levels of BSPL.
- Fig. 3 shows a gel electrophoreis of RNA treated with increasing levels of BSPL as more fully described in Example 3.
- Fig. 4 shows a gel electrophoresis of single stranded DNA treated with increasing levels of BSPL as more fully described in Example 4.
- Fig. 5 shows a gel electrophoresis of E. coli DNA treated with increasing levels of BSPL and T4 endonuclease No. 5 as more fully described in Example 5.
- Fig. 6 shows a gel electrophoreis of E. coli DNA treated with different pulsed wavelengths of the UV visible spectrum.
- Fig. 7 is a density scan of a gel electrophoresis of E. coli DNA treated with BSPL or with various pulsed wavelengths of light.
- Fig. 8 shows a gel electrophoresis of pBR322 DNA treated with BSPL or with various pulsed wavelengths of light.
- Fig. 9 is a density scan of the gel electrophoreis in Fig. 8 and illustrates the percentages of DNA types formed with increased total fluence when pBR322 is treated with BSPL.
- Fig. 10 graphically illustrates the % of degraded E. coli DNA after extraction of the DNA from E. coli treated with increasing levels of BSPL prior to extraction of DNA.
- nucleic acid or “nucleic acid 10 strand” refers to a polymer having two or more nucleotides. This nucleic acid may be single or double stranded DNA or RNA. Further, nucleic acid also includes any modified bases.
- Nucleotide refers to a monomeric unit of DNA or 15 RNA consisting of a sugar moiety (pentose) , a phosphate, and a nitrogenous heterocyclic base.
- the base is linked to the sugar moiety via the glycosidic carbon (l 1 carbon of the pentose) and that combination of base and sugar is called a nucleoside.
- the base characterizes the 20 nucleotide.
- the four DNA bases are adenine ("A"), guanine (“G”), cytosine ("C”), and thymine (“T”).
- the four RNA bases are A, G, C, and uracil ("U”).
- DNA Sequence or "RNA Sequence” refers to a linear array of nucleotides connected one to the other by 25 phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.
- “Plasmid” refers to a nonchromosomal double- stranded DNA sequence comprising an intact "replicon” such that the plasmid is replicated in a host cell.
- a “replicon” is any genetic element (e.g., a plasmid, a chromosome, a virus) that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.
- “Phage” or “Bacteriophage” refers to a bacterial 3 . 5 virus, many of which consist of DNA sequences encapsidated in a protein envelope or coat ("capsid").
- a “vector” is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment.
- An “expression vector” refers to a vector capable of autonomous replication or integration and contains control sequences which direct the transcription and translation of the desired nucleotide sequence in an appropriate host.
- cDNA or “Complementary DNA” refers to DNA which has at some point in its history been made by copying RNA using the enzyme reverse transcriptase.
- PCR Polymerase chain reaction
- PCR refers to technique by which a relatively small piece of DNA of known sequence can be amplified (often from a complex mixture) by successive cycles of strand separation followed by DNA synthesis (using a DNA polymerase purified from a thermophilic bacterium) primed by artificially synthesized oligonucleotide primers (one for each strand) .
- PCR is generally described by U.S. Patent Nos. 4,683,195 and 4,683,202 which are incorporated herein by reference.
- Bioly derived compositions refer to pharmaceutical compositions and compositions such as vaccines, plasma, monoclonal antibodies, protein from genetically engineered mammalian cell lines, gene therapy products, human and/or animal blood derived products, plant derived compositions, hormones, gelatin, biological pharmaceutics such as heparin and/or collagen, bovine serum, sheep blood, peptones/amino acids and/or bovine insulin/transferrin, fermentation broths and mixtures thereof .
- Reagents refers to aqueous solutions, buffers, solvents, and mediums that may be used in the production of biologically derived compositions or in various assays, such as a PCR assay.
- an aqueous solution or compositions refers to water, solvents, suspensions and mixtures thereof.
- an aqueous solution can include a water based buffer and a water based buffer mixed with a solvent such as ethanol .
- an aqueous solution can be in a semi-solid form, such as for example agarose, agar, gelatin and polyacrylaminde .
- “Inactivation of nucleic acids” refers to a method of forming inactive nucleic acids through treatment with BSPL.
- the nucleic acids In order for the nucleic acid to be considered inactive, or an "inactive nucleic acids” the nucleic acids must not be suitable for replication, amplification, or translation. Generally, this will mean that the inactive nucleic acid is not a suitable template for a polymerase as the nucleic acid is too short to serve as a template. Hence, inactive nucleic acids will not be capable of interfering with a PCR assay as they will not replicate or amplify, or not replicate or amplify to a level that would interfere with the assay. Further, an inactive nucleic acid may be degraded, cleaved or neutralized to an extent that it no longer can function biologically as it did prior to treatment with BSPL.
- BSPL is different from continuous, non-pulsed UV light in a number, of ways.
- the spectrum of .BSPL contains UV light, but also includes a broader light spectrum, in particular between about 170 nm and about 2600 nm.
- the spectrum of BSPL is similar to that of sunlight at sea level, although it is 90,000 times more intense, and includes UV wavelengths between 200 and 300 nm which are normally filtered by the earth's atmosphere.
- BSPL is applied in short durations of relatively high power, as compared to the longer exposure times and lower power of non-pulsed UV light.
- BSPL can result in the formation of about 100% of theoretically possible thymine dimers that can be formed in a given nucleic acid strand.
- treatment of with BSPL can result in breaks in double stranded DNA or in single stranded DNA or RNA.
- treatment with BSPL may result in nucleic acid strands having a size of less than about 100 bases.
- Nucleic acid strands having less than about 100 bases in length will not generally interfere with PCR assays and are generally too short to serve as a replication template.
- treatment with BSPL results in a shortening of nucleic acid strands. Further, as shown in Figs. 6 through 9, treatment with BSPL was far more effective than treatment with specific wavelengths of light in eliminating supercoiled DNA and providing linear degraded DNA. In this aspect of the invention, BSPL treatment is effective to eliminate about 100% of the supercoiled nucleic acid from a sample.
- BSPL offers advantages over laser treatment in that lasers are either high intensity or rapid pulsing, but not both, and are significantly more expensive to operate. BSPL is relatively inexpensive and produces high intensity with rapid pulse rates.
- the BSPL Apparatus Various apparatus may be employed to practice the methods of the present invention. Apparatus designed to provide high- intensity, short duration pulsed incoherent polychromatic light in a broad-spectrum are described for example in U.S. Patent Nos. 4,871,559, 4,910,942, 5,034,235, 5,489,442, 5,768,853, 5,786,598 and 5,900,211, each of which is hereby incorporated by reference in its entirety.
- BSPL may be used to inactivate nucleic acids on various products, target objects, packages, water and other fluid, semifluid and solid objects. Such is primarily accomplished by placing the target object into, or passing a target object through, a BSPL device and exposing the object to an appropriate number of flashes of BSPL at an appropriate energy level.
- the sample should be illuminated as fully as possible by the broad-spectrum pulsed light.
- material that is sufficiently transmissive for example at least one percent transmissive, to such broad spectrum pulsed light such that inactivation is reliably achieved.
- suitable materials include, for example, polyolefins, such as polyethylene and polypropylene, nylon, quartz and sapphire.
- U.S. Patent No. 5,786,598, which is hereby incorporated by reference, provides a discussion of various polymers suitable for use in accordance with the method of the present invention.
- the treatment apparatus may employ closely placed thin plates through which the sample composition passes.
- Such plates may be formed of a relatively hard, transmissive material, such as quartz or sapphire.
- the material used to contain the device within the treatment zone of the treatment apparatus may be a pliable polymer material .
- the transmissivity of the sample may be a factor to consider in insuring complete illumination thereof with BSPL.
- the transmissivity of aqueous samples can be adjusted by simple dilution. Appropriate dilution techniques as well as techniques for optional reconcentration following treatment with BSPL are well known.
- the method of the present invention is effective for the inactivation of nucleic acids in waste products and waste streams.
- BSPL to treat waste water is described in U.S. Patent Application Serial No. 09/025,210, which is incorporated herein by reference.
- waste products include solid waste, such as devices used in connection with recombinant DNA research and hospital waste.
- Waste streams may include waste water and manufacturing wastes from production or treatment facilities.
- the waste product or waste stream is passed through or flowed into a treatment zone for illumination.
- the flowing of an aqueous waste stream may be accomplished by containing the water in appropriate pipes, and by pumping the aqueous waste stream into and out of the treatment zone.
- the flowing of aqueous waste may be carried out continuously so that all of the aqueous waste passing through the treatment zone is illuminated before it exits the treatment zone.
- a breakdown of the DNA to fragments smaller than or equal to about 100 bp can be achieved which is also not exceeded by the waste disposal processes (autoclaving) permitted by European regulatory agencies (for example ZKBS) .
- Such fragments are no longer considered to be biologically active and can, therefore, be passed without danger into the waste water.
- BSPL to treat air
- U.S. Patent Application Serial No. 09/025,210 which is incorporated herein by reference.
- air is flowed into a treatment region of an air duct through the use of fans. During its residence in the treatment region, air is exposed to BSPL treatment as described herein.
- treatment facilities that can incorporate the method of the invention include clean rooms, operating rooms, fermentation equipment, production facilities, and military air supply systems, such as in a military tank.
- surfaces in facilities and apparatuses can be exposed to BSPL to inactive any nucleic acids that may be present.
- facilities and apparatuses that can be treated include clean rooms, operating rooms, fermentation equipment, and production facilities.
- BSPL can be utilized to randomly cut nucleic acids to provide various sizes of nucleic acid fragments.
- a nucleic acid sample may be treated with BSPL and subsequently treated with T4 endonuclease No. 5 which cuts at thymine dimers formed during BSPL treatment.
- the method is effective for forming nucleic acid fragments of different lengths ranging from undetectable lengths, less than about 100 bases, up to the size of the starting material.
- Randomly generated fragments of nucleic acids may be useful for a number of applications. For example, shortening of mRNA libraries for use in gene expression arrays, global random shortening of genomic DNA for blots, and for the production of fragments for fingerprinting, similar to RFLP.
- Example 1 Treatment of Ladder DNA with BSPL.
- DNA Ladder from Sigma 25 ⁇ ls of 0.2 g/ ⁇ l was pipetted into a 1 mm quartz holder and exposed to an increasing level of total fluence as described below. Following treatment, samples were removed from the quartz holder, sample buffer containing dyes was added, and approximately 3 ⁇ g of DNA was loaded onto a 1% agarose gel containing ethidium bromide as follows .
- Example 2 Treatment of double stranded DNA with BSPL.
- E. coli DNA from Sigma (25 ⁇ ls of 0.2 ⁇ g/ ⁇ l) was pipetted into a 1 mm quartz holder and exposed to an increasing level of total fluence as described below. Following treatment, samples were removed from the quartz holder, sample buffer containing dyes was added, and approximately 3 ⁇ g of DNA was loaded onto a 1% agarose gel containing ethidium bromide as follows.
- Example 3 Treatment of RNA with BSPL. .
- RNA from Sigma 25 ⁇ ls of 0.2 ⁇ g/ ⁇ l was pipetted into a 1 mm quartz holder and exposed to an increasing level of total fluence as described below. Following treatment, samples were removed from the quartz holder, sample buffer containing dyes was added, and approximately 3 ⁇ g of RNA was loaded onto a 1% agarose gel containing ethidium bromide as follows.
- Single stranded DNA from Sigma (25 ⁇ ls of 0.2 ⁇ g/ ⁇ l) was pipetted into a 1 mm quartz holder and exposed to an increasing level of total fluence as described below. Following treatment, samples were removed from the quartz holder, sample buffer containing dyes was added, and approximately 3 ⁇ g of DNA was loaded onto a 1% agarose gel containing ethidium bromide as follows.
- Example 5 Treatment of double stranded DNA with BSPL and T4 Endonuclease.
- E. coli DNA from Sigma (25 ⁇ ls of 0.2 ⁇ g/ ⁇ l) was pipetted into a 1 mm quartz holder and exposed to an increasing level of total fluence as described below.
- samples were removed from the quartz holder, and divided into equal portions.
- One sample from each BSPL treatment was treated with T4 endonuclease.
- sample buffer containing dyes was added, and approximately 3 ⁇ g of DNA was loaded onto a 1% agarose gel containing ethidium bromide as follows.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Physical Water Treatments (AREA)
- Physical Or Chemical Processes And Apparatus (AREA)
- Apparatus For Disinfection Or Sterilisation (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2002503351A JP2003535613A (en) | 2000-06-20 | 2001-06-08 | Inactivation of nucleic acids using broad-spectrum pulsed light |
AU2001266788A AU2001266788A1 (en) | 2000-06-20 | 2001-06-08 | The inactivation of nucleic acids using broad-spectrum pulsed light |
EP01944368A EP1301217A4 (en) | 2000-06-20 | 2001-06-08 | The inactivation of nucleic acids using broad-spectrum pulsed light |
CA 2412840 CA2412840A1 (en) | 2000-06-20 | 2001-06-08 | The inactivation of nucleic acids using broad-spectrum pulsed light |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US59698700A | 2000-06-20 | 2000-06-20 | |
US09/596,987 | 2000-06-20 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001097868A1 WO2001097868A1 (en) | 2001-12-27 |
WO2001097868A9 true WO2001097868A9 (en) | 2002-10-10 |
Family
ID=24389574
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2001/018571 WO2001097868A1 (en) | 2000-06-20 | 2001-06-08 | The inactivation of nucleic acids using broad-spectrum pulsed light |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1301217A4 (en) |
JP (1) | JP2003535613A (en) |
AU (1) | AU2001266788A1 (en) |
CA (1) | CA2412840A1 (en) |
WO (1) | WO2001097868A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003094978A1 (en) * | 2002-05-08 | 2003-11-20 | Millipore Corporation | Light decontamination of fermentation media |
JP2008272122A (en) * | 2007-04-26 | 2008-11-13 | Kochi Univ Of Technology | Sterilization method by microwave irradiation |
JP2018143223A (en) * | 2017-03-09 | 2018-09-20 | 清水建設株式会社 | Method of deactivating dna in operation room and dna indicator |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU605129B2 (en) * | 1986-11-13 | 1991-01-10 | Purepulse Technologies, Inc. | Preservation of foodstuffs by irradiation |
KR19990067075A (en) * | 1995-10-26 | 1999-08-16 | 웨인 클라크 | Improved Deactivation Method of Organisms Using High Intensity Pulsed Multicolor Light |
AU779193B2 (en) * | 1999-02-13 | 2005-01-13 | Purepulse Technologies, Inc. | Methods of inactivating pathogens using broad-spectrum pulsed light |
-
2001
- 2001-06-08 WO PCT/US2001/018571 patent/WO2001097868A1/en active Application Filing
- 2001-06-08 CA CA 2412840 patent/CA2412840A1/en not_active Abandoned
- 2001-06-08 EP EP01944368A patent/EP1301217A4/en not_active Withdrawn
- 2001-06-08 AU AU2001266788A patent/AU2001266788A1/en not_active Abandoned
- 2001-06-08 JP JP2002503351A patent/JP2003535613A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2001097868A1 (en) | 2001-12-27 |
CA2412840A1 (en) | 2001-12-27 |
AU2001266788A1 (en) | 2002-01-02 |
JP2003535613A (en) | 2003-12-02 |
EP1301217A4 (en) | 2004-03-24 |
EP1301217A1 (en) | 2003-04-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2001236787B2 (en) | Protecting molecules in biologically derived compositions while treating with broad-spectrum pulsed light | |
CA2235244C (en) | Improved deactivation of organisms using high-intensity pulsed polychromatic light | |
CN101238363A (en) | Method and apparatus for sterilizing and disinfecting air and surfaces and protecting a zone from external microbial contamination | |
JP2009077707A (en) | Method for inactivating contaminant in blood product | |
AU779193B2 (en) | Methods of inactivating pathogens using broad-spectrum pulsed light | |
CA2567336A1 (en) | Inactivation of a pathogen in a sample by a treatment with formalin and uv light | |
WO2001097868A9 (en) | The inactivation of nucleic acids using broad-spectrum pulsed light | |
EP1308172A1 (en) | Method for protein-preserving purification of contaminated biological liquids | |
WO1997033629A1 (en) | Ultraviolet purification of biological fluids, blood sera and other contaminated solutions | |
US6635679B2 (en) | Methods and compositions for inactivating viruses | |
WO2000025581A1 (en) | Method for laser inactivation of infectious agents | |
KR102511148B1 (en) | Bee venom sterilization process using E-beam and verification of sterilization process using porcine parvovirus | |
KR102511147B1 (en) | Bee venom sterilization process using E-beam and verification of sterilization process using model virus | |
KR102511149B1 (en) | Bee venom sterilization process using E-beam and verification of sterilization process using Hepatitis A Virus | |
US20030161756A1 (en) | Microdispersion treatment of a protein or pharmaceutical | |
JPS5825437B2 (en) | Biohazard protection method using ozone | |
US20040005694A1 (en) | Light decontamination of fermentation media | |
JPS6037977A (en) | Apparatus for protecting from biohazard |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1/11-11/11, DRAWINGS, REPLACED BY NEW PAGES 1/6-6/6; DUE TO LATE TRANSMITTAL BY THE RECEIVINGOFFICE |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
ENP | Entry into the national phase in: |
Ref country code: JP Ref document number: 2002 503351 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2412840 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2001944368 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2001944368 Country of ref document: EP |