EP1301217A1 - Inactivation d'acides nucleiques par l'utilisation d'une lumiere pulsee a large spectre - Google Patents

Inactivation d'acides nucleiques par l'utilisation d'une lumiere pulsee a large spectre

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Publication number
EP1301217A1
EP1301217A1 EP01944368A EP01944368A EP1301217A1 EP 1301217 A1 EP1301217 A1 EP 1301217A1 EP 01944368 A EP01944368 A EP 01944368A EP 01944368 A EP01944368 A EP 01944368A EP 1301217 A1 EP1301217 A1 EP 1301217A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
inactivated
bspl
treatment
nucleic acids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01944368A
Other languages
German (de)
English (en)
Other versions
EP1301217A4 (fr
Inventor
William Cover
Jeffrey M. Holloway
Mary O'dwyer
Karen Anne Rieger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PurePulse Technologies Inc
Original Assignee
PurePulse Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by PurePulse Technologies Inc filed Critical PurePulse Technologies Inc
Publication of EP1301217A1 publication Critical patent/EP1301217A1/fr
Publication of EP1301217A4 publication Critical patent/EP1301217A4/fr
Withdrawn legal-status Critical Current

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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
    • 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 .
  • FDA U.S. Food and Drug Administration
  • manufacturing processes and methodologies have been developed that are designed to minimize, inactivate or remove nucleic acids from biologically derived compositions. Numerous methods of decontaminating or sterilizing biologically derived compositions are presently available, including physical methods, chemical methods, heat methods, irradiation methods and combinations thereof.
  • 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.
  • 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 an 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 15 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 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.
  • 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 treatment either in a batch process or in a continuous mode type of process . 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 strands.
  • 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.
  • 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 E . 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 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 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 r carbon of the pentose) and that combination of base and sugar is called a nucleoside.
  • the base characterizes the 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 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 virus, many of which consist of DNA sequences encapsidated in a protein envelope or coat ("capsid").
  • a “vector” is a replicon in which another pplynucleotide 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 pro ide 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.
  • 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.
  • 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.
  • Example 4 Treatment of Single Stranded DNA with BSPL.
  • 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 .

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Abstract

L'invention concerne un procédé destiné à inactiver des chaînes d'acide nucléique par l'utilisation d'une lumière pulsée à large spectre. Ces chaînes d'acide nucléique à inactiver peuvent être contenues dans des compositions, des réactifs et des dispositifs d'origine biologique, dans des produits de déchets, et dans l'air.
EP01944368A 2000-06-20 2001-06-08 Inactivation d'acides nucleiques par l'utilisation d'une lumiere pulsee a large spectre Withdrawn EP1301217A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US59698700A 2000-06-20 2000-06-20
US596987 2000-06-20
PCT/US2001/018571 WO2001097868A1 (fr) 2000-06-20 2001-06-08 Inactivation d'acides nucleiques par l'utilisation d'une lumiere pulsee a large spectre

Publications (2)

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EP1301217A1 true EP1301217A1 (fr) 2003-04-16
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WO2003094978A1 (fr) * 2002-05-08 2003-11-20 Millipore Corporation Decontamination lumineuse d'un milieu de fermentation
JP2008272122A (ja) * 2007-04-26 2008-11-13 Kochi Univ Of Technology マイクロ波照射による滅菌方法
JP2018143223A (ja) * 2017-03-09 2018-09-20 清水建設株式会社 作業室内のｄｎａ不活化方法、及びｄｎａインジケータ

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WO2000047240A1 (fr) * 1999-02-13 2000-08-17 Purepulse Technologies, Inc. Inactivation de pathogenes par utilisation de lumiere pulsee a large spectre

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WO2000047240A1 (fr) * 1999-02-13 2000-08-17 Purepulse Technologies, Inc. Inactivation de pathogenes par utilisation de lumiere pulsee a large spectre

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WO2001097868A1 (fr) 2001-12-27
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EP1301217A4 (fr) 2004-03-24
AU2001266788A1 (en) 2002-01-02
WO2001097868A9 (fr) 2002-10-10

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