THE INACTIVATION OF NUCLEIC ACIDS USING BROAD-SPECTRUM PULSED LIGHT

Abstract: The present invention provides a method for inactivating nucleic acid strands using broad-spectrum pulsed light. The nucleic acid strands to be inactivated may be in biologically derived compositions, reagents and devices, waste products and in air.

Description: THE INACTIVATION OF NUCLEIC ACIDS USING BROAD-SPECTRUM PULSED LIGHT 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. BACKGROUND Substantial technical efforts have been directed to inactivating nucleic acids in biologically derived compositions, in reagents and devices for various types of assays, in waste products, and in air treatment. Such efforts have involved both the treatment of products and reagents themselves, and treatment of devices and packaging materials associated with those compositions. Biologically Derived 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. Numerous methods of decontaminating or sterilizing biologically derived compositions are presently available, including physical methods, chemical methods, heat methods, irradiation methods and combinations thereof. While these methods have met with some success, most of these methods require further processing steps that dramatically increase the cost of manufacturing and efficacy of the final product. Further, the potential may exist that a nucleic acid can not be separated from the final product. The use of light having particular characteristics has been found to be effective for treating biologically derived compositions. For example, U.S. Patent No. 2,072,417 describes illuminating substances with ultraviolet rays; U.S. Patent Nos. 3,817,703 and 4,880,512 describe sterilization of light-transmissive material using pulsed laser light; and U.S. Patent No. 3,941,670 describes a method of sterilizing materials, including foodstuffs, by exposing the materials to laser illumination to inactivate microorganisms . However, such methods have various deficiencies, such as limited throughput capacity, adverse effects on the product itself, inefficient energy conversion and economic disadvantages . Reagents and Devices for Assay3 Various reagents, chemical buffers, and devices are often used in connection with the production of biologically derived compositions and in conjunction with carrying out various types of assays. For example, the polymerase chain reaction (PCR) is a technique that mimics nature's way of replicating DNA. First described in 1985, PCR has been adopted as an essential research tool because it can take a minute sample of genetic material and duplicate enough of it for study. PCR has been used to identify human remains, to analyze prehistoric DNA, diagnose diseases and to help make identifications in police investigations, and to establish paternity. As originally developed, the PCR process amplified short (approximately 100-500bp) segments of a longer DNA molecule. Hence, any contaminating nucleic acids having a sequence greater than 100 bp could potentially cause interference with the assay. In this regard, contamination of reactions is an often encountered problem with PCR. Contaminating nucleic acids often result in false positives in the PCR assay. Currently, 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. Therefore, in the case of working with recombinant DNA- containing microorganisms, it is not only necessary to inactivate the organisms but also to destroy the possibly recombinant DNA in the organisms. As a rule, the killing off of microorganisms takes place relatively simply. However, the DNA in the microorganisms is, as a rule, not destroyed by the measures employed. For example, many treatment processes heat waste streams to a temperature of not more than about 60°C. At these temperatures, nucleic acids are not denatured and may not be inactivated. In some instances, sterilization or denaturing of the nucleic acid must take place before the biomass is disposed of or introduced into a treatment facility or clarification plant. Where 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. Thus, 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. Methods previously known and recognized for the sterilization and inactivation (e.g. steam sterilization over 20 minutes at 121°C.) are very expensive and may also require the addition of various chemicals (see U.S. Patent No. 5,118,603). Especially in the case of the treatment of normal industrial volumes, these processes are very cost intensive not only in terms of the equipment requirements but also because of the high energy consumption. Air Treatment In the area of air decontamination, 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. 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. Heretofore, 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. These types of filters provide a significant impediment to air flow, and therefore require the use of high powered fans and the like in order to pump the air through the filters. As more particulate contaminants become trapped in the filters over time, this impediment to air flow increases. In addition, 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. SUMMARY OP THE INVENTION The present invention provides a fast, reliable and efficient method for the inactivation of nucleic acid and/or nucleic acid strands. In an important aspect, 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. Further, 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. In an important aspect, 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. In addition to the pulsed light being of high intensity and short duration, it is characterized in that it is incoherent, polychromatic light in a broad spectrum. In preferred embodiments, 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. For example, 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. Further, 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. Most advantageously, 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. In one aspect, 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 . In accordance with the method of the invention, a biological composition is exposed to a broad- spectrum pulsed light treatment as described above either in a batch process or continuous process. 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. In an important aspect, 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. In another aspect, 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. In accordance with this aspect of the invention, 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. Further exposure of the reagents 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. In another important aspect, the present invention provides a method for inactivating nucleic acid strands in waste products and/or waste product streams. In accordance with this aspect of the invention, 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. In another aspect, the present invention provides a method for inactivating nucleic acid strands in air. In accordance with this aspect of the invention, 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. In an alternative aspect, the present invention provides a method for randomly producing nucleic acid fragments. In accordance with this aspect of the invention, 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. BRIEF DESCRIPTION OF THE FIGURES 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. DETAILED DESCRIPTION Definitions The following definitions are provided in order to provide clarity as to the intent or scope of their usage in the specification and claims. All patents and publications referred to herein are incorporated by reference herein. As used herein "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. "Polymerase chain reaction" or "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. "Biologically 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. "Aqueous" solution or compositions refers to water, solvents, suspensions and mixtures thereof. Hence, an aqueous solution can include a water based buffer and a water based buffer mixed with a solvent such as ethanol . Further, an aqueous solution can be in a semi-solid form, such as for example agarose, agar, gelatin and polyacrylaminde . Inactivation of Nucleic Acid by BSPL "Inactivation of nucleic acids" refers to a method of forming inactive nucleic acids through treatment with BSPL. 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. In this regard, it is believed that there are a number of chemical or physical events caused by BSPL which can alone or in combination results in an inactive nucleic acid. For example, BSPL can result in the formation of about 100% of theoretically possible thymine dimers that can be formed in a given nucleic acid strand. Further, treatment of with BSPL can result in breaks in double stranded DNA or in single stranded DNA or RNA. Hence, in an important aspect of the invention, 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. As shown in all the figures illustrating gels, 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. While not intending to be bound by any theory, it is thought that the combination of chemical and physical events caused by BSPL overwhelms an organisms' nucleic acid repair mechanisms such that the organism can not restore the functionality of its nucleic acids. As shown in the literature (Xue et al . , AEM, Vol. 62, No. 7, p. 2221-2227, 1996), UV treatment alone is not always sufficient to inactivate nucleic acid. Further, 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. In order to most efficiently and reliably inactive nucleic acids in accordance with the methods herein, the sample should be illuminated as fully as possible by the broad- spectrum pulsed light. Thus, it is important that the sample to be treated be contained, at least during treatment, by material that is sufficiently transmissive, for example at least one percent transmissive, to such broad spectrum pulsed light such that inactivation is reliably achieved. Examples of 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. As will be apparent to those of skill in the art, different material may be used for containing sample material depending on the sample type and particular configuration of the pulsed light treatment apparatus. For example, when the method of the invention is used in conjunction with an aqueous sample, such as a biologically derive compositions or waste product streams, 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. Alternatively, where the sample being treated is a device, such as for example an intravenous tube, the material used to contain the device within the treatment zone of the treatment apparatus may be a pliable polymer material . In addition to considerations of the configuration of the BSPL treatment apparatus and the type of sample being treated, certain characteristics of the sample itself should be considered. For example, the transmissivity of the sample may be a factor to consider in insuring complete illumination thereof with BSPL. However, even if a sample has reduced transmissivity to BSPL, it is believed that there will usually be sufficient reflectivity to ensure that adequate illumination is achieved. Moreover, 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. Waste Water Treatment In another embodiment, the method of the present invention is effective for the inactivation of nucleic acids in waste products and waste streams. The use of BSPL to treat waste water is described in U.S. Patent Application Serial No. 09/025,210, which is incorporated herein by reference. Examples of 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. In operation, 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. With the process according to the invention, 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. Air Treatment The use of BSPL to treat air is described in U.S. Patent Application Serial No. 09/025,210, which is incorporated herein by reference. Generally, and in accordance with the method of the present invention, 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. Examples of 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. In another important aspect of the invention, surfaces in facilities and apparatuses can be exposed to BSPL to inactive any nucleic acids that may be present. Examples of facilities and apparatuses that can be treated include clean rooms, operating rooms, fermentation equipment, and production facilities. Producing Nucleic Acid Fragments In another aspect of the present invention, BSPL can be utilized to randomly cut nucleic acids to provide various sizes of nucleic acid fragments. In this aspect of the invention, 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. The following examples illustrate methods for carrying out the invention and should be understood to be illustrative of, but not limiting upon, the scope of the invention which is defined in the appended claims. EXAMPLES Example 1: Treatment of Ladder DNA with BSPL. DNA Ladder from Sigma (25 μls of 0.2 μg/μl) 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 μg of DNA was loaded onto a 1% agarose gel containing ethidium bromide as follows. Total Fluence/Flash Fluence Lane # (J/flash) # Flashes (Joules) 1 0 0 0 2 0.25 1 0.25 3 0.25 2 0.5 4 0.25 4 1.0 5 0.25 8 4.0 6 1.5 2 3.0 7 1.5 3 4.5 8 1.5 4 6.0 9 5.0 5 7.5 10 1.5 10 15 11 1.5 15 22.5 12 1.5 20 30 13 1.5 30 45 14 1.5 40 60 15 1.5 50 75 The resulting gel is set forth in Figure 1. Example 2 : Treatment of double stranded DNA with BSPL. E. coli DNA from Sigma (25 μls of 0.2 μg/μl) 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 μg of DNA was loaded onto a 1% agarose gel containing ethidium bromide as follows. Total Fluence/Flash Fluence Lane # (J/flas :h) JL Flashes (Joules) 1 0 0 0 2 0.25 1 0.25 3 0.25 2 0.5 4 0.25 4 1.0 5 0.25 8 2.0 6 1.5 2 3.0 • 7 1.5 3 4.5 8 1.5 4 6.0 9 1.5 5 7.5 10 1.5 10 15 11 1.5 15 22.5 12 1.5 20 30 13 1.5 30 45 14 1.5 40 60 15 1.5 50 75 16 DNA Size Markers The resulting gel is set forth in Figure 2. Example 3 : Treatment of RNA with BSPL. RNA from Sigma (25 μls of 0.2 μg/μl) 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 μg of RNA was loaded onto a 1% agarose gel containing ethidium bromide as follows. Total Fluence/Flash Fluence Lane ft (J/flash) # Flashes (Joules) 1 0 0 0 2 0.25 1 0.25 3 0.25 2 0.5 4 0.25 4 1.0 5 0.25 8 2.0 6 1.50 2 3.0 7 1.50 3 4.5 8 1.50 4 6.0 9 1.5 5 7.5 10 1.5 8 12 11 1.5 12 18 12 1.5 24 36 The resulting gel is set forth in Figure 3 Example 4 : Treatment of Single Stranded DNA with BSPL. Single stranded DNA from Sigma (25 μls of 0.2 μg/μl) 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 μg of DNA was loaded onto a 1% agarose gel containing ethidium bromide as follows . Total Fluence/Flash Fluence Lane # (J/flash) # Flashes (Joules) 1 0 0 0 2 0.25 1 0.25 3 0.25 2 0.5 4 0.25 4 1.0 5 0.25 8 2.0 • 6 1.5 2 3.0 7 1.5 3 4.5 8 1.5 4 6.0 9 1.5 5 7.5 10 1.5 8 12 11 1.5 16 24 The resulting gel is set forth in Figure . Example 5 ; Treatment of double stranded DNA with BSPL and T4 Endonuclease. E. coli DNA from Sigma (25 μls of 0.2 μg/μl) 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, and divided into equal portions. One sample from each BSPL treatment was treated with T4 endonuclease. After enzyme treatment, sample buffer containing dyes was added, and approximately 3 μg of DNA was loaded onto a 1% agarose gel containing ethidium bromide as follows . Total Fluence/Flash Fluence Enzyme Lane # (j/flash) # Flashes (Joules) Treatment 1 0 0 0 No 2 0 0 0 Yes 3 0.5 1 0.5 No 4 0.5 1 0.5 Yes 5 1.0 1 1.0 No 6 1.0 1 1.0 Yes 7 3.0 1 3.0 No 8 3.0 1 3.0 Yes 9 9.0 1 9.0 No 10 9.0 1 9.0 Yes 11 22 1 22 No 12 22 1 22 Yes The resulting gel is set forth in Figure 5.

Claims: WHAT IS CLAIMED IS: 1. A method for inactivating a nucleic acid strand, the method comprising illuminating the nucleic acid strand with at least one high- intensity, short duration pulse of incoherent polychromatic light in a broad spectrum. 2. The method according to Claim 1, wherein the nucleic acid to be inactivated is in an aqueous solution. 3. The method according to Claim 2 , wherein the aqueous solution containing the nucleic acid is in a semi -solid form. 4. The method according to Claim 1, wherein the nucleic acid to be inactivated is in a suspension. 5. The method according to Claim 1, wherein the nucleic acid to be inactivated is on a surface. 6. The method according to Claim 1, wherein the nucleic acid to be inactivated is an exogenous nucleic acid. 7. The method according to Claim 1, wherein the nucleic acid to be inactivated is endogenous and contained as part of a mammalian cell, a eukaryotic cell, plant cell, biological tissue, tumor cells, chloroplast, cellular organelle, virus, bacteria, fungi, phage, transposon, spores, vaccine, antigen, prion, vector, or mixtures thereof. 8. A method .of inactivating nucleic acids strands in a biologically derived composition, the method comprising illuminating the biologically derived composition containing a nucleic acid with at least one high- intensity, short duration pulse of incoherent polychromatic light in a broad spectrum. 9. The method according to Claim 8 , wherein the nucleic acid to be inactivated is in an aqueous solution. 10. The method according to Claim 9, wherein the biologically derived composition containing the nucleic acid is in a semi-solid form. 11. The method according to Claim 8, wherein the biologically derived composition containing the nucleic acid is a suspension. 12. The method according to Claim 8, wherein the nucleic acid to be inactivated is an exogenous nucleic acid. 13. The method according to Claim 8, wherein the nucleic acid to be inactivated is endogenous and contained as part of a mammalian cell, a eukaryotic cell, plant cell, biological tissue, tumor cell, chloroplast, cellular organelle, virus, bacteria, fungi, phage, transposon, spore, vaccine, antigen, prion, vector, or mixtures thereof . 14. A method of inactivating nucleic acids strands in waste products, the method comprising illuminating the waste product containing a nucleic acid with at least one high- intensity, short duration pulse of incoherent polychromatic light in a broad spectrum. 15. The method according to Claim 14, whe,rein the nucleic acid to be inactivated is in an aqueous solution. 16. The method according to Claim 14, wherein the waste product containing the nucleic acid is in a se i- solid form. 17. The method according to Claim 14, wherein the waste product containing the nucleic acid is a suspension. 18. The method according to Claim 14, wherein the waste product containing the nucleic acid is in a solid form. 19. A method of inactivating nucleic acid strands in air, the method comprising illuminating the air containing a nucleic acid with at least ' one high- intensity, short duration pulse of incoherent polychromatic light in a broad spectrum. 20. The method according to claim 19 wherein the nucleic acid strand is on the surface of an airborne particle. 21. A method for randomly producing nucleic acid fragments, the method comprising illuminating a sample containing nucleic acid strands with at least one high- intensity, short duration pulse of incoherent polychromatic light in a broad spectrum, the method being effective for forming nucleic acid fragments of different lengths .

Inventors: COVER WILLIAM, HOLLOWAY JEFFREY M, O'DWYER MARY, RIEGER KAREN ANNE, COVER, WILLIAM, HOLLOWAY, JEFFREY, M., O'DWYER, MARY, RIEGER, KAREN, ANNE
Applicants: PUREPULSE TECHNOLOGIES INC, PUREPULSE TECHNOLOGIES, INC.
Application year: 2001
Main classification: A61L2
IPC codes: A61L2, A61L9, A61L11, A61M1, B01J19, C02F1, C12N13, C12N15, C12Q1