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  • 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
  • C12Q1/6813—Hybridisation assays
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• • 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

Definitions

• Nucleic acid hybridization assays have become increasingly accepted as diagnostic tools for a wide variety of diseases or illnesses caused by pathogenic unicellular organisms, such as the bacteria Salmonella typhimuri urn , Shigella dysenteriae , Shigella sonnei, Camphylobacter jejuni , or the amoeba Giardia lamhlia , each of which can cause gastrointestinal disorders, including dysentery, in humans.
• Acceptance of hybridization methods is largely due to the high degree of specificity and sensitivity attainable with genus- or species-specific oligonucleotide probes.
• sensitivity to the low picogram-per-milliliter target nucleic acid concentration range is not uncommon, and substantially lower limits of sensitivity have been achieved. See, e.g., Morrissey, D.V. et al., (1989) Analyt. Biochem. 181:354-359. Accordingly, the presence of even very low levels of pathogenic microorganisms can be reliably detected.
• One caveat to this level of sensitivity is an increased risk of introduced error due to factors such as sample carryover or crosscontamination. Thus, there is an ever-present need to maintain the separate integrity of each individual sample to be assayed.
• Sandwich hybridization assays generally involve introducing two nucleic probes to a fluid sample or a fluid extract of a sample suspected of containing the chosen target polynucleotide sequence (DNA or RNA) .
• One of these probes includes a region of nucleotide sequence complementary to a region of the target, and a detectable moiety.
• the other probe referred to as the capture probe, includes a region of nucleotide sequence complementary to a second, distinct region of the target, and an immobilizable moiety. The sample is maintained in the presence of these two probes under conditions sufficient for hybridization of complementary sequences to occur, whereupon a ternary hybridization complex forms, composed of capture probe, target nucleic acid, and reporter probe.
• This ternary hybridization complex is contacted with a solid support or resin to which the immobilizable moiety present in the capture probe binds, whereby the ternary hybridization complex is captured onto the solid support or resin. Thereafter, the captured complex is separated from the fluid sample or extract, and the presence of the target is analyzed by detecting the presence (and optionally the amount) of the reporter probe associated with the solid support or resin.
• the target nucleic acid sequence in the fluid sample or extract is in the interior of intact cells or unicellular organisms present in the fluid, where it is inaccessible to hybridization analysis. Accordingly, it is necessary as a first step to lyse these cells or organisms, thereby releasing their nuclear contents, including nucleic acids, into the sample fluid. Numerous techniques for ly ⁇ ing cells or organisms to be subjected to hybridization analysis have been reported; sonication, or the subjection of the sample to ultrasonic energy, is one such technique.
• Doulah teaches that cells or unicellular organisms disintegrate when the hydrodynamic kinetic energy produced by cavitation exceeds the mechanical strength of their plasma membranes or cell walls. Upon disintegration, the contents of the cells or organisms, including their nucleic acids, spill out into the surrounding medium and become accessible to hybridization analysis.
• Sonication is typically conducted by immersion of a vibrating probe into the sample, or by placing the sample in a bath or liquid-filled vessel which vibrates, transducing the ultrasonic energy to the sample through the liquid.
• Sonication devices or systems appropriate for lysing cells or organisms are available commercially from vendors such as Branson Ultrasonics Corp., Danbury, CT; Heat Systems Ultrasonics, Farmingham, NY; and Raytheon, Co., altham, MA. Both probe-immersion and bath sonication methods carry a significant risk of crossexposure of sample contents, or contamination of the sample contents with the transducing liquid. Additionally, due to the inefficiency of energy transduction, especially with bath-type sonication devices, an excess of ultrasonic energy must be applied in order to produce cavitation. Under these conditions, excessive thermal energy can also be produced, resulting in destruction of the sample or even in melting of the sample container.
• Biochemistry 129:216-223 teaches the use of sonication to generate random double-stranded shear fragments of genomic DNA having lengths suitable for hybridization purposes.
• Deininger cautions that the shear fragments must be rigorously size-fractionated to exclude any which are unsuitably long or short.
• the size distribution of ultrasonic shear fragments of DNA is investigated in Eisner, H.I. and E.B. Lindblad, (1989) DNA 8(10) :697-701. Although ultrasonic degradation of DNA is not wholly random, substantial monodispersity (uniformity of fragment length) is not achieved by conventional sonication methods without a subsequent size-fractionation step.
• sample fluids or fluid extracts for sandwich hybridization assays in the manner described above is time consuming and prone to introduced error.
• This invention relates to a method for simultaneously shearing and denaturing a nucleic acid, in which an aqueous mixture of the nucleic acid and a chaotropic agent is subjected to the noninvasive transfer of a sufficient level of ultrasonic energy to produce cavitation in the aqueous mixture, thereby producing single-stranded nucleic acid shear fragments which are of substantially the same (equal) length.
• Suitable chaotropic agents for use in the present method include, but are not limited to, guanidiniu thiocyanate, guanidinium hydrochloride, urea, perchloric acid, trichloroacetic acid, sodium thiocyanate, and sodium iodide.
• This invention is particularly advantageous for preparing sample nucleic acids to be analyzed by hybridization methods, because the present method results in the formation of single- stranded nucleic acid shear fragments which are of a substantially uniform length, the length being suitable for hybridization analysis. The resulting single- stranded fragments are accessible for hybridization with nucleic acid probes.
• the method described herein is used for the preparation ⁇ of samples wherein the target nucleic acid sequence is found in the interior of intact cells or unicellular organisms.
• cells or organisms are lysed and nucleic acids present within them are fragmented and rendered single- stranded; the single-stranded nucleic acid shear fragments produced are of substantially the same length.
• the invention presently described simultaneously accomplishes three objectives, which could previously only have been achieved after several distinct steps. Therefore, the present method provides for a substantially more reliable and more rapid preparation of samples for nucleic acid hybridization analysis than has hitherto been possible.
• the present method is particularly advantageous for the analysis of clinical, veterinary, foodstuff, water supply, and environmental samples.
• BRIEF DESCRIPTION OF THE DRAWING The Figure is a graphic representation of the sandwich hybridization results observed when target polynucleotide-containing samples are prepared by subjecting them to noninvasive sonication in the presence of varying concentrations of a chaotropic agent (guanidinium thiocyanate) , with and without a heat-denaturation step prior to hybridization analysis.
• a chaotropic agent guanidinium thiocyanate
• the invention described herein relates to a method for producing single-stranded nucleic acid shear fragments of substantially the same length (i.e., onodisperse single-stranded nucleic acid shear fragments) .
• the method of the present invention is particularly useful because it is a rapid, reliable method for preparing nucleic acid-containing samples for hybridization analysis.
• the present method is particularly useful where sample preparation or handling is critical to the reliability of hybridization results reported for human clinical, veterinary, foodstuff, water supply, or environmental samples.
• the present method involves subjecting a sample suspected of containing a particular nucleic acid sequence to noninvasive sonication. More specifically, a sample is prepared by subjecting it to noninvasive sonication in the presence of a sufficient concentration of a chaotropic agent to lyse intact cells (including unicellular organisms) , shear nucleic acids released from the lysed cells into fragments having substantially uniform lengths, and denature the fragments into single-stranded polynucleotides, which are, as a result, available for hybridization analysis.
• a chaotropic agent to lyse intact cells (including unicellular organisms)
• shear nucleic acids released from the lysed cells into fragments having substantially uniform lengths shear nucleic acids released from the lysed cells into fragments having substantially uniform lengths
• denature the fragments into single-stranded polynucleotides which are, as a result, available for hybridization analysis.
• the advantage conferred by the method of the present invention is particularly relevant to the analysis of genomic DNA (prepared by techniques such as those described generally in Current Protocols in Molecular Biology, F. M. Ausubel et al . eds., Sarah Greene, pub.), nucleic acid containing extracts or fractions of cells (including unicellular organisms) , and human clinical, veterinary, foodstuff, water supply, and environmental samples.
• a non-invasive sonication device and sample vessel or cuvette appropriate for practicing nucleic acid sample preparation according to the present invention are described in Li, M.K., et al., (1989) European Patent Application No. 0 337 690, the teachings of which are incorporated herein by reference.
• a feature of the sample cuvette which is essential for successful noninvasive transduction of ultrasonic energy is that the interior wall of the cuvette has a surface discontinuity, such as a crack or groove, which transduces the ultrasonic energy to the sample fluid when the cuvette is contacted with an ultrasonic oscillator. See, e.g., Ringrose, A., (1988) European Patent Application No. 0 271 448.
• Noninvasive sonication offers distinct advantages over conventional sonication techniques. For example, it is not necessary to wash or decontaminate the probe after sonicating each sample. Therefore, there is no significant risk of carryover from one sample to the next. Noninvasive sonication is also unlike bath sonication, in that it does not rely on a liquid to transduce energy. Therefore, splash or bath liquid contamination is not an error source. Moreover, completely enclosed disposable cuvettes can be used, further ensuring both sample integrity and, for example in a clinical setting, safety for the analyst or practitioner.
• the shear fragments produced are of approximately uniform lengths, which are suitable for hybridization analysis. Typically the fragments are from about 400 bp to about 600 bp in length. Fragments of this length are well-suited to hybridization analysis, in that stearic hinderance with the capture of ternary hybridization complexes (capture probe- target sequence-reporter probe) is not a complicating factor.
• the production of shear fragments of approximately uniform lengths suitable for sandwich hybridization analysis by noninvasive sonication is more particularly described in Example 1.
• double-stranded polynucleotide shear fragments must also be denatured, producing single- stranded polynucleotides.
• a chaotropic agent is, generally, a chemical substance which, upon dissolution in an aqueous liquid, produces an ion capable of disrupting the structure of water, thereby facilitating such processes as the dissolution of nonpolar substances in the aqueous liquid.
• Typical chaotropic agents include, for example, guanidinium thiocyanate (GuanSCN) , guanidinium hydrochloride (GuanHCl) , trichloroacetic acid (TCA) , perchloric acid, sodium thiocyanate (NaSCN) , sodium iodide (Nal) and urea.
• Example 2 there is a particular minimum concentration of the chaotrope which is needed to promote the denaturation of polynucleotide shear fragments into single strands when the sample/chaotrope mixture is subjected to noninvasive sonication under conditions sufficient to produce cavitation in the mixture.
• This minimum concentration is not the same as that required to denature double-stranded DNA. Either the addition of heat, or additional chaotrope, is required to achieve denaturation in the absence of noninvasive sonication.
• this concentration is not merely that which is sufficient to denature polynucleotide shear fragments: it is additionally defined by the presence of a level of energy sufficient to produce cavitation, delivered by noninvasive means.
• This minimum concentration is herein defined as the concentration of the chosen chaotrope which is sufficient to promote, the denaturation of a substantial portion of the polynucleotide shear fragments produced by noninvasive sonication when the chaotrope-sample mixture is subjected to noninvasive sonication in the manner presently described. Determining this threshold concentration can be carried out using known methods and requires no more than routine experimentation. For example, it can be determined by conducting an analysis similar to that described in Example 2. As can be seen by reference to the Figure, in the case of GuanSCN, this "threshold" concentration is at least in excess of about 2.5 M.
• the invention can be practiced at GuanSCN concentrations between about 2.5 M and 3.0 M, in general it is practiced using concentrations between about 3.0 M and about 3.5 M. In one preferred embodiment, the GuanSCN concentration used is about 3.5 M. Little further advantage is conferred by conducting noninvasive sonication in the presence of a chaotrope concentration substantially in excess of the threshold (e.g., 4.2 M GuanSCN).
• a chaotrope concentration substantially in excess of the threshold e.g., 4.2 M GuanSCN.
• the threshold concentration of GuanSCN sufficient to produce ⁇ ingl ' e- stranded polynucleotide shear fragments according to the instant invention is also sufficiently high to promote the chemical or osmotic lysis of intact cells or unicellular organisms.
• the use of high concentrations of chaotropic agents to induce lysis is described in J.M. Chirgwin, A.E. Przybyla, R.J. MacDonald, and W.J. Rutter, (1979) Biochemistry 18(24) :5294-5299, 1979. M. Verma, (1988) Biotechni ⁇ u ⁇ s 6(9) :848-853. Cox, et al.
• lysis may be initiated even prior to subjecting the sample to ' noninvasive sonication. This premature lysis will not adversely affect the practice of the instant invention.
• Practitioners in the art will readily appreciate the advantages offered by the method of the present invention in preparing samples for sandwich hybridization analysis of DNA sequences suspected of being present in the sample. DNA sequences are typically encountered in double-stranded form, and must be denatured prior to analysis. This invention is also advantageous for preparing other double-stranded polynucleotide sequences for hybridization analysis.
• RNAs or RNA sequences having a secondary or tertiary structure can be advantageously prepared for sandwich hybridization analysis according to the method described herein.
• ribosomal RNA rRNA is known to exhibit such structural characteristics; see C.R. Woese, et al . (1980) Nucleic Acids Research. 8(10) :2275-2293.
• the present method is particularly well-suited to the preparation of samples for the analysis of contamination by pathogenic unicellular organisms.
• the method described herein involves minimal exposure of the practitioner to samples suspected of containing harmful organisms, and minimizes the risk that the contents of two or more samples may become intermingled during preparation.
• the rapidity and reliability of the present method make it particularly useful for preparing human clinical samples, veterinary clinical samples, foodstuff samples, water supply samples, and environmental samples for hybridization analysis of the presence of pathogenic unicellular organisms such as those capable of causing gastro ⁇ intestinal disorders (e.g., bacterial or amoebic dysentery) in humans.
• gastro ⁇ intestinal disorders e.g., bacterial or amoebic dysentery
• a four-point timecourse was constructed to examine the fragment lengths of sheared DNA produced by noninvasive sonication.
• Buffer A (0.01 M Tris pH 7.5, containing 0.001 M EDTA
• a chaotropic agent was not used, as the presence of such an agent is known to interfere with the electrophoretic analysis of polynucleotides.
• Approximately 10 ⁇ g of purified genomic Shigella ⁇ onnei DNA was added to each cuvette. Sonication was conducted using a device similar to that described in Li, M.K.
• Duty cycle is herein defined as the amount of time a device operates, as opposed to the idle time.
• the term applies to a device such as a sonicator, that normally runs intermittently, rather than continuously. It is conventional to allow any heat produced by sonication to dissipate by briefly interrupting the delivery of ultrasonic energy.
• sonicator that normally runs intermittently, rather than continuously. It is conventional to allow any heat produced by sonication to dissipate by briefly interrupting the delivery of ultrasonic energy.
• ultrasonic energy is actually delivered to the sample for 60% of the time period indicated.
• the sonicated genomic DNA was found to be sheared into fragments having an average length of about 400-600 bp (basepairs) , indicating that a substantially onodisperse population of fragments had been produced. This population remained stable; fragment length was not further reduced even after 2 minutes of sonication.
• This finding is in direct contrast with control results obtained with a conventional immersion probe sonicator,' such as the Vibra CellTM brand immersion probe sonication device (Sonics & Materials, Inc., Danbury, CT) .
• the lengths of genomic DNA shear fragments obtained ranged from about 500 bp to about 4000 bp.
• monodispersity was not achieved by sonication with this conventional procedure and device, even when the sample was subjected to conditions at least as rigorous as those described above for noninvasive sonication.
• Example 2 Production of Single-Stranded Shear Fragments by Noninvasive Sonication in the Presence of Guanidinium Thiocyanate
• a four-point concentration curve of guanidinium thiocyanate was constructed to test the effects of this chaotropic agent on the denaturation of DNA shear fragments in the Shigella ⁇ onnei genomic DNA model system. The extent of denaturation was analyzed in a sandwich hybridization assay by comparing the results of duplicate concentration series, wherein one series was directly analyzed, and the other series was heat- . denatured by boiling prior to analysis.
• Eight molded plastic sonication cuvettes (A-H) were prepared by adding the components listed below in • Table l, from concentrated stocks. Values given are final concentrations, in a total volume per cuvette of 500 ⁇ L.
• Table 1 Components of sonication cuvettes (in 500 ⁇ l)
• the prepared samples were sonicated for 60 seconds at 60 KHz and 60% duty cycle using the noninvasive sonication device. Promptly thereafter, the samples were , prepared for sandwich hybridization analysis.
• Eight polypropylene microtubes were prepared, one corresponding to each sonication cuvette, by adding to each tube 175 ⁇ L sonicate (equivalent to about 28 ⁇ g S. sonnei DNA) , an S. sonnei-specific dA-tailed capture probe having the target-binding sequence TTGCAGCGCC- TCTACTACCGGATACAGCCTCCATT (SEQ ID NO:l); and an S.
• soJinei-specific 32 P labelled reporter probe having the target binding sequence CCTCCTTCAGGGCGGATTCCAGCCGTTC- ACATTGT (SEQ ID NO:2).
• Appropriate amounts of GuanSCN and/or Buffer A were added to the tubes to adjust each to a final volume of 355.8 ⁇ l and a final concentration of GuanSCN of 2.5 M.
• one concentration curve series (A-D) was boiled for 5 minutes to heat-denature the samples; the other series (E-H) was not.
• NAME Galloway, Norval B.

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