EP0555449A1

EP0555449A1 - Simultaneous shear and denaturation of nucleic acids

Info

Publication number: EP0555449A1
Application number: EP19920918431
Authority: EP
Inventors: Charlotte Ruth Williams
Original assignee: BP Corp North America Inc
Current assignee: BP Corp North America Inc
Priority date: 1991-07-31
Filing date: 1992-07-28
Publication date: 1993-08-18
Also published as: JPH06502549A; WO1993003150A1

Abstract

Procédé permettant, simultanément, le découpage d'acides nucléiques en fragments sensiblement monodispersés de longueur appropriée aux fins d'analyse d'hybridation en sandwich, et la dénaturation de ces fragments en polynucléotides monobrins disponibles pour l'hydridation. Le procédé est en outre utile pour effectuer la lyse de cellules intactes ou d'organismes unicellulaires dans lesquels des séquences d'acides nucléiques sont présentes. Le procédé de l'invention consiste à appliquer une énergie sonique non invasive à un échantillon en présence d'un agent chaotropique en concentration suffisante pour induire la dénaturation des fragments découpés de polynucléotides double brin sur application de l'énergie sonique, et sans étape ultérieure de dénaturation thermique. Cette invention est particulièrement utile pour l'analyse d'échantillons cliniques humains, vétérinaires, alimentaires, d'alimentation en eau, ainsi que des échantillons de l'environnement par hybridation "sandwich" d'acides nucléiques.Process allowing, simultaneously, the cutting of nucleic acids into substantially monodispersed fragments of appropriate length for the purpose of analysis of sandwich hybridization, and the denaturation of these fragments into single-stranded polynucleotides available for hydridation. The method is further useful for the lysis of intact cells or single-celled organisms in which nucleic acid sequences are present. The method of the invention consists in applying a non-invasive sonic energy to a sample in the presence of a chaotropic agent in sufficient concentration to induce denaturation of the cut fragments of double-stranded polynucleotides on application of sonic energy, and without subsequent step. thermal denaturation. This invention is particularly useful for the analysis of human, veterinary, food, water supply clinical samples as well as environmental samples by "sandwich" nucleic acid hybridization.

Description

SIMULTANEOUS SHEAR AND DENATURATION OP NUCLEIC ACIDS

BACKGROUND 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. For example, 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, however, 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.

One type of nucleic acid hybridization assay which is advantageously employed for detecting the presence of pathogenic organisms is the sandwich assay. 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, referred to as the reporter probe, 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.

Frequently, 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 lyεing 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.

Sonication as a method of inducing cell lysis is taught in Doulah, M.S., (1977) Biotechnology and Bioenσineerinσ 19:649-660. As ultrasonic waves pass through a fluid, areas of rarefaction and compression are produced. In the areas of rarefaction, the ultrasonic wave produces a local decrease in pressure sufficient to induce a liquid-to-vapor phase change. As a result, microscopic bubbles are produced, and subsequently collapse upon contact with a region of compression. Collapse converts the ultrasonic energy into hydrodynamic energy, including the generation of shock waves and eddy currents. Thermal energy is also generated. This phenomenon is termed cavitation, and is encountered at ultrasonic frequencies up to about 1 MHz. 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.

Once any intact cells or unicellular organisms present in the sample have been lysed, it is important for achieving optimum sensitivity and specificity to convert the target nucleic acid into fragments having lengths suitable for hybridization with the probes referred to above. Very large fragments (e.g., genomic DNA, which is frequently obtained in fragments up to 20 kilobases (kb) in length) may be difficult to immobilize in sufficient number to yield accurate results in a sandwich hybridization assay. This is due to εtearic hinderance during the capture of ternary hybridization complexes, both with the binding of the immobilizable moiety to the solid support, and with access of the ternary hybridization complexes to adjacent sites on the solid support. To overcome this adverse effect, several chemical, physical, and enzymatic methods for producing shear fragments of genomic DNA have been developed. Sonication is one physical method which has been employed for these purposes as well as for the lysis of cells or unicellular organisms. For example, Deininger, P.L. (1983) Analytical

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. However, 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.

Even if fragments of appropriate length have been obtained, hybridization of double-stranded shear fragments with capture and/or reporter probes cannot proceed until the shear fragments are denatured to produce single-stranded polynucleotides. The thermal energy generated during immersion probe or bath sonication is insufficient to denature more than about 30% of the shear fragments produced. Eisner and Lindblad, at 699. Therefore, in order to render the^' majority, (at least about 70%) of the shear fragments available for hybridization analysis, the sample is typically boiled to heat-denature double-stranded shear fragments.

Preparing sample fluids or fluid extracts for sandwich hybridization assays in the manner described above is time consuming and prone to introduced error. A need exists for a rapid method of sample preparation which does not involve multiple steps, each of which can result in crossconta ination and/or loss of sample materials prior to analysis.

SUMMARY OF THE INVENTION 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. In a preferred embodiment, 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. When processed by the method described herein, 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.

DETAILED DESCRIPTION OF THE INVENTION 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. The simultaneous accomplishment of these three objectives in a single procedural step is a significant advantage to one desirous of preparing nucleic acids for hybridization analysis in such a manner that results are reliable. Currently available methods, in contrast, involve multiple procedures and have numerous potential limitations, such as sample carryover, splash error, external contamination, or volume loss. 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.

When noninvasive sonication is condμcted according to the present invention, a surprising result is observed: 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.

In order for the sandwich hybridization analysis to be possible, double-stranded polynucleotide shear fragments must also be denatured, producing single- stranded polynucleotides. Surprisingly, when noninvasive sonication is conducted in the presence of a sufficient concentration of a chaotropic agent, the double-stranded polynucleotide shear fragments produced are also denatured - without the application of thermal energy as - demonstrated in Example 2. 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.

As described more particularly in Example 2 with respect to GuanSCN, 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. Thus, 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, referred to as the threshold 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. While 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).

It will be apparent that the threshold concentration of GuanSCN sufficient to produce εingl^'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) Biotechniσuβs 6(9) :848-853. Cox, et al. (May 1983) FEBS Letters, 155(1) :73-78. Thus, in the present method, 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. For example, RNAs or RNA sequences having a secondary or tertiary structure can be advantageously prepared for sandwich hybridization analysis according to the method described herein. In particular, ribosomal RNA (rRNA) is known to exhibit such structural characteristics; see C.R. Woese, et al . (1980) Nucleic Acids Research. 8(10) :2275-2293.

It will readily be appreciated that the present method is particularly well-suited to the preparation of samples for the analysis of contamination by pathogenic unicellular organisms. Specifically, 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. In addition, 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. The method of the invention described herein has wide applicability to the ^" preparation of the above-listed samples for hybridization analysis, in that it can be used for samples which are (1) obtained as fluids, (2) capable of being extracted with a suitable fluid, or (3) capable of being suspended in a suitable fluid carrier. The invention will now be further illustrated by the following examples, which are not to be viewed as limiting in any way. Example 1: Assessment of Lengths of Shear Fragments Produced by Noninvasive Sonication

A four-point timecourse was constructed to examine the fragment lengths of sheared DNA produced by noninvasive sonication. Four plastic molded sonication cuvettes similar to those described in Ringrose, A. , (1988) European Patent Application No. 0 271 448, were prepared by adding 90 μL of Buffer A (0.01 M Tris pH 7.5, containing 0.001 M EDTA) to each. 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 μg of purified genomic Shigella εonnei DNA (average size greater than 20 kb) was added to each cuvette. Sonication was conducted using a device similar to that described in Li, M.K. , et al., (1989) European Patent Application No. 0 337 690, at 60 KHz and 60% duty cycle for the following times, in seconds: 20, 40, 60, or 120. 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. Thus, at 60% duty cycle, ultrasonic energy is actually delivered to the sample for 60% of the time period indicated.

Twenty μL (approximately 2 μg) of the sonicated DNA was loaded onto a 0.8% agarose gel and electrophoresed according to the method of J. Sambrook,

E.F. Fritsch, and T. Maniatis, Molecular Cloning, A Laboratory Manual, 2d ed. , Cold Spring Harbor Laboratory, 1989. The electrophoretic band pattern generated by the sonicated timecourse samples was compared to unsonicated DNA and phage lambda DNA digested with Hind III.

By the first timepoint (20 seconds) , 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 Cell™ brand immersion probe sonication device (Sonics & Materials, Inc., Danbury, CT) . After two minutes of sonication using this device at power level 5 and 80% duty cycle, the lengths of genomic DNA shear fragments obtained ranged from about 500 bp to about 4000 bp. Thus, 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 εonnei 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 μL.

Table 1: Components of sonication cuvettes (in 500 μl)

l. Tris(hydroxymethyl)aminomethane

2. (Ethylenedinitrilo)tetraacetic acid

3. dithiothreitol

4. sodium lauryl sarkosyl

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 μL sonicate (equivalent to about 28 μg 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 ³²P labelled reporter probe having the target binding sequence CCTCCTTCAGGGCGGATTCCAGCCGTTC- ACATTGT (SEQ ID NO:2). The derivation and characteristics of these probes are described in more detail in a copending patent application U.S. Serial Number 07/738,800, filed July 31, 1991, the teachings of which are incorporated herein by reference. Appropriate amounts of GuanSCN and/or Buffer A were added to the tubes to adjust each to a final volume of 355.8 μl and a final concentration of GuanSCN of 2.5 M. As indicated above, one concentration curve series (A-D) was boiled for 5 minutes to heat-denature the samples; the other series (E-H) was not. The samples were then allowed to hybridize for 30 minutes at 37°C, in a manner similar to that described in the published European Patent Application 0 357 306 of Parados, K. et al., hereby incorporated by reference. Triplicate 100 μl samples were then withdrawn from each tube, and allowed to capture on poly (dT)-coated, blocked polystyrene wells for one hour at room temperature. The wells were washed in SSC (saline with sodium citrate; see J. Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning. A Laboratory Manual. 2d ed. , Cold Spring Harbor Laboratory, 1989), and counted in Quantafluor® brand scintillation fluid (Mallinkrodt, Inc.) in a Beckman model LS 1801 scintillation counter. The results of this analysis are summarized in the Figure. It will be seen that when sonication is conducted in the presence of a guanidinium thiocyanate concentration in excess of the threshold concentation (e.g., see the 3.35 M GuanSCN data point on the figure) little further advantage is conferred by boiling the sample. Boiling is necessary, however, to denature samples sonicated in the presence of 2.5 M or lower concentrations of guanidine thiocyanate.

EQUIVALENTS Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other such equivalents are intended to be encompassed by the following Claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Williams, Charlotte

(ii) TITLE OF INVENTION: Simultaneous Shear and Denaturation of Nucleic Acids

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Amoco Corporation

(B) STREET: 200 East Randolph Drive, P.O. Box 87703

(C) CITY: Chicago

(D) STATE: Illinois

(E) COUNTRY: USA

(F) ZIP: 60680-

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) PRIORITY APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/738,799

(B) FILING DATE: 31-JUL-1991

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Galloway, Norval B.

(B) REGISTRATION NUMBER: 33,595

(C) REFERENCE/DOCKET NUMBER: GTR90-03 PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 312-856-7180

(B) TELEFAX: 312-856-4972

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TTGCAGCGCC TCTACTACCG GATACAGCCT CCATT 35 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 CCTCCTTCAG GGCGGATTCC AGCCGTTCAC ATTGT

Claims

LA1MS 1. A method for simultaneously shearing and denaturing a nucleic acid, comprising the steps of:

(a) providing an aqueous mixture comprising the target nucleic acid and chaotropic agent; and

(b) subjecting the aqueous mixture of (a) to the noninvasive transfer of a sufficient level of ultrasonic energy to produce cavitation therein, thereby producing single-stranded nucleic acid shear fragments which are of substantially the same length.

2. The method of Claim 1 wherein the chaotrope is present in a concentration sufficient to denature polynucleotide shear fragments upon noninvasive exposure to a sufficient level of ultrasonic energy to produce cavitation in the aqueous mixture.

3. The method of Claim 1 wherein the chotropic agent is selected from the group consisting of: guanidinium thiocyanate, guanidinium hydrochloride, urea, perchloric acid, trichlorocetic acid, sodium thiocyanate, and sodium iodide

4. The method of Claim 1 wherein the nucleic acid is double-stranded.

5. The method of Claim 4 wherein the nucleic acid is a genomic DNA sequence.

6. A method for simultaneously shearing and denaturing a polynucleotide, comprising the steps of:

(a) providing an aqueous mixture of the polynucleotide and a sufficient concentration of a chaotropic agent to denature double-stranded polynucleotide shear fragments upon noninvasive exposure to a sufficient level of ultrasonic energy to produce cavitation in the aqueous mixture; and

(b) subjecting the aqueous mixture of (a) to the noninvasive transfer of a sufficient level of ultrasonic energy to produce cavitation in the aqueous mixture, thereby producing single-stranded polynucleotide shear fragments of substantially the same length, the length being suitable for hybridization analysis.

SUBSTITUTE SHEET

7. The method of Claim 6 wherein the polynucleotide is double-stranded.

8. The method of Claim 7 wherein the polynucleotide is a genomic DNA sequence.

9. The method of Claim 6 wherein the chaotropic agent is selected from the group consisting of: guanidinium thiocyanate, guanidinium hydrochloride, urea, perchloric acid, trichloroacetic acid, sodium thiocyanate, and sodium iodide.

10. The method of Claim 6 wherein the polynucleotide is a genomic DNA sequence; the chaotropic agent is guanidinium thiocyanate at a concentration of at least about 3.35 M; and the aqueous mixture of (a) is subjected to the noninvasive transfer of ultrasonic energy ultrasonic energy at a resonance of at least about 60 KHz, for at least about 20 seconds, at a duty cycle of at least about 60%.

11. A method of detecting the presence of a nucleic acid sequence in a sample, comprising the steps of: a) combining the sample with a chaotropic agent, whereby a mixture is produced wherein the chaotropic agent is present at a concentration of at least the threshold concentration sufficient to produce single-stranded nucleic acid from double-stranded nucleic acid shear fragments during noninvasive sonication of the sample; and b) subjecting the mixture of (a) to noninvasive sonication under conditions sufficient to produce cavitation in the mixture, thereby simultaneously: i) lysing intact cells present in the mixture, ii) shearing nucleic acids present in the mixture into fragments of substantially the same length, wherein the length is suitable for hybridization analysis, whereby single-stranded and double-stranded nucleic acid shear fragments are produced, and iii) denaturing double-stranded nucleic acid shear fragments to form single-stranded nucleic acid shear fragments; c) combining the product of (b) and at least one nucleic acid probe capable of hybridizing to the target nucleic acid sequence, under

SUBSTITUTE SHEET conditions sufficient for the hybridization of the complementary nucleotide sequences to occur; and d) monitoring the mixture of (c) for hybridization of complementary nucleotide sequences wherein hybridization is an indication of the presence of the nucleic acid sequence.

12. The method of Claim 11 wherein the chaotropic agent is selected from the group consisting of: guanidinium thiocyanate, guanidinium hydrochloride, urea, perchloric acid, trichlorocetic acid, sodium thiocyanate, and sodium iodide.

13. The method of Claim 12 wherein the chaotropic agent in the aqueous mixture is guanidinium thiocyanate at a concentration of at least about 3.35 M.

14. The method of Claim 13 wherein noninvasive sonication is conducted at a frequency of at least about 60 kHz for at least about 20 seconds using at least about 60% duty cycle.

15. The method of Claim 11 wherein the nucleic acid sequence is present in genomic DNA.

16. The method of Claim 11 wherein the nucleic acid sequence is present in an RNA having a secondary or tertiary structure.

17. The method of Claim 16 wherein the nucleic acid sequence is present in ribosomal RNA.

18. The method of Claim 11 wherein the sample is an aqueous fluid, fluid extract, or suspension and the nucleic acid sequence is present in the interior of an intact cell.

19. The method of Claim 18 wherein the sample is selected from the group consisting of human clinical samples, veterinary clinical samples, foodstuff samples, water supply samples, and environmental samples.

SUBSTITUTE SHEET