WO1994003642A1 - ADN A FAIBLE VALEUR C0t UTILISE COMME SONDE D'EMPREINTE GENETIQUE D'ADN, ET PROCEDE DE PREPARATION - Google Patents

ADN A FAIBLE VALEUR C0t UTILISE COMME SONDE D'EMPREINTE GENETIQUE D'ADN, ET PROCEDE DE PREPARATION Download PDF

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Publication number
WO1994003642A1
WO1994003642A1 PCT/US1993/007293 US9307293W WO9403642A1 WO 1994003642 A1 WO1994003642 A1 WO 1994003642A1 US 9307293 W US9307293 W US 9307293W WO 9403642 A1 WO9403642 A1 WO 9403642A1
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Prior art keywords
dna
probe
low
repetitive
sequence
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PCT/US1993/007293
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English (en)
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Frederick C. Leung
Darrell P. Chandler
Xiao-Zhou Shen
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Battelle Memorial Institute
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Priority to AU48006/93A priority Critical patent/AU4800693A/en
Publication of WO1994003642A1 publication Critical patent/WO1994003642A1/fr

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism

Definitions

  • This invention relates to a novel probe, and a method of making the probe, for DNA fingerprinting to enable accurate identification of a subject organism. Specifically, this invention uses multiple repetitive sequences or low C 0 t DNA (comprised of all or a majority of the repetitive sequences within the genome of the reference organism) as the DNA probe.
  • C 0 t is defined, as- in the book entitled Gene IV, by B. Lewin, IV edition, 1990, page 468, to be the product of initial DNA concentration (C Q ) and incubation time (t) .
  • DNA material having a C Q t that is low implies a small amount of time for DNA component association and an associated low complexity in terms of number of base pairs per repetitive sequence.
  • DNA fingerprinting assay techniques are gaining in importance as their reliability and cost effectiveness are improved. These techniques are already used in a number of medical and forensic applications, and an even larger number of potential uses have been identified. For example, these techniques are used (i) to identify perpetrators of crimes based on body tissue/fluids left at the scene of a crime, (ii) to establish or refute paternity/maternity, (iii) to identify the origin of a posttransplant cell population after a bone marrow transplant to monitor engraftment, (iv) to identify poached wild game, etc.
  • SUBSTITUTESHEET Specificity is the key to DNA fingerprinting. In other words, finding a probe that will combine with a DNA sequence of interest to produce an autoradiogram having a number of bands, thereby providing resolution sufficient for further comparisons is of prime importance.
  • probe development is one of the key steps to the widespread use and application of this technology. Many probes have been developed and are commercially avail ⁇ able, and new probes are being developed. Because development of a new probe is a laborious and expensive process, the vast majority of potential uses of DNA fingerprinting has not yet been undertaken for lack of probes. Potential uses, include but are not limited to:
  • a probe is a sequence of DNA material that may or may not be characterized. However, the probe combines or hybridizes with other DNA material in consistent and repeatable ways. When the hybridization results in the ability to obtain an autoradiogram showing distinct bands, the probe is useful for that combination. If, on the other hand the probe/other material hybridization leads to an autoradiogram from which bands are not distinguishable, then that combination is not useful and another probe must be found.
  • Probes are of two types, single locus and multi- loci probes.
  • Single locus probes are those containing a single repetitive sequence that when hybridized with other DNA material, produce one single or double dark band having a distinct vertical position on an auto- radiogram.
  • Multi-loci probes also contain a single repetitive sequence, but they produce multiple dark bands with intervening light bands.
  • a probe that combines so completely with another material so that there are no intervening light bands produces a solid dark vertical area and is of limited value since there is no discern- able pattern.
  • a probe that does not combine at all with another material produces no dark band at all and is also of limited utility. Multiple single locus probes have been used for DNA fingerprinting analysis.
  • probes have different specificities, where specificity refers to the characteristic of combining or hybridizing with other DNA material.
  • a probe of high specificity will hybridize with only a few other DNA materials while a probe of low specificity will hybridize with many other DNA materials.
  • the specificity of the DNA assay techniques currently in use depends on the polymorphism of the genome and the detection assay depends on the use of restriction enzymes and DNA probes.
  • the hypervariable region of DNA are the repetitive sequences, and consist of core tandem repeat sequences within the DNA.
  • probes used in profiling may hybridize to either a single locus or many loci simultaneously (i.e., single-locus or multi-locus probes) .
  • the identification and charac ⁇ terization of an unknown genome is done using selected repetitive sequences as probes.
  • These probes are developed by making a genomic library and screening or extracting clones of a particular repetitive sequence. Because only a single repetitive sequence is used for probing an unknown genome, only a very small fraction of the unknown genome is evaluated or characterized.
  • Pre- ferred sequences are those having a high copy number, for example the Alu family of repetitive sequences.
  • Other sequences used as probes include dinucleotide and trinucleotide repetitive sequences.
  • FIG. 1 is an autoradiogram of three bacteria species, using E. coli as a probe.
  • FIG. 2 is an autoradiogram of three bacteria species, using P . putida as a probe.
  • FIG. 3 is an autoradiogram of three bacteria species, using F199 as a probe.
  • FIG. 4 is an autoradiogram of three bacteria species and four E. coli strains, using E. coli as a probe.
  • FIG. 5 is an autoradiogram of five species of Fusarium oxysporum , using F . o . f . sp . con ⁇ lutinans as a probe.
  • FIG. 6 is an autoradiogram of five species of Fusarium oxysporum f using F.o . f .sp. lycoperisici as a probe.
  • FIG. 7 is an autoradiogram of five species of Fusarium ox ⁇ sporum . using F. o . f . sp. phaseoli as a probe.
  • FIG. 8 is an autoradiogram of five species of Fusarium oxysporum . using F. o . f .sp. raphini as a probe.
  • FIG. 9 is an autoradiogram of six species of Fusarium oxysporum , using F. o . f . sp. cubense as a probe and Hae III as the enzyme.
  • FIG. 10 is an autoradiogram of six species of Fusarium ox ⁇ sporum. using F.o . f .sp. cubense as a probe and Hinf I as the enzyme.
  • the present invention comprises both a novel probe for use in DNA fingerprinting analysis, and processes for making the probe and for using the probe.
  • the novel probe com ⁇ prises a quantity of low C Q t DNA which has been isolated from a reference organism wherein the low C 0 t DNA has a plurality of repetitive DNA sequence types.
  • the low C Q t DNA multiple sequence probe enables the identification or exclusion of the subject organism.
  • the process for producing the probe useful herein comprises the steps of isolating, shearing and denaturing a quantity of DNA from a reference organism, renaturing the DNA to an intermediate C Q t, separating for further use the double stranded portions of the repetitive sequence low C Q t renatured DNA from the nonrepetitive sequence DNA, and labeling the low C Q t DNA.
  • the method of use of the novel probe requires hybridizing the labeled probe with DNA from a subject organism.
  • the present invention comprises both a novel probe for use in DNA fingerprinting analysis, and processes for making the probe and for using the probe.
  • the probe comprises a quantity of low C Q t DNA which has been isolated from a reference organism wherein the low C 0 t DNA has a plurality of repetitive DNA sequence types.
  • the probe contains from two to all DNA repetitive sequence types of low C Q t DNA of a genome. In lower life forms, including but not limited to bacteria and fungi, all low C Q t DNA may be used because of the relatively low fraction of repetitive sequence regions in these forms. Fewer specific but significant portions of low C 0 t repeti ⁇ tive sequences may be used in higher life forms, includ ⁇ ing but not limited to higher animals and higher plants wherein the fraction of repetitive sequence regions is relatively high.
  • repetitive sequence DNA may contain genomic information related to both host-pathogen recognition and genetic phenotypic markers, permitting the identification of genes controlling pathogenicity and host specificity.
  • the method of making and using the low C 0 t probe according to the present invention requires three main steps, DNA extraction, low C Q t DNA extraction, and DNA hybridization.
  • the DNA extraction procedure varies from organism to organism. Hence any DNA extraction technique appropriate to the organism to be studied is appropriate.
  • the low C 0 t DNA extraction procedure is well known for making subtractions from a genomic library to facilitate the identification of a particular non- repetitive or repetitive sequence of a genome.
  • a plurality of the extracted low C Q t sequences are labeled for use as a probe.
  • the plurality of sequences may comprise all or part of the extracted low C 0 t sequences.
  • DNA hybridization techniques are known and may be applied to the present invention. Again, however, instead of hybridizing a single sequence, a plurality of the labeled low C 0 t sequences are used in the hybridization according to the present invention.
  • a sample of 100 micrograms of genomic DNA are sheared by sonication for 30 seconds and the volume adjusted to 1 milliliter with water.
  • the DNA is then heat denatured in a boiling water bath for 15 minutes followed by a quick chill on ice.
  • Sodium chloride is added to a final concentration of 0.18 M.
  • the DNA is allowed to renature over a 16-18 hour period at a temperature of 60 C.
  • Hydroxyapatite "columns" are made of glass test tubes containing 0.25 grams of hydroxyapatite (Bio-Gel HTP, BIORAD, Richmond, California) . Four (4) milliliters of 0.12 M phosphate buffer is preheated to 60 C then added to the renatured DNA. The buffered renatured DNA is then placed within the hydroxyapatite column for l hour with intermittent shaking. After 1 hour, shaking ceases and the contents of the column are allowed to settle. The supernatant is removed and the column is washed 6 times with 1 milliliter of 0.12 M phosphate buffer and 3 times with 1 milliliter of 0.4 M phosphate buffer. Washes are monitored with ultraviolet spectrophotometry for salt content.
  • washes having high salt content are combined and desalted in a filter unit (Millipore Ultrafree-MC 30,000 NMWL, Millipore Products Division, Bedford, Massachusetts) .
  • DNA retained in the filter unit is recovered with washes of a mixture of 10 micromolar Tris and 1 micromolar ethylenediaminetetraacetic acid (EDTA) having a pH of 7.8. Concentrations of recovered DNA are determined by Hoechst 33258 staining.
  • Restriction enzymes from the group of Hae III, Hinf I, Alu I, Sau3A-I, and Cfo I are used. Restriction enzymes are obtained from BRL, Life Technologies, Inc., Grand Island, New York.
  • Samples of 2 micrograms of DNA are digested with restriction enzymes to completion according to the manufacturer's directions.
  • the digested DNA is separated by electrophoresis through 1% agarose gels in a buffer.
  • the agarose gels are from FMC Bioproducts, Rockland, Maine and are known as Seakem GTG.
  • the buffer is a mixture of 89 millimolar Tris, 89 millimolar boric acid, and 2 millimolar EDTA.
  • the electrophoresis potential is 2 V/cm and separation is carried out for 22 to 24 hours.
  • the separated DNA is treated by acid depurination, alkaline denaturization, neutralization, then transferred to Nylon membranes (tradename Nytran, company Schleicher & Schuell Inc.
  • the fixed DNA on the Nytran membrane is prehybridized by addition of an aqueous solution and subsequent heating in a hybridization oven (Robbins Scientific, Sunnyvale, California) for a time sufficient for aqueous solution to completely wet the membrane.
  • the aqueous solution is 0.25M NaH 2 P0 4 , pH 7.4; 7% by volume sodium dodecyl sulfate (SDS) ; 1% by volume bovine serum albumin; 1 millimolar EDTA, pH 8.0.
  • hybridization begins when the aqueous solution is decanted and replaced with 10 microliters per square centimeter identical solution and containing heat denatured low C 0 t DNA.
  • the denatured low C g t DNA has been previously labeled to greater than (10) 9 counts per minute per microgram. Labeling is done with the random primer method as described by Feinberg and Vogelstein (Analytical Biochemistry 132:6-13, 1983).
  • Hybridization continues overnight at a temperature of 65°C.
  • hybridized DNA samples still on the membrane, are washed. Washes are 4 times for a period of twenty minutes each with 0.1% by volume SDS,
  • Exposure may be with or without intensifying screens (Cronex Lighting-plus) and is for a period of from 1 to 7 days at a temperature of -80°C.
  • EXAMPLE 1 An experiment was conducted wherein separate batches of low C Q t DNA were isolated from each of (a) Escherichia coli. (b) Pseudomonas putida , and (c) F199 (a gram negative aerobic heterotroph) . Bacterial DNA were isolated using well known methods either by cesium chloride preparation or by phenol/chloroform procedures as described in sections 2.4.3 and 2.4.1, respectively, in Current Protocols in Molecular Biology, Vol. 1, ed. by AUSUBEL et al. 1990, Published by Wiley Inter Science.
  • Each batch of low C 0 t DNA was further extracted and hybridized for assay according to the methods described above.
  • Each assay included DNA samples from all three bacterial species and were done using three restriction enzymes, Hae III, Hinf I, and Alu I.
  • FIG. 1 is an autoradiogram of DNA fingerprints of genomic DNA from the three bacterial species. Genomic DNA from each of the three species were digested with each of the three restriction enzymes and mixed for hybridization with the 32 P labeled low C 0 t DNA from E. coli . From FIG. 1, one sees that the E. coli low C 0 t DNA probe hybridized to multiple restriction fragments in the E. coli genomic DNA cut with the three enzymes, but that the E. coli low C Q t DNA probe did not hybridize with the other two bacterial species.
  • FIG. 2 shows results of the same general procedure as was performed to obtain FIG. 1, but using low C Q t DNA from P. putida . Again, hybridization occurred only for species matched DNA and hybridization did not occur for E. coli and F199.
  • FIG. 3 shows results of the same general procedure as was performed to obtain Figures 1 and 2, but using low C Q t DNA from F199. Again, hybridization occurred only for species matched DNA and hybridization did not occur for P. putida and E. coli .
  • Example 2 A second experiment was done according to the procedures described in Example 1, wherein E. coli low C Q t DNA was labeled with P as a probe. Three additional strains of E. coli . HB 101, JM 109, and DH5 ⁇ genomic DNA were digested with restriction enzymes Alu I, Sau 3A-1, and Hind III. Each additional strain/restriction enzyme combination was individually hybridized with E. coli low C 0 t probe.
  • the procedure of the experiment was generally identical to that of Examples 1 and 2 except that low C Q t DNA was obtained from the strains of Fusarium ox ⁇ sporum which required a different DNA extraction method, and that the restriction enzymes used were Hinf I, Hae III, and Cfo I.
  • Filtered F. ox ⁇ sporum cultures were frozen in liquid nitrogen, lyophilized to dryness and stored at -80°C. An amount of 50 milligrams of the dried culture was ground to a fine powder in a mortar and pestle and resuspended in 500 microliters of extraction buffer (200 millimolar Tris, pH 8.0; 25 millimolar EDTA, pH 8.0; 250 millimolar NaCl; 2% by volume SDS.
  • extraction buffer 200 millimolar Tris, pH 8.0; 25 millimolar EDTA, pH 8.0; 250 millimolar NaCl; 2% by volume SDS.
  • Tris-buffered phenol and 150 microliters water-saturated chloroform were mixed into the resuspended culture by inversion and the mixed suspension extracted overnight (about 12 hours) by shaking.
  • RNAse A concentration of 10 milligram per milliliter (Sigma Chemical Co., St. Louis, Missouri) and incubated at 37°C for 15 minutes. Incubated suspensions were serially extracted with equal volumes of phenol:chloroform (50:50) and chloroform only followed by centrifugation for 10 minutes at 16,000g. DNA was recovered by adding 0.6 volumes (300 microliters isopropanol, incubating for 10 minutes at room temperature and centrifuging for an additional 10 minutes at 16,000g. Nucleic acid pellets were washed once with 70% by volume ethanol, dried under vacuum and resuspended in sterile water.
  • Results for use of low C Q t DNA from F. o . f . sp. 1 ⁇ co peri ⁇ ici are shown in FIG. 6 in an autoradiogram.
  • the low C 0 t DNA hybridized to multiple bands of genomic DNA digested with all three enzymes.
  • the probe revealed an number of bands that are shared between all five Fusarium ox ⁇ sporum strains as well as a number of bands specific to F.o. f.sp. l ⁇ coperi ⁇ ici .
  • results for use of low C Q t DNA from F.o . f .sp. phaseoli are shown in FIG. 7 in an autoradiogram.
  • the low C 0 t DNA hybridized to multiple bands of genomic DNA digested with all three enzymes.
  • the probe revealed an number of bands that are shared between all five Fu ⁇ arium oxysporum strains as well as a number of bands specific to F.o . f .sp. phaseoli .
  • Results for use of low C Q t DNA from F. o . f . sp. raphani are shown in FIG. 8 in an autoradiogram.
  • the low C Q t DNA hybridized to multiple bands of genomic DNA diges ⁇ ted with all three enzymes.
  • the probe revealed an number of bands that are shared between all five Fu ⁇ arium oxysporum strains as well as a number of bands specific to F.o . f .sp. raphani .
  • Results for use of low C 0 t DNA from F. o . f . sp. cuben ⁇ e are shown in FIG. 9 in an autoradiogram.
  • the low C Q t DNA hybridized to multiple bands of genomic DNA diges ⁇ ted with restriction enzyme Hae III.
  • the probe revealed an number of bands that are shared between all six Fu ⁇ arium oxysporum strains as well as a number of bands specific to F. o . f . ⁇ p. cuben ⁇ e .
  • results for use of low C 0 t DNA from F.o . f . ⁇ p. cuben ⁇ e are shown in FIG. 10 in an autoradiogram.
  • the low C Q t DNA hybridized to multiple bands of genomic DNA digested with restriction enzyme Hinf I.
  • the probe revealed an number of bands that are shared between all six Fu ⁇ arium ox ⁇ porum strains as well as a number of bands specific to F. o . f . ⁇ p. cuben ⁇ e .
  • the data in this example demonstrate that low c Q t DNA is useful as a probe for fingerprinting or identifi ⁇ cation of specific species of Fu ⁇ arium ox ⁇ porum.

Abstract

La présente invention se rapporte à la fois à une sonde utilisée dans l'analyse d'empreinte génétique d'ADN, et à des procédés de préparation et d'utilisation de la sonde. Cette dernière comprend une certaine quantité d'ADN à faible valeur C0t (concentration initiale d'ADN x temps d'incubation), qui a été isolée à partir d'un organisme de référence dans lequel l'ADN à faible valeur C0t présente une multiplicité de types de séquences d'ADN répétitives). Ladite multiplicité de types de séquences répétitives est composée d'un moins deux des séquences répétitives à faible valeur C0t ou de toutes ces séquences. Lorsque cette sonde à séquences multiples d'ADN à faible valeur C0t est utilisée selon le procédé de l'invention, elle permet l'identification ou l'exclusion de l'organisme à étudier. Le procédé de préparation de la sonde décrite consiste à isoler, soumettre à un cisaillement et dénaturer une certaine quantité d'ADN à partir d'un organisme de référence, à renaturer l'ADN à une valeur C0t intermédiaire, à séparer les parties double brin de l'ADN à faible valeur C0t et à séquence répétitive de l'ADN à séquence non répétitive afin de les utiliser ultérieurement, et à marquer l'ADN à faible valeur C0t.
PCT/US1993/007293 1992-08-07 1993-08-03 ADN A FAIBLE VALEUR C0t UTILISE COMME SONDE D'EMPREINTE GENETIQUE D'ADN, ET PROCEDE DE PREPARATION WO1994003642A1 (fr)

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AU48006/93A AU4800693A (en) 1992-08-07 1993-08-03 Low c0t dna as dna fingerprinting probe, and method of manufacture

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US92673092A 1992-08-07 1992-08-07
US07/926,730 1992-08-07

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0186271A1 (fr) * 1984-11-12 1986-07-02 THE LISTER INSTITUTE OF PREVENTIVE MEDICINE Royal National Orthopaedic Hospital Méthode de caractérisation d'un échantillon d'ADN génomique
EP0298656A1 (fr) * 1987-07-10 1989-01-11 Stephen Thomas Reeders Sondes polynucléotides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0186271A1 (fr) * 1984-11-12 1986-07-02 THE LISTER INSTITUTE OF PREVENTIVE MEDICINE Royal National Orthopaedic Hospital Méthode de caractérisation d'un échantillon d'ADN génomique
EP0298656A1 (fr) * 1987-07-10 1989-01-11 Stephen Thomas Reeders Sondes polynucléotides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEUNG, F. ET AL: "use of repetitive sequences for the identification of species-specific DNA fragments in fassarium-oxysporium", PHYTOPATHOLOGY, vol. 82, no. 10, 1992, pages 1065 *
LEWIN,B.: "Gene IV", 1990, OXFORD UNIVERSITY PRESS *

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