WO1994003642A1

WO1994003642A1 - LOW C0t DNA AS DNA FINGERPRINTING PROBE, AND METHOD OF MANUFACTURE

Info

Publication number: WO1994003642A1
Application number: PCT/US1993/007293
Authority: WO
Inventors: Frederick C. Leung; Darrell P. Chandler; Xiao-Zhou Shen
Original assignee: Battelle Memorial Institute
Priority date: 1992-08-07
Filing date: 1993-08-03
Publication date: 1994-02-17
Also published as: AU4800693A

Abstract

The present invention comprises both a for use in DNA fingerprinting analysis, and processes for making the probe and for using the probe. The probe comprises a quantity of low C0t DNA which has been isolated from a reference organism wherein the low C0t DNA has a plurality of repetitive DNA sequence types. The plurality of repetitive sequence types is at least two to all of the repetitive low C0t sequences. When used in the process of the present invention, the low C0t 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 C0t, separating for further use the double stranded portions of the repetitive sequence low C0t renatured DNA from the nonrepetitive sequence DNA, and labeling the low C0t DNA.

Description

LOW C₀t DNA AS DNA FINGERPRINTING PROBE, AND METHOD OP MANUFACTURE

FIELD OF THE INVENTION

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 C₀t DNA (comprised of all or a majority of the repetitive sequences within the genome of the reference organism) as the DNA probe.

As used herein, the term C₀t 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_Qt 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.

BACKGROUND OF THE INVENTION

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. There¬ fore, 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:

* identification of strains and/or races of path- ogens including but not limited to bacteria, fungi, and viruses which are not practically resolvable by currently available morphologic or DNA techniques;

* diagnostics for difficult to identify human blood and tissue infections; * bio arkers for use in compiling a history of environmental exposure to, for example, chemicals or radiation;

* assessment of genetic relatedness in populations including but not limited to microorganisms, plants, animals, and humans;

* elucidation of race and ethnic origins of individuals and/or isolated human populations;

* elucidation of utational spectrum frequency resulting from specific environmental or occupational contaminants; etc.

The limiting factor in the widespread use of many DNA fingerprinting techniques thus far is succinctly stated by Kirby in his book, "DNA Fingerprinting". published 1992 by .H. Freeman & Co. : "Perhaps the greatest constraint at present is the availability of probes specific to different [animal] groups . " Page 5. 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. Different 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 (minisatellites, or vari- able number tandem repeats) are the repetitive sequences, and consist of core tandem repeat sequences within the DNA. Depending on probe specificity and the stringency of the analysis conditions, probes used in profiling may hybridize to either a single locus or many loci simultaneously (i.e., single-locus or multi-locus probes) .

Traditionally, 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.

It is precisely the difficulties in acquiring appropriate probes and evaluating large fractions of genomes that makes the present invention so useful—a probe is available for manufacture in virtually every situation where DNA fingerprinting is to be practiced.

BRIEF DESCRIPTION OF THE FIGURES

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 . conσlutinans 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 oxγsporum . 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 oxγsporum. using F.o . f .sp. cubense as a probe and Hinf I as the enzyme.

SUMMARY OF THE INVENTION

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.

In its simplest embodiment, the novel probe com¬ prises a quantity of low C_Qt DNA which has been isolated from a reference organism wherein the low C₀t DNA has a plurality of repetitive DNA sequence types. In other words, instead of further isolating a single repetitive low C₀t sequence, for example Alu, at least two to all of the repetitive low C_Qt sequences are retained and labeled as a probe. When used in the process of the present invention, the low C_Qt 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_Qt, separating for further use the double stranded portions of the repetitive sequence low C_Qt renatured DNA from the nonrepetitive sequence DNA, and labeling the low C_Qt DNA.

The method of use of the novel probe requires hybridizing the labeled probe with DNA from a subject organism.

The subject matter of the present invention is particularly pointed out and distinctly claimed in the concluding portion of this specification. However, both the organization and method of operation, together with further advantages and objects thereof, may best be understood by reference to the following description taken in connection with accompanying figures.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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_Qt DNA which has been isolated from a reference organism wherein the low C₀t DNA has a plurality of repetitive DNA sequence types. The probe contains from two to all DNA repetitive sequence types of low C_Qt DNA of a genome. In lower life forms, including but not limited to bacteria and fungi, all low C_Qt DNA may be used because of the relatively low fraction of repetitive sequence regions in these forms. Fewer specific but significant portions of low C₀t 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. Because of the nature of the repetitive sequences and their inheritability, they can be used to characterize species, subspecies and races of organisms for which conventional identification techniques would be inadequate. As an example, 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 C₀t probe according to the present invention requires three main steps, DNA extraction, low C_Qt 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 C₀t 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. However, according to the present invention, instead of proceeding to identify a single sequence, a plurality of the extracted low C_Qt sequences are labeled for use as a probe. The plurality of sequences may comprise all or part of the extracted low C₀t sequences.

Similarly, 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 C₀t sequences are used in the hybridization according to the present invention.

The environments in which the probes of the present invention are almost limitless—and the following examples are intended for illustrative purposes only. A number of the possible uses of these probes are set forth above, and any recitation herein is not deemed to be limiting of any possible use of the invention.

LOW C₀t DNA EXTRACTION

In the examples that follow, the same low C_Qt DNA extraction technique was used. The technique is summarized here.

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.

DNA HYBRIDIZATION The hybridization technique used in the following examples is summarized here.

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. , Keene, New Hampshire) on a vacuum blotting system (tradename VacuGene, company Pharmacia LKB Biotechnology, Piscataway, New Jersey) according to the manufacturer's directions. The treated and transferred DNA is fixed to the membrane by baking at 80°C for 1.5 hour.

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 NaH₂P0₄, pH 7.4; 7% by volume sodium dodecyl sulfate (SDS) ; 1% by volume bovine serum albumin; 1 millimolar EDTA, pH 8.0.

Once the membrane is wet, hybridization begins when the aqueous solution is decanted and replaced with 10 microliters per square centimeter identical solution and containing heat denatured low C₀t DNA. The denatured low C_gt 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.

After hybridization, 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,

0.1X SSC (where 2OX SSC = 3M NaCl, 0.3M sodium citrate, pH 7.0) at a temperature of 65°C.

After washing, the DNA containing membrane are wrapped in plastic and exposed to photographic film (Kodak XAR-5) . 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_Qt 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 C₀t 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 ³²P labeled low C₀t DNA from E. coli . From FIG. 1, one sees that the E. coli low C₀t 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_Qt 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_Qt 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_Qt DNA from F199. Again, hybridization occurred only for species matched DNA and hybridization did not occur for P. putida and E. coli .

The data in this example demonstrate that low C₀t DNA is useful as a probe for species specific fingerprinting or identification in bacteria.

EXAMPLE 2

A second experiment was done according to the procedures described in Example 1, wherein E. coli low C_Qt DNA was labeled with P as a probe. Three additional strains of E. coli . HB 101, JM 109, and DH5α 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 C₀t probe.

P. putida and F-199 were included as control samples. Results presented in FIG. 4 is an autoradiogram showing that the P. putida and the F-199 again did not hybridize with the E. coli low C₀t. While all four strains of E. coli hybridized with the E. coli low C_Qt, each hybridization was unique to each strain. The data in this example demonstrate that low C_Qt DNA is useful as a probe for strain specific finger¬ printing or identification within a species in bacteria.

EXAMPLE 3

An experiment was conducted using species of the genus Fusarium oxγsporum as listed in Table 1.

TABLE l. Fusarium oxγsporum species

F. o . lγcopersici F. o . pisi F. o . conσlutinanε F. o . phaseoli

F. o . raphani F. o . cubense

These species of Fusarium oxγsporum were obtained from the American Type Culture Collection with the exception of F. o . conσlutinans that was obtained from Dr. Paul H.

Williams, Department of Plant Pathology, University of

Wisconsin, Madison Wisconsin.

The procedure of the experiment was generally identical to that of Examples 1 and 2 except that low C_Qt DNA was obtained from the strains of Fusarium oxγsporum which required a different DNA extraction method, and that the restriction enzymes used were Hinf I, Hae III, and Cfo I.

DNA extraction for F. oxγεporum was done using a fungal DNA extraction procedure as described by Raeded and Broda (Letters in Applied Microbiology, 1:17-20, 1985) , and briefly described here.

Filtered F. oxγsporum 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.

A solution of 350 microliters 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.

The extracted mixed suspensions were centrifuged at room temperature for 1 hour at 16,000g. The supernatant was transferred to a new tube containing 15 microliters RNAse A at 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.

Low C₀t DNA extraction and hybridization were the same as earlier described herein. Results for use of low C_Qt DNA from F.o . f .sp. conglutinans are shown in FIG. 5 in an autoradiogram. The low C₀t DNA hybridized to multiple bands of genomic DNA digested with all three enzymes. The highest number of bands are shown for F. o . f .sp . conglutinans and the probe revealed an number of bands that are shared between all five Fusarium oxysporum strains as well as a number of bands specific to F. o . f . sp. conglutinans .

Results for use of low C_Qt DNA from F. o . f . sp. 1 γco peri εici are shown in FIG. 6 in an autoradiogram. The low C₀t 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 oxγsporum strains as well as a number of bands specific to F.o. f.sp. lγcoperiεici .

Results for use of low C_Qt DNA from F.o . f .sp. phaseoli are shown in FIG. 7 in an autoradiogram. The low C₀t 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 Fuεarium oxysporum strains as well as a number of bands specific to F.o . f .sp. phaseoli .

Results for use of low C_Qt DNA from F. o . f . sp. raphani are shown in FIG. 8 in an autoradiogram. The low C_Qt 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 Fuεarium oxysporum strains as well as a number of bands specific to F.o . f .sp. raphani . Results for use of low C₀t DNA from F. o . f . sp. cubenεe are shown in FIG. 9 in an autoradiogram. The low C_Qt 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 Fuεarium oxysporum strains as well as a number of bands specific to F. o . f . εp. cubenεe .

Results for use of low C₀t DNA from F.o . f . εp. cubenεe are shown in FIG. 10 in an autoradiogram. The low C_Qt 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 Fuεarium oxγεporum strains as well as a number of bands specific to F. o . f . εp. cubenεe . The data in this example demonstrate that low c_Qt DNA is useful as a probe for fingerprinting or identifi¬ cation of specific species of Fuεarium oxγεporum.

CLOSURE

Until the advent of the present invention, there was not an expeditious way to use DNA fingerprinting to distinguish species of Fuεarium oxγεporum . With this ability, farmers can test their fields for pathogenic strains that are host specific to the planned crops and take action prior to crop failure. Many other opportunities beyond and including those set forth above in the Background of the Invention are now possible.

While a preferred embodiment of the present invention has been shown and described, it will be apparent to those skilled in the art that many changes and modifications may be made without departing from the invention in its broader aspects. The appended claims are therefore intended to cover all such changes and modifications as fall within the true spirit and scope of the invention.

Claims

We Claim:

1. A DNA fingerprinting probe comprising a plurality of repetitive DNA sequences in low C_Qt DNA isolated from a reference organism, and labeled.

2. The probe of Claim 1, wherein the labeled DNA is thermally denatured.

3. The probe of Claim 1, wherein the denatured DNA is labeled with a radioactive material.

4. The probe of Claim 3, wherein the radioactive material is phosphorus.

5. The probe of Claim 1, wherein the probe is utilized in hybridization analysis of DNA from a target organism.

6. A process for producing a probe useful in DNA fingerprinting analysis, comprising:

(a) isolating, shearing and denaturing a quantity of DNA from a reference organism;

(b) renaturing the DNA to an intermediate c₀t; (c) separating for further use the double stranded portions of the repetitive sequence low C₀t renatured DNA from the nonrepetitive sequence DNA; and (d) labeling the low C_Qt DNA.

7. The process of Claim 6, further comprising thermally denaturing the DNA.

8. The process of Claim 6, further comprising labeling the low C₀t DNA with a radioactive material.

9. The process of Claim 8, further comprising labeling the low C₀t DNA with phosphorus.

10. The process of Claim 6, further comprising labeling the low C_Qt DNA with a non-radioactive material.

11. A DNA fingerprinting process comprising the steps of:

(a) producing a labeled low C_Qt DNA probe having a plurality of repetitive sequences from a reference organism; and (b) hybridizing the labeled probe with DNA from a subject organism.