WO1987002065A1 - Determination d'identite entre deux organismes - Google Patents

Determination d'identite entre deux organismes Download PDF

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Publication number
WO1987002065A1
WO1987002065A1 PCT/GB1986/000210 GB8600210W WO8702065A1 WO 1987002065 A1 WO1987002065 A1 WO 1987002065A1 GB 8600210 W GB8600210 W GB 8600210W WO 8702065 A1 WO8702065 A1 WO 8702065A1
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Prior art keywords
dna
organisms
organism
genomic dna
probe
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PCT/GB1986/000210
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English (en)
Inventor
Barry Gordon Dimitri Hall
James Howard Slater
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Biotal Limited
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Priority claimed from GB08500450A external-priority patent/GB2153597A/en
Application filed by Biotal Limited filed Critical Biotal Limited
Publication of WO1987002065A1 publication Critical patent/WO1987002065A1/fr

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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to determining to an ascertainable degree of probability whether a first and second organism, the identity of one of which is known, * are identical. It is especially applicable to three main categories of organism: (1) those which reproduce principally by asexual means, in particular microorganisms such as bacteria, fungi and viruses, (2) those where it is important to identify a unique individual and (3) inbred populations which are reproducing sexually, such as some plant varieties.
  • Plasmids usually reduce the fitness of microorganisms. Consequently, such plasmids are often lost during mass culture procedures.
  • the easiest kind of mutational markers to introduce are mutations to drug resistance. Howewever, these mutations are often themselves disadvantageous. Further, both types of markers can be altered with relative ease.
  • a new approach has now been developed to the problem of identifying organisms, utilising unique properties of the organism that cannot be altered.
  • the present invention takes advantage of the natural genetic variation that occurs in a species.
  • the present invention provides a method of determining to an ascertainable probability whether a first and a second organism, the identity of one of which is known, are identical, which method comprises:
  • steps (i.) to (iv) being effected using an amount of probe DNA and one or more restriction endonucleases such that sufficient bands are revealed by the hybridisation in step (iii) to achieve a sufficiently low probability (X) that, when the comparison in step (iv) reveals that the two organisms appear identical, the two organisms will have failed to have been distinguished as genuinely different and unrelated as determined by:-
  • F is a fraction representative of the proportion of DNA fragments which are identical between restriction endonuclease digests of genomic DNA of pairs of independently-obtained organisms of the same species as the first and second organisms and q is the number of positions revealed by the probing in step (iii) .
  • q is the total number of common positions of DNA digest fragments revealed by the pairwise comparison of the first and second organisms in step (iv) , when the two organisms have identical maps.
  • steps (i) to (iv) are effected by
  • steps (i 1 ) to (iv') can be effected two or more times using a different restriction endonuclease each time.
  • the present invention depends upon the degree to which the failure to detect a difference between hybridisation patterns for two organisms can be taken as evidence of identity.
  • two genomes are digested by a restriction endonuclease
  • F fraction of conserved fragments
  • n is the number of base pairs in a restriction endonuclease recognition site.
  • F is the probability that a fragment will be conserved. Consequently F q is the probability that, if a digest produces q fragments, all the fragments will be conserved. This may then be used to determine how many different restriction endonucleases and different DNA probes need be employed in the present invention to determine whether two organisms are identical beyond reasonable doubt. Fragments produced when genomic DNA is digested by a restriction endonuclease can be detected by a labelled DNA probe after separation of the genomic DNA fragments by electrophoresis and hybridisation. The greater the amount of probe DNA and the more restriction endonuclease digests examined, the more DNA fragments will be revealed. In other words q increases. As F is a value less than 1, F q becomes smaller as q increases. Thus the probability becomes less that two organisms, which show identical hybridisation patterns but which are in fact genuinely different, will fail to be distinguished.
  • F and q are as defined above.
  • F is estimated experimentally for a number of independently-obtained organisms ("standard organisms") known to be of the same species.
  • standard organisms a number of independently-obtained organisms known to be of the same species.
  • hydridization patterns can be obtained for an appropriate number of non-clonally derived organisms of the same species, for example as determined by conventional taxonomic criteria.
  • Sufficient standard organisms are used to obtain a statistically significant F value for the species, i.e. a F value representative for the species.
  • F values may be determined from the definition of F, the fraction of conserved fragments:
  • N is the total number of common bands between a pair of organisms under comparison and a and b are the respective numbers of fragments detected from all the restriction endonuclease digests and probes of genomic DNA of the two organisms of the pair.
  • the experimental determination of F for a given species of organism enables users of this invention to predetermine the number of genomic DNA digests needed to be undertaken for a given amount of probe DNA in order to obtain a required level of confidence (C) or probability (X) that identity will not be wrongly claimed. Two organisms, therefore, can be analysed to determine the probability that they genuinely differ or the level of confidence that they are identical.
  • band patterns are different then the two organisms cannot be identical and cannot have been derived clonally or by asexual reproduction or by sexual reproduction of appropriate highly in-bred organisms. If the band patterns appear to be identical a quantitative probability can be assigned that identity will be wrongfully claimed. Very accurate results can be achieved (see Example 3) .
  • the present method involves digesting genomic - DNA of the two organisms being analysed with restriction enzymes and separating the DNA fragments in the resulting digests by electrophoresis.
  • the mobilities of the DNA fragments which are detected by a probe can be measured for each organism. Since the same restriction endonuclease has been used and electrophoresis has been conducted in the same way, if the hybridised fragment mobilities (or band patterns) are the same the organisms may be, but not necessarily are, identical. However, where the fragment mobilities are different, the two organisms must be different.
  • band patterns are the same, it is possible to determine whether two organisms are identical with more certainty by employing more than one restriction endonuclease and/or more than one DNA probe.
  • Type II restriction endonucleases cut DNA at specific sites that depend upon the DNA sequence at that site. This high specificity means that a particular enzyme cuts (digests) the DNA from a particular organism into a specific set of DNA fragments. A different endonuclease with a different recognition sequence will produce a different set of fragments. The average length of these fragments depends upon the length of DNA required for recognition by a particular restriction endonuclease. For example, restriction endonucleases recognising 6-base sequences generate fragments of an average length of 4096 base pairs (bp) whilst those recognising 4-base sequences generate fragments of an average length of 256 bp. However, the distribution of recognition sites results in a large actual array of fragment lengths. On electrophoresis, the large amount of DNA in a cell combined with the variation in fragment length produces a smear of fragment sizes with little or no recognizable pattern.
  • DNA will bind specifically to DNA with the same sequence to produce a set of patterns unique to a particular organism. If a random fragment of DNA from an organism is cloned into a suitable vector and subsequently labelled, the cloned fragment acts as a probe which is capable of hybridising with DNA that has the same sequence. The fact that the probe is labelled enables its position to be detected.
  • the comparison of two organisms according to steps (i) to (iv) of the method of the invention can be illustrated as follows. A random fragment of DNA from a first strain of an organism or a specific individual is used to make a labelled probe. The total genomic DNA from the first strain (or individual) is digested with a restriction endonuclease.
  • the DNA fragments are separated by electrophoresis on a gel. If visualised, the digested DNA would be seen only as a smear.
  • the digested DNA is hybridised with the probe.
  • the probe will bind only to those fragments that show a certain degree of homology with the sequences in the probe.
  • several bands can be detected by means of the label on the probe at the sites of hybridisation of homologous DNA sequences. The positions of these bands are determined by the fragment sizes.
  • the total genomic DNA from a second strain or individual is digested by the restriction endonuclease and subjected to electrophoresis in an identical manner, and hybridised with the same probe.
  • the positions of the bands will be different, since digestion of the genomic DNA will produce fragments of different sizes due to the position of different restriction sites. If the positions are the same, the probe will have hybridised with identical DNA fragments and appear in identical positions in the gel. The strains or two individuals may therefore be the same. Further tests employing different restriction endonucleases and/or different probes will establish beyond reasonable doubt whether there is identity between the two strains or individuals.
  • the genomic DNA in step (i) may be digested separately with one or more restriction endonucleases. Respective portions of the DNA sample can be treated with different restriction endonucleases. Typically, the sample of genomic DNA may be divided into five portions each of which is treated with a different restriction endonuclease. Any suitable restriction endonuclease may be employed in the present invention. Restriction 5 endonucleases recognising 4-, 5- or 6-base sequences on the genomic DNA can be employed. Suitable endonucleases are Sau3A, PstI, Ba HI, XhoII, Hindlll, EcoRl, Neil, Bgll, Accl, Sail, Avail, Asp700 and Clal.
  • the DNA fragments obtained on digestion with a lOrestriction endonuclease are separated on a gel by electrophoresis in step (ii) .
  • the DNA fragments derived from the organisms being compared with one another are subjected to electrophoresis in an identical manner, for example at the same time under the same conditions. This 5 is preferably achieved by subjecting the fragments to electrophoresis side-by-side on the same gel. Any suitable method of electrophoresis may be adopted.
  • the DNA fragments may be run on an agarose gel.
  • the probe employed in step (iii) comprises a 0 randomly derived fragment of DNA. The fact that the probe DNA is derived randomly is important.
  • the DNA for the probe is not specifically selected from functional genes which are under strong selection for conservation and consequently which will tolerate relatively few base 5 substitutions as mutations. It is mutations which alter the base sequence of a recognition site for a restriction endonuclease that provide the variability which is detected by this invention. Genes which are under pressure for selection because of their genetic functions 0 are likely to contain fewer variations. Consequently, such genes may be conserved between two different strains of an organism, particularly between two closely related organisms, and are not representative of the natural genetic variation between the two strains. On the other hand, by randomly choosing DNA for use as a probe in the present invention, the probe is likely to contain genes which are not under a strong pressure for selection. Consequently, these genes can tolerate many more mutations, are considerably less likely to be retained from strain to strain of the species in question and are representative of the natural genetic variation of individual strains.
  • the DNA for a probe can be produced by digesting, preferably partially digesting, the genomic DNA of a strain to be identified or a strain from the same species with a restriction endonuclease.
  • the DNA fragments thus obtained can then be introduced into suitable vector plasmids to generate a bank of potential probes.
  • the plasmids are screened and a suitable number, for example five, that contain different sequences of the original ' genomic DNA are chosen.
  • the length of DNA fragment in the plasmid is about 5 Kb or more, preferably 10 Kb or more.
  • the length of the DNA fragment may vary depending upon, for example, the length of DNA that can comfortably be inserted in a particular plasmid.
  • the total amount of DNA may typically be about 30 Kb or more, for example about 50 Kb (for example five fragments of about 10 Kb each) .
  • Any vector system can be used in this invention, another suitable example being cosmids.
  • the organism from the genomic DNA of which the DNA fragment incorporated in the probe is randomly derived is of the same species as an organism to which the ⁇ present invention is being applied. Consequently, where a first organism is being compared with a second organism, then the DNA fragment in the probe must have been derived from the genomic DNA an organism of the same species as the first or second organism and preferably has been derived from genomic DNA of the first or second organism or of one of the standard organisms employed in an experimental determination of an F value.
  • radioactively labelled probes may be prepared by nick translating with [ ⁇ - 32 P]dATP or [ - 32 P]dCTP.
  • biotinylated probes ' can be prepared by nick translating with biotinylated dUTP.
  • the advantage of biotinylated probes is that they can be stored for future use whereas radioactive probes, generally, must be used within a short time of preparation.
  • the positions ' of the DNA fragments which bind to a probe can be determined in any suitable manner.
  • the DNA fragments on each gel following electrophoresis may be transferred by the capillary method of Southern (E. Southern, 1975, J. Mol. Biol. S>8_, 503-517) to nitrocellulose membranes or to diazotised aminobenzyloxy ethyl (DBM) paper, or nylon membrane (e.g. Amersham pic's Hybond N) .
  • DNA fragments may be more rapidly transferred by transverse electrophoresis to charge-modified nylon membranes such as Zeta Probe of Biorad Laboratories Ltd.
  • the DNA fragments are then contacted with a labelled probe.
  • the position of any DNA fragments which hybridise with the probe is then detected by suitable means.
  • a pattern of these positions can then be produced and compared with a pattern produced for a second organism or-, indeed, further organisms.
  • Several such band patterns can be plotted for each organism using different restriction endonucleases and different probes. Where any of the patterns for two organisms are different, it follows that the two organisms are themselves different. Where a sufficient number of patterns have been compared and all are the same, then it can reasonably be assumed that the two organisms are identical. Indeed, the probability that two organisms will fail to be distinguished can be ascertained (equation (1)) .
  • a sufficient amount of probe DNA and a sufficient number of restriction endonucleases which may be 1) , a probability that two organisms will fail to be
  • a F value for a species of an organism may be determined experimentally by:
  • steps (e) optionally repeating steps (b) to (d) for one or more further portion of the digest for each of the independently-obtained organisms but using a said labelled probe comprising a different said fragment of DNA each time.
  • steps (a) to (e) can be effected two or more times using a different restriction endonuclease each time.
  • the same set of restriction endonucleases as used in steps (i) and (iv) will be used in determining the species F value. However, this need not be the case and different sets of restriction endonucleases may be used in step (a) .
  • the organisms used in step (a) must be different strains of the same species. They must be independently-obtained, i.e. non-clonally derived. They must belong to the same species as the two organisms which it is wished to analyse for identity. Indeed, one of those two organisms preferably is included in the set of standard organisms. A sufficient number of standard organisms should be employed so that the F value that is obtained is statistically significant and can be considered as representative for the species in question.
  • Restriction endonuclease digests are prepared from samples of the genomic DNA of the standard organisms.
  • the restriction endonucleases mentioned previously may be employed.
  • the genomic DNA of each organism is digested with the same restriction endonuclease(s) .
  • a genomic DNA sample of each organism is treated with a single restriction endonuclease.
  • a panel of samples each digested by different restriction endonuclease can thus be built up for an organism.
  • the digests are subjected to electrophoresis to separate the DNA fragments in them. This may be achieved as described above.
  • Digests for the same restriction endonuclease(s) are subjected to electrophoresis under identical conditions. Preferably, this is achieved by running them side-by-side on the same gel.
  • the or each labelled probe used in step (c) preferably comprises a fragment(s) of genomic DNA from one of the standard organisms or one of the two organisms under test.
  • the probes may be constructed as described above. The same criteria apply. If more than one probe is used, the probes must contain different DNA fragments.
  • the probes hybridise with DNA fragments amongst those which have been subjected to electrophoresis which possess DNA sequences homologous to those of the probe. For a given restriction endonuclease digest, a hybridisation pattern is therefore revealed for each of the standard organisms. This permits a pairwise comparison of the patterns to be undertaken.
  • the hybridisation patterns of each and every possible pairwise combination of the standard organisms are examined to determine the fraction of the hybridised DNA fragments which are revealed by the probing which are identical for each pair. This fraction is the F value for that pair: see equation (5) above.
  • the F values for all the pairs may be used to deduce the weighted average F value for that particular restriction endonuclease digest.
  • a mean value of F for all the restriction endonuclease digests can be ontained from a weighted average of the F values of individual restriction endonuclease digests.
  • F F ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • Fav is a number less than 1, the more bands revealed the lower will be the probability that the two organisms, if different, will have failed to have been distinguished.
  • the number of bands examined can be increased by increasing the amount of probe DNA. It can also be increased by repeating steps (i) to (iv) for further restriction endonucleases.
  • a mean value of F for all the restriction endonucleases can be obtained from a weighted average of the F values of the individual restriction enzyme digests and the total number of bands in the hybridisation patterns obtained for the restriction endonucleases summed (or pooled) so as to obtain a yet more accurate prediction of whether the two organisms will have failed to have been distinguished. That is, the greater the value of q the less will the probability be that erroneous identity is claimed.
  • the or each probe employed in steps (iii) and (iv) need not be the same as the or each probe used in the determination of the F value; however, preferably probe(s) will be used in both cases.
  • probe(s) will be used in both cases.
  • the invention can be applied to any organism. It will be appreciated that the term "organism” applies to a population of individuals which are clonally derived, e.g. microorganisms such as bacteria, fungi and viruses.
  • the conventional taxonomic identity of one of the first and second organisms being compared must be known. As a minimum, this means that the species of one of the two organisms must be known so that an appropriate F value can be chosen/determined.
  • the invention is particularly applicable to organisms that reproduce by asexual means, for example microorganisms such as bacteria, fungi and DNA viruses.
  • the invention can be applied to such microorganisms which are naturally occurring or are mutants which have been derived in the laboratory or, indeed, have been engineered _in_ vitro by the insertion of a plasmid or other genetic manipulations to genomic DNA.
  • RNA viruses cDNA obtained by use of a reverse transcriptase can be employed as the genomic DNA.
  • the invention is also applicable to inbred populations which are reproducing sexually, such as some plant varieties, and to organisms where it is important to identify a unique individual.
  • a tissue sample may be obtained from the individual either shortly after birth, or at any subsequent time. Genomic DNA is prepared from that tissue sample and stored until such time as a question concerning identity arose. At such a time DNA is prepared from the individual in question, and that DNA is compared with the original DNA sample. In this case, the two DNA samples are compared by digestion with a series of restriction enzymes and hybridization to a set of probes containing random DNA fragments from an organism of the same species. Identity of the patterns of the two samples can be employed to establish the probability that the individual in question is the same as the individual from which the original DNA sample was taken. In this way the invention may be applied * to humans and to animals, for example horses, especially race horses, and cattle, particularly stud bulls. This invention may also be employed by plant breeders.
  • the present invention therefore has wide application. If a strain of organism is thought to be identical to a particular strain this can quickly and unequivocally be established employing the present invention. A pattern for each organism can be built up employing different restriction endonucleases and different probes in the present invention. Where the patterns are the same, the probability that the two organisms will have failed to have been distinguished can be determined.
  • the present invention can also be employed to permit clinical identification of an unknown pathogen. This may be achieved by preparing a series of probes incorporating randomly derived fragments of the genomic DNA of the pathogen.
  • the genomic DNA of the pathogen and the genomic DNA of various microorganisms to which it is thought to be identical can be digested with restriction endonucleases and subjected to electrophoresis under identical conditions in accordance with the invention.
  • band patterns of the pathogen and of the organisms against which it is being tested can then be built up and compared for identity. Subsequently after determining the F value the confidence of correctly identifying the pathogen may be stated.
  • the present method may be augmented by subjecting cellular enzymes of the first and second organisms to electrophoresis to compare the electrophoretic mobilities of the enzymes. Differences in electrophoretic mobility indicate that the two organisms are different. However, if the electrophoretic mobility of enzymes of the two organisms is the same then the two organisms may be identical. The more enzymes that are tested the greater the chance of detecting whether two organisms are indeed identical. However, this means of establishing identity between two organisms is not as powerful as the restriction endonuclease analysis of the genomic DNA of the two organisms.
  • electrophoretic mobility of enzymes of two organisms may be compared quite simply.
  • a crude cell extract of each organism is subjected to an electric field in a gel, for example a starch or polyacrylamide gel.
  • each gel is immersed in a solution containing a substrate for the enzyme in question. Degradation of the substrate results in the appearance of a band at the position of the enzyme in the gel. If the enzymes from two organisms differ by one or more charged amino acids they are likely to migrate to different positions in the gel and a different band pattern results.
  • a further technique for supplementing the present method of identifying an organism is to compare the respective protein patterns in gels of two organisms. Proteins can be separated by charge by isoelectric focussing. Proteins can also be separated by size, for example on SDS gels. These two techniques can be combined to provide a two dimensional separation of proteins on a single gel. The proteins appear on the gel as a pattern of spots. Where identical patterns of spots are produced for two organisms, the organisms may be identical. However, if the patterns are different then the two organisms are different.
  • the three figures show band patterns produced in
  • one of the lanes contained as a standard lamda DNA (which had been digested with restriction enzymes EcoRI and Hindlll) and pBR322 DNA (which had been digested with restriction enzyme Sau 3A) .
  • Figure 1 is a band pattern revealed for genomic DNA of 12 Lactobacillus plantarum strains, and lamda DNA, digested with restriction enzyme Accl, bound to nylon filters (Southern blot) and probed with nick-translated
  • [ 32P]-labelled plasmid pBTL30 The filter was autoradiographed at -70°C for 1 day to produce the X-ray film shown. The bands correspond to positions of genomic fragment and probe DNA hybridisation.
  • Figure 2 is a band pattern produced as for Figure 1 except that the restriction enzyme used was Asp700 and the probe was pBTL29.
  • Figure 3 is an X-ray autorad iograph o f a g el o f type C (Section 8 of Example 3) , that is DNA d igested with r estr iction enzyme Asp700 , bound to nylon f i l ters
  • genomic DNA from a strain A. Do partial digests of the genomic DNA with Sau3A restriction enzyme to give fragments of an average size of about 10 kbp. Ligate these fragments into the BamHI site of the plasmid pBR322. Transform a suitable host, selecting Ap transformants, and isolate 10 to 20 Tc s transformants.
  • digest genomic DNA from strain A separately with a respective restriction endonuclease, five endonucleases having 6-base recognition sequences and five having 4-base recognition sites. The choice of enzymes should be dictated by the degree to which the enzyme is known to have randomly distributed recognition sites.
  • digest genomic DNA from a strain B which is to be compared with strain A. For each of the ten digests, carry out electrophoresis by running - the digest for strain A in a lane next to the digest for strain B on an agarose gel. All twenty digests 5 can be run on a single gel. Run five such identical gels.
  • fragments of an average size of about 10 kb The fragments are ligated with the Pst I site of plasmid pBR322.
  • the ligated plasmid now containing a piece of strain A DNA is transformed into a suitable host, selecting for Tc r transformants and isolate
  • E. coli strain SJ84R/pBR322 (as a source of plasmid pBR322 for constructing the probes)
  • E. coli RL57 (as the organism which could be transformed by the ligated probes and be analysed) .
  • the genomic DNA was isolated as follows. Organisms were harvested (7000 rpm, 10 min) , washed once in cold TES buffer (30 mM-Tris HCl, pH8.0; 5mM EDTA and 50mM-NaCl) and the pellets resuspended in 1.5 ml 0.1M-EDTA, 0.15M-NaCl with 100 ⁇ l of a solution of 70 mg/ml pronase E (Sigma, Registered Trade Mark) and incubated at 37°C for lh.
  • Organisms were harvested (7000 rpm, 10 min) , washed once in cold TES buffer (30 mM-Tris HCl, pH8.0; 5mM EDTA and 50mM-NaCl) and the pellets resuspended in 1.5 ml 0.1M-EDTA, 0.15M-NaCl with 100 ⁇ l of a solution of 70 mg/ml pronase E (Sigma, Registered Trade Mark) and incuba
  • the DNA was extracted twice with CHCl ⁇ , on ice, the aqueous phase was removed and 0.1 volume of 3M-sodium acetate, pH 5.2, added, followed by O. ⁇ mM isopropanol (room temperature). Precipitated DNA was spooled off. The spooled DNA was dipped in cold 70, 80, 90 and 100% ethanol sequentially (to remove the acetate) and dissolved in 500 ⁇ l TE buffer and subsequently dialysed against TE buffer. The purified DNA was quantified and ready for use in probe construction or digestion as appropriate. This method was based on that described by Marmur (1961, J. Mol. Biol. 3_, 208-218).
  • minipreps For small scale preparation (so-called minipreps) the boiling method of Maniatis e_t al ⁇ (1982) (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, U.S.A.) was used, followed by two isopropanol precipitations at room temperature. For large scale preparations the method of Hansen and Olsen (1978, J. Bact. 135, 227-238) was used.
  • restriction enzymes The restriction enzymes used for constructing the probes or digesting genomic DNA were obtained from Bethesda Research Laboratories (BRL) or Boeringher Mannheim (BM) or Pharmacia. All were used in accordance with the manufacturers' instructions for use.
  • Genomic DNA from Lactobacillus plantarum strain BTLSl was partially digested with restriction enzyme Pstl to give a majority of fragments in the size range 5-15 Kb DNA.
  • the Pstl fragments were ligated with Pstl digested plasmid pBR322 DNA.
  • Plasmid DNA was treated with 5 units of calf intestinal alkaline phosphatase for 60 min at 37°C in 0.OlM-Tris-HCl, pH 9.0 buffer containing 1.0mM-ZnCl 2 and l.OmM-MgCl,.
  • This preliminary treatment prevented self ligation and contributed to a successful ligation between plasmid pBR322 DNA and the genomic DNA of Lactobacillus plantarum strain BTLSl.
  • the ligation step was carried out using T4 ligase from BRL according to the specified instructions for use.
  • the ligation mixture following ligation of genomic and plasmid DNA, was used to transform E. coli strain RL57.
  • Transformed E. coli strain RL57 cells were selected by plating on solidified L-broth containing 20 ⁇ g/ml tetracycline (Tc) . This ensured that only those cells now containing introduced pBR322 DNA grew.
  • Tc r cells were checked for ampicillin resistance (Ap r ) and sensitivity (Ap ) by plating on solidified L-broth with ampicillin at 50 ⁇ g/ml. Isolates which showed Ap were retained since these strains would have L. plantarum DNA ligated into the Pstl site which occurs within the ampicillin resistance gene of plasmid pBR322.
  • Mini-preps of plasmid DNA from Tc r Ap s strains of transformed E. coli RL57 were made and digested with restriction enzyme Pstl.
  • the digested DNA was subjected to electrophoresis in 0.7% (w/v) agarose gels to estimate the size of the Lactobacillus plantarum DNA which had been ligated into the plasmid pBR322 DNA.
  • the sizes of the DNA inserted were estimated by comparison with a standard DNA digest with fragments of known size, namely bacteriophage lambda DNA digested with Hindlll and EcoRI restriction endonucleases. The following sizes were obtained: for pBTL8, 8.8 Kb of L. plantarum DNA had been inserted; for pBTL23, 8.0 Kb DNA; for ⁇ BTL29, 5.7 Kb DNA; and for pBTL30, 10.4 Kb DNA. These four plasmids constituted the probes to be used for examining the genomic DNA of all the Lactobacillus plantarum strains.
  • the probes In order to use the probes for genomic DNA investigations the probes needed to be labelled either with biotin-11-dUTP (for "biotinylated” probes) or with deoxycytidine 5'-[o- 32P]-triphosphate (herein referred to as [ 32P]-dCTP (for "radioactively labelled” probes) ) .
  • [ 32P]-dCTP was used to produce radioactive probes using the nick translation procedure and kit supplied by Amersham International pic.
  • the labelled probes were separated from unincorporated [ 32P]-dCTP by passage down a Sephadex (Registered Trade Mark) G-50 column previously equilibrated with 50mM-Tris HCl, ImM-EDTA and 0.1% (w/v) SDS. The leading radioactive peak (detected by a Gieger Muller monitor) was collected, and contained the [ 32 P]-labelled probes.
  • the four plasmid probes contain pBR322 DNA as well as the cloned L. plantarum DNA. It was shown that nick translated [ 32 P]-labelled pBR322 DNA did not hybridise significantly with digested L. plantarum DNA under the conditions described here. Thus any hybridisation observed are due to genomic L.
  • the five replicate filter blots were separately hybridised with the four pBTL probes and a probe of nick translated lambda DNA according to the procedure supplied by Amersham pic.
  • the probed and hybridized filters once dried, were wrapped in Saran Wrap. Filters were then autoradiographed for between 4 hours and 2 days at -70°C using fast tungstate intensifying screens.
  • the band patterns produced are exemplified by Figures 1 to 3 of the accompanying drawings.
  • the bacteriophage lambda probe was used to monitor the completeness of the genomic DNA restriction enzyme digests. From a complete lambda DNA digest a known number of restriction fragments will be generated when hybridised with nick translated [ 32P]-labelled lambda DNA. The appearance of additional restriction fragments after autoradiography indicates an incomplete or partial digestion of lambda DNA and hence an incomplete digestion of L. plantarum genomic DNA. That is gel C is used as a control to assess that the samples have been completely digested. Those lanes which showed incomplete digestion were ignored.
  • the X-ray films (or autoradiographs) produced from the filters hybridised with [32P]-labelled DNA probes showed a series of bands corresponding to fragments of DNA from the DNA samples which hybridised with the probes.
  • Hybridising fragments were distinguished on the basis of their relative mobilities in the agarose gel. Thus, the largest hybridising fragments gave rise to bands on the autoradiograph which were closest to the gel's origin (as marked on the corresponding autoradiograph) .
  • Tables 2.1-2.4 and 3.1-3.4 below show the bands present for genomic DNA digestions with restriction enzymes Bgll and Aval respectively.
  • the total number of bands for each set of probes for the Bgll and the Avail digests are shown in Table 1.
  • pairwise comparisons of the band patterns determined the number of common fragments (2N) between each pair of digests with a total of a and b fragments respectively.
  • Tables 2.5-2.8 and 3.5-3.8 below show the pairwise comparisons for genomic DNA digestion with Bgll and Avail respectively.
  • Tables 2.5-2.8 and 3.5-3.8 From the individual pairwise comparisons (i.e. Tables 2.5-2.8 and 3.5-3.8) a set of six tables (only two - Tables 2.9 and 3.9 - shown here) showing the pairwise comparison for the total probed DNA were constructed. Tables 2.9 and 3.9 show the summed pairwise comparison for genomic DNA digested with Bgll and Avail, respectively. Finally, Table 4 shows the complete pairwise comparisons between all the strains for the complete set of four probes and six restriction endonuclease digests.
  • the fraction of * conserved fragments (F) for each pairwise comparison can be calculated (according to equation (5)) using the data in Tables 2.9 and 3.9 (for a pairwise determination of F for the complete set of four probes using only one genomic DNA digestion) and in Table 4 (for a complete pairwise determination of F for the total set of four probes and six genomic DNA digests) . These values of F are shown in Tables 5, 6 and 7 respectively. As expected since the twelve strains were independently isolated the values for F varied, showing that the degree of relatedness varied. In some instances, at the lower level of genomic DNA analysis (i.e.
  • test strain X a thirteenth strain, denoted the "test strain X" is compared with the standard set of twelve organisms and shown to produce an identical band pattern with one of the strains previously analyzed.
  • test strain X a thirteenth strain
  • the range of confidences with the present data may best be considered by examining the F values calculated for the maximum, minimum and average F values calculated from the complete pairwise analysis, taking the number of bands for test strain X to be the maximum, minimum and average values determined for the twelve strains analysed (Table 8).
  • This table shows that the closer F for the population approaches 1 then the larger the value of F and hence the lower the confidence level that identity between two observed patterns will be interpreted as. showing clonal identity between the two sources of DNA (strain X and the strain with which it is compared). That is, if strain X was probed with the 4 probes used in this study and the genomic DNA's only analysed with restriction enzyme Bgll, then if the highest F value was used, the confidence level C would be 0.29.
  • strain BTLSl was taken as an "unknown" thirteenth strain for comparison with a "known" sample of BTLSl.
  • the "unknown" strain was designated strain Y.
  • Strain Y had been reisolated from a natural source which had been inoculated with BTLSl.
  • Genomic DNA from strain Y and from BTLSl was digested with the restriction enzyme Bgll and hybridised with probes pBTL8, pBTL23, pBTL29 and pBTL30 as above. Identical band patterns were obtained.
  • the four probes revealed a total of 24 identically-positioned bands for the Bgll digests of each of strain Y and BTLSl (Table 1) .
  • F_ma_. tenux, Fmm. and Fav values deduced for the standard set of twelve strains of L. plantarum (Table 5) it could be predicted from equations (1) and (4) that the probabilities that strain Y and BTLSl were in fact different were:
  • Genomic DNA from strain Y and from BTLSl was digested with restriction endonuclease Avail and hybridised with probes pBTL8, pBTL23, pBTL29 and pBTL30 as above to try to achieve a lower probability (greater confidence) that the two strains are different.
  • Identical band patterns were revealed with 35 common bands per strain (Table 1) .
  • Restriction endonucleases that recognise 4-base sequences produce fragments of about 0.25 kbp average length and those that recognise 6-base sequences produce fragments of about 4 kbp average length.
  • F 2 may be derived by assuming values for P in equation (3), the probability (X) that two different organisms will fail to be distinguished is:
  • Enzyme TOTAL NUMBER OF BRANDS VISUALISED FOR STRAINS used for digestion BTLSl 8531 6105 8299 5914 8026 8016 6376 11974 6461 8102 7720
  • Table 2.1 DNA from 12 Lactobacillus plantarum strains digested with restriction enzyme Bgl I and probed with pBTL8. The bands are numbered in sequence from the origin of the agarose gel.
  • Table 2.4 DNA from 12 Lactobacillus plantarum strains digested with restriction enzyme Bgl I and probed with pBTL30. The bands are numbered in sequence from the origin of the agarose gel,
  • Table 2.2 determined as indicated for Table 2 .

Abstract

Le procédé permettant de déterminer l'identité entre deux organismes consiste à soumettre un digesté d'endonucléase de restriction d'ADN génomique du premier organisme à une électrophorèse; à déterminer les positions des fragments d'ADN ainsi séparés qui s'hybridisent avec un ou plusieurs agents d'investigation d'ADN marqués; et à comparer ces positions avec les positions de fragments d'ADN fixés sur chaque agent d'investigation et produits à partir d'ADN génomique du deuxième organisme de manière analogue. Une quantité de l'agent d'investigation d'ADN et une ou plusieurs endonucléases de restriction sont utilisées de sorte que des bandes suffisantes sont révélées par l'investigation pour obtenir une probabilité (X) suffisamment faible que les deux organismes n'aient pas été distingués lorsqu'ils se révèlent identiques par rapport à leurs configurations de bandes, selon la formule X = Sq, où F est une fraction représentative de la proportion de fragments d'ADN qui sont identiques entre les digestés d'endonucléase de restriction de l'ADN génomique de pairs d'organismes obtenus de manière indépendante et de la même espèce que les premier et deuxième organismes, et q est le nombre de positions révélées par l'investigation.
PCT/GB1986/000210 1985-01-08 1986-04-14 Determination d'identite entre deux organismes WO1987002065A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0405376A2 (fr) * 1989-06-24 1991-01-02 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Caractérisation de rhinovirus humains
WO1992005280A1 (fr) * 1990-09-21 1992-04-02 Imperial Cancer Research Technology Limited Identification d'organismes
AU626530B2 (en) * 1987-07-17 1992-08-06 Schering Biotech Corporation Human granulocyte-macrophage colony stimulating factor and muteins thereof
GB2262987A (en) * 1990-09-21 1993-07-07 Imp Cancer Res Tech Identification of organisms

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984000979A1 (fr) * 1982-08-24 1984-03-15 Wistar Inst Procede pour determiner si un virus de l'herpes est a l'etat virulent ou a l'etat latent
GB2135774A (en) * 1983-02-28 1984-09-05 Actagen Inc Identification of individual members of a species
WO1984003716A1 (fr) * 1983-03-21 1984-09-27 John A Webster Jr Procede d'identification et de caracterisation d'organismes
WO1984004758A1 (fr) * 1983-05-26 1984-12-06 Plant Resources Inst Procede de relevement topographique genetique et d'hybridation pour plantes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984000979A1 (fr) * 1982-08-24 1984-03-15 Wistar Inst Procede pour determiner si un virus de l'herpes est a l'etat virulent ou a l'etat latent
GB2135774A (en) * 1983-02-28 1984-09-05 Actagen Inc Identification of individual members of a species
WO1984003716A1 (fr) * 1983-03-21 1984-09-27 John A Webster Jr Procede d'identification et de caracterisation d'organismes
WO1984004758A1 (fr) * 1983-05-26 1984-12-06 Plant Resources Inst Procede de relevement topographique genetique et d'hybridation pour plantes

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU626530B2 (en) * 1987-07-17 1992-08-06 Schering Biotech Corporation Human granulocyte-macrophage colony stimulating factor and muteins thereof
EP0405376A2 (fr) * 1989-06-24 1991-01-02 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Caractérisation de rhinovirus humains
EP0405376A3 (en) * 1989-06-24 1992-11-25 Boehringer Ingelheim International Gmbh Characterization of viruses
US5340713A (en) * 1989-06-24 1994-08-23 Boehringer Igelheim International Gmbh Process for the characterization of human rhinoviruses
WO1992005280A1 (fr) * 1990-09-21 1992-04-02 Imperial Cancer Research Technology Limited Identification d'organismes
GB2262987A (en) * 1990-09-21 1993-07-07 Imp Cancer Res Tech Identification of organisms

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