EP0467883A1 - Methode pour cartographier physiquement des elements genetiques - Google Patents

Methode pour cartographier physiquement des elements genetiques

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Publication number
EP0467883A1
EP0467883A1 EP19890906527 EP89906527A EP0467883A1 EP 0467883 A1 EP0467883 A1 EP 0467883A1 EP 19890906527 EP19890906527 EP 19890906527 EP 89906527 A EP89906527 A EP 89906527A EP 0467883 A1 EP0467883 A1 EP 0467883A1
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European Patent Office
Prior art keywords
cassette
sequence
dna
genome
vector
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German (de)
English (en)
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EP0467883A4 (en
Inventor
Michael J. Lane
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GENMAP Inc
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GENMAP Inc
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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
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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]

Definitions

  • the present invention relates to novel DNA
  • This method incorporates several technologies, including incorporation of synthetic and/or natural DNA sequences into genomic DNA, generation of rare restriction enzyme cutting sites, and size resolution of DNA fragments up to and greater than the million base pair size range.
  • genomic maps can be used to create a map of genomic DNA. Once a genomic map has been created, it can be used to create a map of genomic DNA. Once a genomic map has been created, it can be used to create a map of genomic DNA. Once a genomic map has been created, it can be used to create a map of genomic DNA. Once a genomic map has been created, it can be used to create a map of genomic DNA. Once a genomic map has been created, it can be
  • the method is useful in locating genetic lesions or alterations in the primary DNA sequence by comparison of such maps. This comparative method is thus capable of detecting genetic disorders, diseases,
  • polymorphic loci polymorphic alleles
  • el ⁇ ctrophorese DNA through gel matrices such as, but not limited to, agarose gels by employing pulsing electric fields (Schwartz, B. and Cantor, C, Cell 37:67, 1984; Snell and Wilkins, 1986). These techniques make it possible to resolve and analyze DNA fragment sizes orders of magnitude greater in size than was possible through historical gel matrices such as, but not limited to, agarose gels by employing pulsing electric fields (Schwartz, B. and Cantor, C, Cell 37:67, 1984; Snell and Wilkins, 1986). These techniques make it possible to resolve and analyze DNA fragment sizes orders of magnitude greater in size than was possible through historical
  • restriction enzyme cleavage sequence Appropriate choice of the correct methylation system therefore allows generation of very large restriction fragments.
  • the human genome is approximately 3x10 9 base pairs in length covering an estimated 3300 centimorgans (White et al., Nature 313:101-
  • An effective map suitable for general diagnostic and prognostic testing would require far more information than the 100 markers cited above. Ideally the map would have markers spaced every 50 kilobases of DNA or less, or would consist of upwards of 10 4 -10 5 markers. Generation of this many ordered markers is not feasible using current techniques. While advances have been made in constructing genetic maps by the implementation of various molecular biology techniques, at present count, less than a thousand genes, spanning only a small portion of the human genome, have been cloned (Willard et al., Cytogenet. Cell Genetics 80, 1985). These cloned genes have been used as probes to identify restriction fragment length polymorphisms (RFLPS) in genomic DNA and have proven useful in diagnosing some genetic disorders.
  • RLPS restriction fragment length polymorphisms
  • invention to be able to generate a map or partial maps of a cell's or organism's genomic DNA.
  • the present invention involves a method and biological tools for mapping genomic DNA. This mapping technique can be used as a diagnostic test for detecting genetic disease and polymorphic loci and as a prognostic test.
  • the present mapping method comprises integrating
  • rare restriction sequence or site it is meant one which does not occur, or occurs at very low frequency in the genome to be mapped, or can be made to be cleaved preferentially over genomic sites by any means rare, so long as its frequency allows partial mapping away from the rare restriction sequence into the genome of the host cell.
  • the "unique DNA sequences”, hereinafter referred to as DNA A, and optionally, DNA B, need not be restriction sequences, but rather are simply sequences capable of being identified uniquely in a
  • the cassette is inserted into the host cells by way of a vector, preferably a vector which will accomplish gene transfer through a single random integration of the
  • the invention also provides novel DNA cassettes comprising a restriction sequence rare for the genome to be tested, flanked on at least one side by a nucleotide
  • cassettes for use in integrating the cassette into the host cell genome.
  • the nature of the cassette and vector will generally differ depending on the source of the genome to be mapped.
  • any genome which does not have A methylation, and has an appropriate genomic utilization can be mapped utilizing a cassette comprising a rare Clal/Clal overalapping restriction sequence flanked on one or both sides by a retroviral sequence. Examples of organisms which would fall into such a category include but are not limited to mammals (humans), birds, and Drosophila; this cassette can be transmitted to the host cell by way of a retroviral vector.
  • Similar other constructs also can be created for mapping any other genomes, for example, other vertebrates, and invertebrates, yeast, plants, and bacteria.
  • the present invention also provides cell cultures or organisms, into the genome of which have been integrated the novel cassettes described above.
  • resolvable distance between RFLP markers at present is no better than several million base pairs.
  • the present method does not require an extensive pedigree study; also, resolvable distances are not limited by RFLP markers, but rather are dependent only upon the available cleavage and resolution technology.
  • the method provides high mapping accuracy with a rapidity heretofore uncontemplated in the art.
  • Figure 1 Flow diagram illustrating a genomic insertion mapping procedure for mapping mammalian cells.
  • Each arrow indicates a step in the procedure with the expected DNA structures shown in boxed insets.
  • Figure 2 shows maps of the vector pZipNeo
  • Fig. 2a specifically shows the pZipNeo vector containing the Clal/Clal/Dpnl sites;
  • Fig. 2b illustrates a pZipNeo vector having multiple copies of the NotI recognition sequence.
  • Figure 3 schematically illustrates a procedure for locating the position of a particular gene or DNA
  • FIG. 4 illustrates the presence and in situ
  • Lane 1 lambda Hind III markers; Lane 2 - MClal/Dpnl:SstI digest; Lane 3 - BamHI:SstI digest; Lane 4 - MClaI:ClaI digest control; Lane 5 - minus enzyme control; Lane 6 - AM Neo minus control.
  • the lower 3.3 kb band represents the actual neo gene while the two larger bands derive from the pBR322 section of transfected DNA. Note that both BamHI:SsT I digestion and MClal: Dpnl:SstI
  • Figure 5 illustrates the use of lambda phage concatemers as pulsed field electrophoresis size markers with whole yeast chromosomes on the outside lane as a reference. Twelve distinct bands are resolved in the outside lanes in the figure containing all 17 chromosom(es (6 bands represent doublets). 5. DETAILED DESCRIPTION OF THE INVENTION
  • genomic DNA cam be mapped by inserting into said genomic DNA a DNA sequence which is a rare cleavage site in the context of the host DNA with which it is integrated.
  • This rare restriction sequence is flanked at one end by a uniquely identifiable
  • unique DNA A DNA sequence
  • unique DNA B DNA sequence
  • cassette DNA sequence
  • the cassette may contain a sequence or sequences which facilitate
  • the cassette may also contain a high affinity protein binding site.
  • the ⁇ repressor binding sequence can be used in conjunction with a
  • DNA affinity column composed of covalently bound repressor monomers. In this way, DNA fragments containing the unique
  • DNA A or unique DNA B sequence can be readily isolated for subsequent manipulation.
  • the cassette can contain genetic functionalities that allow it to be maintained as a plasmid in E. coli or other appropriate host, thus facilitating ready isolation of the unique DNA A or unique DNA B and flanking genomic DNA for subsequent manipulation.
  • the actual sequences of the rare restriction sequence, unique DNA A, and unique DNA B are not critical.
  • sequences should be different from one another, and, in a preferred embodiment, the sequences should occur infrequently, if at all (i.e., they are underepresented) in the host genomic DNA. It is possible that a similar sequence or sequences exists in the host organism. All that is required in this procedure is to differentiate between the inserted DNA and the endogenous DNA.
  • the identity of the rare restriction site will differ depending upon the host organism whose genome is to be mapped, since a particular sequence may be rare in one organism, but not another.
  • the term "rare" in the present context can best be defined operationally. An initial consideration in choosing an appropriate sequence is what will be the preferred fragment size resulting from cleavage. The preferred fragment size is not dictated by any
  • “Large” is, of course, determined relative to the total size of the genome to be mapped. Smaller fragments are just as acceptable functionally, but require many more repetitions of the procedure in order to get an equivalent map. For this reason, large fragments are preferred.
  • the choice of a rare cutter can be made in a number of ways.
  • One approach is to simply treat the DNA with an appropriate enzyme (appropriate to be defined below), and observe the size of the fragments produced. If the fragment size is acceptable in accordance with the guidelines noted above, then a useful sequence has been chosen, and may be used in the present procedure.
  • an appropriate sequence may be predicted empirically by reference to the overall nucleotide composition of the genome to be mapped. For example, a general knowledge of the approximate GC content of the genome provides a
  • the average fragment size produced by cleavage of the restriction sequence ATCGAT in this genomic environment is estimated to be 3086 base pairs. Given the initial estimation of the desired fragment size for the genome of choice, it is readily apparent whether or not the chosen site is acceptable for the purpose.
  • the selection of a rare site can be taken an additional step, by modification of the sequence in a manner which renders it even less likely to be cut than it would be in its unmodified state.
  • Selective methylation of a particular sequence may, depending on the organism, result in the production of a highly specific cleavage site which is only rarely cut in the genome of choice (McClelland et al., PNAS USA 81:983- 987, 1984).
  • the chosen cleavage site can be arranged in tandem arrays. This will normally result in a preferential cleavage of the chosen site within the
  • the initial sequence need not even be particularly uncommon in the host genome, but merely need to be
  • a preferred sequence when a human genome is being mapped, can be the overlapping
  • This site is of particular utility since it is subject to selective methylation by the enzyme MClal (McClelland et al., PNAS USA 81:983-987, 1984).
  • This methylation renders a rare 10-base sequence cleavable, since mammalian DNA is not routinely methylated at Clal.
  • the methylated 10-base Clal sequence is subject to selective cleavage by the restriction endonuclease Dpnl (or Cful), which cuts only DNA which is methylated at adenine in both strands of the recognition site.
  • Dpnl restriction endonuclease
  • An additional benefit can be obtained by constructing this rare site, within the cassette, in tandem repeats.
  • the selected oligonucleotide restriction sites can be readily prepared synthetically. 5.1.2. UNIQUE DNA AND VECTOR SELECTION
  • unique DNA flanking the rare restriction site is to provide a basis for detecting the cassette amidst the genomic DNA.
  • unique DNA A and unique DNA B need only be distinguishable, by some detectable means, from the host DNA.
  • the sequences can be generated by fragmentation and isolation of genomic DNA derived from an organism genetically distant from the host organism.
  • unique DNA can be derived from procaryotic i.e., bacterial or viral, genomic DNA.
  • the unique DNA is then detectable by virtue of, for example, hybridization with a labelled complementary DNA probe, or the presence of a selectable marker.
  • the unique DNA is unique to the unique DNA
  • sequences are chosen in association with a vector used to transform the host cells.
  • the vector chosen is preferably one sufficiently distinct genetically from the host cell to permit detection of the vector DNA after its integration into the host cell genome.
  • a replication-defective amphotrophic virus vector into which the rare restriction sequence has been ineerted, such as that described by Sorge et al. (Mol. Cell Biol. 4:1730-1737, 1984), is used to infect a mammalian cell line. These viruses are capable of infecting cells, but once genomically integrated, are incapable of post-insertion replication, preventing reinsertion by the virus into other segments of the genome. 5.1.3. CASSETTE INTEGRATION
  • the cassette constructed, as outlined above, must be integrated with the genomic DNA to be mapped. This integration can be achieved by any method useful in
  • attaining DNA transfer includes, but is not limited to, the use of electroporation, micrinjection, infection or ligation into a cloning vector.
  • integration in the present context means the association of the cassette with the genomic DNA in a continuous piece of DNA.
  • the cassette is integrated into the genomic DNA to be analyzed by use of a vector which inserts the cassette into a host cell.
  • the vector is preferably a
  • transposon-like element i.e., one capable of being
  • sequence into a properly chosen vector automatically flanks the.,restriction sequence with a distinct sequence which, upon integration into the host cell genome, will be readily detectable, provided the vector sequence is sufficiently distinguished from the host cell.
  • transposable elements has long been recognized (Kleckner, Cell 11:11-23, 1977; Calos et al., Cell 20:579-595, 1980), and provide a means for sequence neutral integration of the type necessary to attain insertion of the restriction site.
  • Ty elements of yeast are also similar in structure, and to some extent, function, to the prokaryote transposons (Boeke et al., Cell 40:491-500, 1985).
  • P elements have been routinely used to introduce cloned sequences into the organism (Steller et al., EMBO J. 4:167-171, 1985).
  • the use of Ti plasmids to introduce exogenous DNA is now relatively routine technology (Chilton, M.
  • virral vectors can be employed for integration of DNA into mammalian cells.
  • virral vectors can be employed for integration of DNA into mammalian cells.
  • Particularly preferred as a vector for mammalian cells are retroviral vectors. Alternate choices for vectors will be readily apparent to those skilled in the art.
  • the trait of being "uniquely identifiable” is intended to convey that, by some means, the presence of the cassette by detection of the unique DNA, can be verified.
  • a convenient method of achieving this is by the use of a vector which is genetically distant from the host; in this way, insertion of the cassette can be verified by
  • the cassette can be constructed so as to include a particular selectable marker which allows the identification of the presence of cassette DNA.
  • the host cells are divided into single cell solutions. This can be accomplished by, for example, dilution or cell sorting. The cells are then propagated in a manner consistent with culture conditions required for the the cell line selected. Organisms will be treated by whatever means necessary so as to effect the same result.
  • cassette DNA into mammalian cells, the cells are serially diluted in order to produce cultures containing a single cell. These clones are then propagated in the presence of a selectable agent which will prevent growth of cells which have not integrated a copy of the virus.
  • a selectable agent which will prevent growth of cells which have not integrated a copy of the virus.
  • This can be achieved by insertion, with the vector, of a selectable marker, such as antibiotic resistance.
  • a selectable marker such as antibiotic resistance.
  • Similar screening procedures can be achieved with whole organisms, e.g., whole plants.
  • vectors can also be constructed to carry a selectable marker, and plant cell cultures transformed thereby. Plants regenerated from culture can be screened on a selective medium at an early stage of development, and the surviving plants represent those which have integrated cassette DNA.
  • a single insertion is preferable, but not critical, to the present method.
  • Insertion of additional cassettes, of different structure from the first is also contemplated.
  • the clonal populations which contain the genomic DNA to be mapped are then lysed in any manner which is suitable for the DNA separation method selected.
  • These techniques can include, but are not limited to, prior suspension of cells in agarose, e.g., agarose microbead technique (Cook, P. EMBO Jour. 3:1837, 1984) and agarose block technique (Schwartz, B. and Cantor, C., Cell 37:67, 1984 and U.S. Patent No. 4,473,452).
  • agarose microbead technique Cook, P. EMBO Jour. 3:1837, 1984
  • agarose block technique Rosartz, B. and Cantor, C., Cell 37:67, 1984 and U.S. Patent No. 4,473,452
  • These techniques allow in situ cell lysis by enzymes, detergents and proteins diffused into the agarose while maintaining DNA integrity. Any method of DNA isolation which leaves the DNA in a state available for subsequent treatment is acceptable (see, e.g. Maniatis
  • the genomic DNA is then treated so as to produce fragments suitable for mapping.
  • the DNA will be cleaved with a restriction enzyme having specificity for the rare restriction site, and at least one secondary restriction enzyme.
  • the rare restriction site is first treated with a site-specific methylating enzyme, in order to render the restriction site more rare, and then followed by cutting with methylation dependent restriction enzyme.
  • this initial enzyme treatment will preferentially cut within the cassette, and will produce little or no cleavage within the genomic DNA.
  • the DNA is also partially digested with one, or independently, a series or mixture of secondary restriction enzymes, so that each DNA sample will be digested with the restriction enzyme specific for the rare site, and partially digested with the secondary restriction enzymes.
  • These secondary enzymes will be specific for various sequences within the genomic DNA.
  • the identity of the enzyme used is not critical, and can be any restriction enzyme which cuts within the genome to be mapped. However, if it is desired to produce larger fragments for mapping, the chosen enzyme will preferably be one which cuts a relatively uncommon site. Many such enzymes are available as commercial
  • the genomic DNA restriction fragments are separated, either according to size or molecular weight. This separation may be achieved by any method which is capable of resolving fragments of the size produced. A majority of the current techniques rely on electrophoretic separation. The choice of technique will to a large extent govern the ultimate resolution that can be obtained in the mapping procedure. Any technique that allows measurement of the distance from the rare site to the mapping site is acceptable. For example, HPLC methods are not particularly suited to separation of large fragments. In a preferred embodiment of the present method, fragments are separated via pulsed field electrophoresis, as
  • the genomic fragments are contained in a suitable medium, preferably a gel medium and the DNA fragments subjected to pulsing electric fields.
  • sequences are identified. This can be achieved by any method capable of distinguishing the unique cassette DNA from the genomic DNA background of the host cell organism.
  • a convenient method for specifically identifying the unique sequences is by probing the fragments with a unique cassette DNA-specific, labelled, cloned DNA fragment substantially homologous to one of the unique flanking DNA sites.
  • the complementary sequence will be labelled with a radioactive, fluorescent or color indictor.
  • the separated DNA fragments will be blotted onto a support membrane, such as, but not limited to, nylon or nitrocellulose, prior to hybridization, in accordance with the method of Southern.
  • a support membrane such as, but not limited to, nylon or nitrocellulose
  • This procedure results in an end- label of only those fragments containing the introduced restriction site, producing a ladder of fragments giving the genomic restriction site pattern away from the integration site in one direction.
  • the same blot can be probed with a sequence homologous to the other side of the restriction site, (i.e., DNA B, if present), producing a fragment pattern representing the genomic restriction sites on the other side of the integrated cassette.
  • mapping procedure is as follows:
  • One fragment will represent that portion of the genomic DNA from unique DNA A to a first secondary
  • a second fragment will represent the distance from unique DNA A to a second secondary
  • restriction enzyme cut Deducting the first distance from the second distance generates the distance from the first to the second secondary cuts.
  • the above procedure will have to be repeated a number of times, the number of times being dependent on the length of the genome to mapped.
  • the clonal maps are then compared in order to ascertain overlapping portions.
  • the Clal/Clal overlapping sequence ATCGATCGAT has an estimated frequency of occurrence in the human genome of once every 2 x 10 8 base-pairs.
  • This sequence is inserted into the DNA of a defective amphotropic retrovirus.
  • the DNA recombinant is then transfected into a cell line harboring a trans acting, replication defective copy of the retrovirus (See, e.g., Sorge et al., Mol. Cell. Biol. 4:1730, 1984; Cohn et al., PNAS 63:49, 1981). This allows assembly of RNA containing viral particles, which are then exported from the cell.
  • This type of construction has the demonstrated capability to infect cell lines, but is incapable of post-insertional replication. These particles are used to infect,
  • the DNA is partially digested with a second restriction enzyme and then electrophoresed next to appropriate DNA size markers, for example, pulse field electrophoresis with a partially annealed ⁇ phage ladder as size standard.
  • electrophoresis is carried out under conditions which allow resolution of large DNA molecules in order to acquire as much mapping information as possible. If the gel is then southern blotted and probed with nick translated, cloned, viral DNA from one side of the Clal/Clal introduced site, this will result in an end label of only those fragments containing the introduced Clal/Clal site, producing a ladder of
  • map density is defined as the number of genome equivalents in DNA base pairs mapped. If 50 lanes may be run on each gel, such a map could be created by running 30 pulsed field gels. Once the first map is created, mapping can be done comparatively.
  • Embryos obtained from a Canton S (Brown University) strain are injected (Zalokar, Microscopica Acta 84:231, 1981; Zalokar, Experientia 37:1354, 1981;
  • Positive P element containing flies are selected by addition of G418 to the growth media as
  • DNA can be prepared from overnight collections of embryos which have been dechorionated (Santamaria, in Drosophila, A Practical Approach, D.B. Roberts, ed., IRL Press, 1986) prior to embedding in agarose plugs (Schwartz and Cantor, Cell 37:67, 1984) for lysis.
  • Lysis is performed in a Tris buffer containing 1 mg/ml proteinase K and 1% sarcosine (5-10 ⁇ l) of each agarose plug containing the now-purified DNA is then rinsed in buffer composed of 10 mM Tris/1 mM Na2EDTA for one hour, then the agarose slice washed in 10 ml of the same buffer containing 200 ⁇ l of 100 mM PMSF (phenylmethyl sulfonyl fluoride). This procedure is repeated once again, then the PMSF is washed out of the slice by a one-hour incubation in the 10 mM Tris/1 mM EDTA solution. The DNA is then clean enough for further
  • the method also has utility in identifying the position of any genomic DNA fragment, for example, to locate the map position of a cloned DNA segment. From rudimentary localization of contigs determined by in situ hybridization, it is possible to determine specific localization of a gene or genes known to fall in given chromosomal regions by cleaving the cell lines representing the appropriate contig to completion with both MClal/Dpnl and Notl. The procedure is outlined in Figure 3. DNA so cleaved can be pulse- electrophoresed, blotted and probed sequentially with probes hybridizable to the unique DNA to either side of the
  • MClal/Dpnl site This identifies MClal/DpnI-NotI fragment sizes.
  • the same blot can then be probed with the gene of interest. This procedure will identify the cell line in which the genomic Notl-NotI fragment containing the gene has been interrupted by retroviral insertion, and to which side of the MClal/Dpnl site the gene falls. This localizes the gene to that region of the contig. Failure to identify such a sequence would suggest repeating the procedure
  • the strategy is to utilize a defective retroviral vector to insert a rare restriction site into random positions in the human host cells.
  • the rare restriction site selected is the Clal/Clal overlapping sequence, which has been estimated to occur at a frequency in the human genome of about once every
  • the oligonucleotide is inserted, both singly and in tandem arrays, at the unique BamHI into the murine (MuLV) retroviral shuttle vector pZipNeo originally described by Cepko (Cell 37:1043, 1984).
  • This vector contains a pBR322 origin of replication, an SV40 origin of replication, and a selectable marker, the resistance gene for G418 (neomycin).
  • Figure 2 shows the map of the vector pZipNeo.
  • Tandem arrays of the sequence are created by ligating the insert to itself in the presence of T4 kinase, 32 P ATP and T4 ligase. Products corresponding to 3 and 6 ligation events are isolated by elution from an 8% polyacrylamide gel following autoradiography. 6.2. CASSETTE INSERTION
  • the recombinant DNA's are then transfected into a cell line harboring a trans-acting, replication defective copy of the retrovirus ( ⁇ AM) which allows assembly of recombinant RNA containing infective viral particles.
  • ⁇ AM trans-acting, replication defective copy of the retrovirus
  • pZipNeo DNA can be transfected by, for example, the scrape loading technique (Fechheimer, PNAS USA 84:8463, 1987) into the amphotrophic packaging line.
  • the viral particles produced are used to infect, monotonically, clonal human embryo fibroblast line MRC-5, by incubation of the packaging cell line media with the fibroblast cells (Conn & Mulligan, supra).
  • Treated cells are selected for neomycin resistance by culturing with G418 for a period of 2-3 weeks.
  • Clones are evident at this time, and are subsequently picked and grown up in the presence of G418.
  • the cells are
  • Plugs are treated for MClal/Dpnl digestion, first by cutting into 1/4 pieces. They are twice washed with Tris/EDTA buffer, then twice in Tris/EDTA buffer containing 1 mM PMSF and finally twice more in Tris/EDTA buffer. Plugs then are cut into 1/3 slivers and washed in microfuge tubes with Tris/EDTA
  • the reactions are terminated, by addition of fresh lysis solution (e.g., 1 mg/ml Proteinase K/10 mM Tris (pH 9.0)/0.5 M disodium EDTA/1% sarcosine), and then pulse- electrophoresed on a 1% agarose gel, then blotted and probed with a Xhol-Xhol (Neo) probe created from pZipNeo DNA by random priming (BRL) of the fragment.
  • fresh lysis solution e.g., 1 mg/ml Proteinase K/10 mM Tris (pH 9.0)/0.5 M disodium EDTA/1% sarcosine
  • Wild type whole lambda phage (Ci857sam7) are dialyzed after purification on a cesium chloride gradient and subsequently diluted in PBS and mixed with an equal volume of 1.5% of low gelling agarose (FMC lot #12276). The molten agarose solution is mixed and then pipeted into plastic forms and allowed to solidify making agarose 'plugs' (Schwartz, D.C. et al., CSHSQB 47:189,
  • Agarose plugs are suspended in a solution composed of 0.5 M disodium EDTA/1% sarcosine/1 mg/ml proteinase K/10 mM Tris-Cl (pH 9.0) and incubated at least 4 hours at 55oC with gentle shaking. Samples are loaded as described
  • the separated, labelled blot of the fragments are exposed to film, the film used to assign molecular weights and the order in which they appear to the fragments, and then examined to recognize restriction pattern overlaps, from which a complete genome map can be determined.
  • this invention provides for a fast and easy way to generate maps of complex genomes, including human genomes.
  • Maps of genomes can be compared to each other in order to detect any differences between them. For example, a genome can be mapped and compared to a standard map of that genome. This procedure will be important in such areas as prenatal diagnosis of inherited genetic disease,
  • a genomic map is generated and placed in a data base. Thereafter, any other genomic maps generated are compared to the first map by use of an
  • the map can also be used to locate specific genes, and to identify normal genes.
  • genomes from various cells in the same organism can be mapped and compared to detect for differences between them. This will allow for greater specificity, since most, if not all, of the genomes should be identical to each other, and detailed maps can be
  • a standard map can be prepared from a normal human cell, and that can be compared to map of a neoplastic cell from the same individual. This procedure will indicate what genetic changes a human cell undergoes as and when it becomes cancerous.
  • the technique can be used to create a library of marked cell lines, or marked whole organisms (plant or animal), each of which represent a particular part of the genome.
  • This invention will also find ready application in other fields, such as anthropology and evolutionary biology. Maps of genomes from various organisms can be generated and compared in order to further the study of evolution. Other fields will benefit as well, such as horticulture, animal husbandry and genetic engineering.
  • the present method also has particular significant advantage in its ability to effect map closure by non-random extension, at lower resolution, of maps produced from contig ends.
  • the inability to close a map is a drawback of other types of mapping techniques, and, in fact, the present method can be used to close maps prepared by these other methods. 7.

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Abstract

L'invention concerne une cassette d'ADN, laquelle comprend une séquence à restriction rare bordée d'une séquence d'ADN A unique et/ou d'une séquence d'ADN B unique. On peut insérer la cassette d'ADN dans l'ADN génomique. L'invention concerne également un procédé utilisant ladite cassette afin de cartographier physiquement l'ADN génomique.
EP19890906527 1989-04-14 1989-05-09 Method of physically mapping genetic material Withdrawn EP0467883A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US337990 1982-01-08
US33799089A 1989-04-14 1989-04-14

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EP0467883A1 true EP0467883A1 (fr) 1992-01-29
EP0467883A4 EP0467883A4 (en) 1992-04-22

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EP (1) EP0467883A4 (fr)
AU (1) AU3730689A (fr)
WO (1) WO1990012891A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017269A1 (fr) * 1990-05-03 1991-11-14 Ig Laboratories, Inc. Procede de representation cartographique d'un chromosome eucaryote
US5162514A (en) * 1990-05-15 1992-11-10 Board Of Regents, University Of Texas High molecular weight dna compositions for use in electrophoresis of large nucleic acids
ZA915422B (en) * 1990-07-11 1993-04-13 Genetype Ag Genomic mapping method by direct haplotyping using intron sequence analysis variations.
US5851762A (en) * 1990-07-11 1998-12-22 Gene Type Ag Genomic mapping method by direct haplotyping using intron sequence analysis
WO1999010540A1 (fr) * 1997-08-29 1999-03-04 Lopez Osvaldo J Etablissement de genotype au moyen de la methyltransferase d'adn

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
US4473452A (en) * 1982-11-18 1984-09-25 The Trustees Of Columbia University In The City Of New York Electrophoresis using alternating transverse electric fields

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
* abstract * *
* the whole document * *
GENE. vol. 57, 1987, AMSTERDAM NL pages 193 - 201; R.LATHE ET AL.: 'Plasmid and bacteriophage vectors for excision of intact inserts' *
GENE. vol. 67, 1988, AMSTERDAM NL pages 169 - 182; K.D.TARTOF ET AL.: 'New cloning vectors and techniques for easy and rapid restriction mapping' *
NUCLEIC ACIDS RESEARCH. vol. 17, no. 2, 25 January 1989, ARLINGTON, VIRGINIA US page 817; C.L.SMITH ET AL.: 'Insertion of rare cutting sites nearby genes allows their rapid physical mapping: localization of the E. coli map locus' *
See also references of WO9012891A1 *

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EP0467883A4 (en) 1992-04-22
WO1990012891A1 (fr) 1990-11-01

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