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  • 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
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  • 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 cassettes and a method of mapping genetic material in procaryotes and eucaryotes, including humans, using such cassettes.
• This method incorporates several technologies, including incorporation of synthetic and/or natural DNA sequences into genomic DNA, generation of rare restrictio enzyme cutting sites, and size resolution of DNA fragment up to and greater than the million base pair size range.
• These technologies can be used to create a map of genomic DNA. Once a genomic map has been created, it can be compared to subsequently generated maps of cellular or organismal DNA.
• 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) , and genetic alterations.
• 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 4-105 markers. Generation of this many ordered markers is not feasible using current techniques. _. « .
• 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 DNA of the organism whose genome is to be mapped with a cassette of DNA containing a rare restriction sequence or site, the rare sequence or site being flanked on one or bot sides by a unique DNA sequence.
• 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.
• DNA A and optionally, DNA B
• DNA A need not be restriction sequences,- but rather are simply sequences capable of being identified uniquely in a background of host cell genome, which preferably do not occur in the host cell genome, and which differ from each other as well as from the rare sequence.
• 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 cassette into the host cell. Independently derived. _ ⁇ _
• the unique DNA sequence(s) flanking the rar restriction sequence serves as a marker within the genomic DNA.
• the clonal cells are propagated independently, and then the restriction fragment pattern produced by each is examined. Fragments are generated by cutting at the rare restriction site, and at least one secondary restriction site, and then separated. The generated fragments are identified by virtue of the presence of the uniquely identifiable DNA sequences adjacent to the rare restricti site. Distance between the unique sequence and secondary sites on each fragment are measured, and then, by comparison, distances between restriction sites calculate From this information, a regional restriction map can be constructed; repetition of this process ultimately permit construction of a total genomic map, by recognition of secondary restriction pattern overlap.
• the invention also provides novel DNA cassette comprising a restriction sequence rare for the genome to tested, flanked on at least one side by a nucleotide sequence which is uniquely identifiable in the genome to tested? also provided are novel vectors containing the cassettes, for use in integrating the cassette into the h cell genome.
• the nature of the cassette and vector will generally differ depending on the source of the genome to mapped. In one embodiment, for example, any genome which does not have A methylation, and has an appropriate genom utilization can be mapped utilizing a cassette comprising rare Clal/Clal overalapping restriction sequence flanked one or both sides by a retroviral sequence.
• Examples of organisms which would fall into such a category include b are not limited to mammals (humans), birds, and Drosophil this cassette can be transmitted to the host cell by way 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 culture or organisms, into the genome of which have been integrate the novel cassettes described above.
• the method of the present invention provides ma significant advantages over currently known mapping techniques.
• the present methods of choice rely on the identification of naturally occurring RFLP markers, and constructing linkage maps by pedigree analysis this requires observation of numerous generations of individuals.
• the average 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.
• FIG. 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.
• the defective retroviruses and the trans packaging cell lines are described in Watanabe, S. and Temin, H.J., Mol. Cell Biol. _: 224A--i9 , 1983? Mann et l ⁇ .. Cell 3_3_:153-59, 1984? Sorge, et al., Mol. Cell Biol. 4:1730-37, 1984.
• the uniqu DNAs, A and B correspond to the retroviral sequences at t 5' and 3' sides, respectively, of the Cla I/Cla I inserted sequence.
• This sequence, ATCGATCGAT will be inserted in non-essential region of the defective retrovirus so as to allow subsequent replication and packaging by factors provided in trans.
• Figure 2 shows maps of the vector pZipNeo indicating the sequence and position of the inserted oligonucleotide restriction site.
• Fig. 2a specifically shows the pZipNeo vector containing the Clal/Clal/Dpnl sites?
• Fig. 2b illustrates a pZipNeo vector having multipl copies of the NotI recognition sequence.
• Figure 3 schematically illustrates a procedure for locating the position of a particular gene or DNA segment once a map is constructed. Description of the method is found in Section 5.7, infra.
• Figure 4 illustrates the presence and in situ MClal/Dpnl cleavability of insertion mapping vector pZipNeo28.
• 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?
• 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 digestion produce the same band pattern indicating successful cleavage by the rare cutting strategy.
• Figure 5 illustrates the use of lambda phage concate ers 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 chromosomes bands represent doublets) .
• 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
• DNA sequence termed unique DNA A
• unique DNA B a DNA sequence that is at the to other end by a second uniquely identifiable DNA sequence, termed unique DNA B.
• unique DNA sequences can be natural or synthetic.
• This combination of sequences, i.e., the rare restriction sequence, unique DNA A and optional unique DNA B, is termed a cassette.
• the cassett is termed a cassette.
• the cassette 15 may contain a sequence or sequences which facilitate subsequent isolation of the genomic DNA flanking the cassette.
• the cassette may also contain a hig affinity protein binding site.
• the ⁇ repressor binding sequence can be used in conjunction with
• DNA affinity column composed of covalently bound repressor
• DNA A or unique DNA B sequence can be readily isolated for subsequent manipulation.
• the cassett can contain genetic functionalities that allow it to be maintained as a plasmid in E_ coli or other appropriate
• 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 35 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 cleavag The preferred fragment size is not dictated by any particular requirement, but rather is a matter of convenience: the larger the fragment size produced, generally, the easier the mapping procedure will be. "Large” is, of course, determined relative to the total si 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 cutte can be made in a number of ways.
• One approach is to simpl treat the DNA with an appropriate enzyme (appropriate to b defined below) , and observe the size of the fragments produced. If the fragment size is acceptable in accordan with the guidelines noted above, then a useful sequence h 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.
• a general knowledge of the approximate GC content of the genome provides a convenient means by which the expected average fragment size, in base pairs, generated by cleavage at any given restriction site can be predicted.
• Information relating to GC content of various organisms is readily available in the literature (e.g.. Hill, J. Gen. Microbiol. 44:419-437, 1966) , or is readily deter inable by known techniques (Owen, R.J. and Pitcher, D. (1985) In "Chemical Methods in Bacterial Systematics" pp.
• AT content can also be determined (fraction GC + fraction AT - 1). Assuming random order of dinucleotides/trinucleotides , then average fragment size (AFS) generable by cleavage of a given recognition sequence can be calculated by the following formula
• the average fragment size produced by cleavage of the restriction sequence ATCGAT in this genomi 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 th 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 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 cassette, over cleavage of the same sequence in a genomic site, in turn producing greater efficiency in large fragmen production.
• incorporation of a DNA molecule modified to cleave, such as a D-loop, or a triple helix (Strobel et al. , J. Am. Chem. Soc. 110:7927-7929, 1988) wil achieve substantially the same effect.
• the initial sequence need not even be particularl uncommon in the host genome, but merely need to be modifiable in such a way as to render them "rare" in the present context.
• a preferred sequence when a human genome is being mapped, can be the overlapping Clal/Clal sequence
• the selected oligonucleotide restriction sites can be readily prepared synthetically. 5.1.2. UNIQUE DNA AND VECTOR SELECTION
• the purpose of the uniquely identifiable DNA flanking the rare restriction site is to provide a basis f detecting the cassette amidst the genomic DNA.
• unique DNA A and unique DNA B need only be distinguishable, by some detectable means, from th host DNA.
• the sequences can be generated by fragmentation and isolation 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 sequences are chosen in association with a vector used to transform the host cells.
• the vector chos is preferably one sufficiently distinct genetically from t 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. Cel Biol. :1730-1737, 1984), is used to infect a mammalian ce line. These viruses are capable of infecting cells, but once genomically integrated, are incapable of post-inserti replication, preventing reinsertion by the virus into othe 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? this includes, but is not limited to, the use of electroporation, icrinjection, 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 integrated into the genome essentially "at will".
• the use of a vector provides a convenient source of uniquely identifiable DNA: insertion of the rare restriction 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.
• DNA is now relatively routine technology (Chilton, M. et al. , Cell 11:263-271, 1977) and plant virus vectors may al be used.
• vir vectors can be employed for integration of DNA into mammalian cells.
• Particularly preferred as a vect 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 th cassette by detection of the unique DNA, can be verified.
• 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 hybridization with a vector-specific probe, which probe wi not hybridize with the nonhomologous host genomic DNA.
• the cassette can be constructed so as to include a particular selectable marker which allows the identification of the presence of cassette DNA.
• the cells when using a viral vector to integrate cassette DNA into mammalian cells, the cells ar serially diluted in order to produce cultures containing single cell. These clones are then propagated in the presence of a selectable agent which will prevent growth cells which have not integrated a copy of the virus.
• a selectable agent which will prevent growth 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.
• 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.
• DNA ISOLATION 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 _ l9 _
• 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 render the restriction site more rare, and then followed b 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 restrictio enzymes, so that each DNA sample will be digested with the restriction enzyme specific for the rare site, and partial 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 i 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 products, such as, for example, Xmalll and XmnI (New England Biolabs, Inc.)
• 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 0 size produced. A majority of the current techniques rely on ele ⁇ trophoretic 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 5 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 described, for example in Schwartz and Cantor, Cell 37:67, 1984, and U.S.
• Patent No. 4,473,452 This technique is 0 particularly well suited to separation of the large fragments which are preferred for this method.
• the genomic fragments are contained in a suitable medium, preferably a gel medium and the DNA fragments subjected to pulsing electric fields. 5
• genomic fragments Once the genomic fragments have been separated, those fragments which contain the unique DNA A/DNA B sequences are identified. This can be achieved by any 0 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 cassett
• DNA-specific, labelled, cloned DNA fragment substantially 5 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 ont a support membrane, such as, but not limited to, nylon or nitrocellulose, prior to hybridization, in accordance with the method of Southern. This procedure results in an end- label of only those fragments containing the introduced restriction site, producing a ladder of fragments giving th 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 restriction enzyme cut. 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 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 genom to mapped.
• the clonal maps are then compared in order to ascertain overlapping portions. By aligning these overlapping portions, a complete map of the genomic DNA can be generated.
• the Clal/Clal overlapping sequence ATCGATCGAT has an estimated frequency of occurrence in the human genome of once every 2 x 10 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 3:49, 1981). This allows assembly of RNA containing viral particles, which are then exported from the cell.
• 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.
• 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 0 gels. Once the first map is created, mapping can be done comparatively.
• 3'-GTAGCTAGCTACCTAG-5' was synthesized and inserted in tandem arrays into the retroviral vector pZipNeo as described above for mammalian mapping.
• the same oligonucleotide 0 can be inserted into the P element vector, pUChsneo (Steller and Pirotta, EMBO J 4_:167, 1985).
• the polylinker cloning site of pUChsneo is opened with BamHI, the phosphorylated linker added, and closed with ligase.
• the ligation products cake are used to transform the E_ j _ coli strain GM2929 (dam -) .
• Positive clones are grown up and insert size (e.g., number of Clal/Clal sites inserted) determined by double digestion of each positive plasmid with Smal and Sail. Each has unique sites flanking the insert (Steller, H. and Pirrotta, v., supra) . The insert sizes are then resolved on a native polyacrylamide gel against size standards. Plasmids containing 6, 8, and 10 Clal/Clal sites, respectively are grown up preparatively by standard techniques (Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory, 1982) . These plasmids are used to microinject Canton S embryos.
• insert size e.g., number of Clal/Clal sites inserted
• Embryos obtained from a Canton S (Brown University) strain are injected (Zalokar, Microscopica Acta 84_:231, 1981? Zalokar, Experientia 37:1354, 1981? Santamajria, Dev. Bio. 96:285, 1983) with the P element construct generated as described above along with the transposase positive p-25.7wc (Karess and Rubin, Cell 3_8:135, 1984).
• Positive P element containing flies are selected by addition of G418 to the growth media as described (Steller and Pirotta, supra) . Positive fly stocks are maintained under G418 selection for several generations in order to ensure stable P element insertion into the germ line.
• P element insertion can be verified by isolation of DNA from each positive line and spot blotting (Kafatos et al., Nucl. Acid Res. 7:1541, 1982) followed by hybridization with [32]P labeled P element plasmid.
• 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 3_7:67, 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 g 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/l mM EDTA solution. The DNA is then clean enough for further manipulation. 0
• 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 5 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 0 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 5 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 0 a sequence would suggest repeating the procedure substituting a lower frequency cutting system for Notl. (e.g., MTaq/Dpnl? McCleland and Nelson, supra) .
• 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 200,000,000 base pairs (McClelland and Nelson, supra) .
• the following overlapping oligonucleotide is synthesized, flanked by BamHI cohesive ends:
• 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 Cep o (Cell 37J1043, 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.
• a number of constructs can be-made: a vector containing a single copy o the oligonucleotide inserted (pZipNeo28? n*l) ?
• Tandem arrays of the sequence are created by ligating the insert to itself in the presence of T4 kinase, 32P ATP and T4 ligase. Products corresponding to 3 and 6 ligation events are isolated by elution from an 8% polyacrylamide ge following autoradiography.
• the recombinant DNA's are then transfected into cell line harboring a trans-acting, replication defective copy of the retrovirus ( ⁇ AM) which allows assembly of recombinant RNA containing infective viral particles.
• pZipNeo DNA can be transfected by, for example, the scrape 0 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 packagin 5 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 0
• Clones are evident at this time, and are subsequently picke and grown up in the presence of G418.
• the cells are harvested at confluence and cast in 1% low melting agarose
• Plugs are 5 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 slivers and washed in microfuge tubes with Tris/EDTA 0 containing 1 mM s-adenosylmethionine for 1 hour. MClal reactions were incubated at 37*C, usually overnight, in th presence of 40% glycerol, 1 mM SAM, 5 mM DTT in 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
• Lambda phage concatemers are employed as electrophoresis size markers. 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, 1982) .
• 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 55'C with gentle shaking.
• Samples are loaded as described (Schwartz, D.C. et al. , supra) onto a 1% agarose gel for electrophoresis. The gel shown was run for 62 hours at 8.5 v/cm with a pulse time of 150 seconds on an apparatus made in this laboratory after a modified design of Schwartz and Cantor. (Waterbury and Lane, Nucl. Acids Res. 15:1940, 1987).
• the separated, labelled blot of the fragments a 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, includin human genomes.
• Maps of genomes can be compared to each other i 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 are as prenatal diagnosis of inherited genetic disease, identification of induced (acquired) genetic disease, as well as in prognostic applications.
• 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 appropriate computer algorithm.
• 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 generated without having to account for variability.
• 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 genom .
• 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.
• the pZipNeo vector containing a Clal/Clal overlapping restriction sequence is deposited, in an E ⁇ col host, with the NRRL and has been assigned the accession number B-18490
• Ad ⁇ a a .1 dapaattary ImWuM.n (in « * «d.na paalal «*d* and caa-itk ⁇ r. »

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