BACKGROUND OF THE INVENTION Genes are routinely characterized by mapping at the molecular level. Physical maps of polynucleotides are generated by cutting the polynucleotide at specific points and determining the distance between the breaks. The map generated by this process is referred to as a restriction map. Restriction mapping of a polynucleotide can be accomplished using multiple restriction enzyme digests. Typically, complete digests with two or more restriction enzymes and partial mixed enzyme digests are performed. The size of the fragments provided by the complete digests are compared with the various sized fragments of the partial digest to determine a putative order of the restriction fragments.

This approach has been adequate for mapping plasmids and small stretches of polynucleotide. However, when this method is attempted using larger lengths of polynucleotide, it becomes increasingly difficult to unambiguously size fragments. Thus, reliance on restriction fragment size limits the utility of these methods for use with longer stretches of polynucleotide.

Genotypic markers are of significant value in endeavors involving the distinction and identification of genotypes or characteristics associated with those genotypes. Genetic marker systems based on restriction maps face the limitations of conventional restriction mapping. The inability of traditional restriction mapping to deal with larger polynucleotide segments and its reliance on fragment size limits the utility of methods of genotype-specific marker identification based on conventional restriction maps. What is needed in the art is a method of identifying genotype-specific markers which does not rely on conventional methods of restriction mapping. The present invention provides this and other advantages.

SUMMARY OF THE INVENTION Generally, it is an object of the present invention to provide methods of characterizing a polynucleotide.

In one embodiment, the present invention provides a method of restriction site profiling a polynucleotide. In this method, a partial digest of a sample of polynucleotide molecule is obtained using a restriction enzyme. A second partial digest is obtained from another sample of the same polynucleotide molecule using a different restriction enzyme which is not an isoschizomer of the restriction enzyme used to obtain the first partial digest. This process can be repeated any number of times with different samples and different non-isoschizomeric restriction enzymes. The linear order of restriction sites along the polynucleotide molecule is determined from the partial digests and provides the restriction site profile.

In another embodiment, the present invention provides a method of physically mapping polynucleotides of an organism. In this method, the polynucleotide molecule is characterized as described for the method of restriction site profiling a polynucleotide. A restriction site profile is obtained for a different polynucleotide of the same organism using at least two of the same restriction enzymes used to obtain the restriction profile of the first polynucleotide. A determination of the contiguity of the two polynucleotides is made using the restriction site profiles. Polynucleotides representing contiguous regions are physically mapped.

Another embodiment of the present invention provides a method of determining the phylogenetic relationship between two organisms of the same species. In this method, a restriction site profile is obtained for a polynucleotide from an organism. Another restriction site profile is obtained for a polynucleotide of another organism of the same species using at least two of the same restriction enzymes used to obtain the first restriction site profile. When the RSPs overlap, they are compared in those overlapping regions to determine the phylogenetic relationship between the organisms.

In yet another embodiment, the present invention provides a method of identifying genotype-specific markers. Restriction site profiles of a polynucleotide are obtained from each genotype to be compared. The restriction site profiles are compared to identify differences in the presence or absence of restriction sites in the overlapping segments of polynucleotide. Restriction sites specific to a genotype are identified as genotype-specific markers.

Definitions The terms defined below are more fully defined by reference to the specification as a whole. Units, prefixes, and symbols may be denoted in their SI accepted form. Numeric

ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range.

The term"partial digest"refers to the detectable products of cleavage of a polynucleotide with at least one restriction enzyme under conditions in which the restriction enzyme does not cleave at every potential restriction site of the polynucleotide.

Thus, this cleavage yields a nested series of different lengths of nucleic acid products resulting from single or multiple restrictions of a polynucleotide. Those which are detectable make up the partial digest.

As used herein the term"restriction enzyme"includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves at a specific polynucleotide sequence. Exemplary restriction enzymes include but are not limited to Types II and III restriction enzymes, lambda terminase, Cre recombinase, FLP recombinase and certain cleaving peptide nucleic acids such as those described in US Patent No. 5,641,625.

By"isoschizomer"is meant a restriction enzyme having the same recognition site and the same restriction site as the restriction enzyme of which it is said to be an isoschizomer. For example, Bal I is an isoschizomer of MluN I because they have the same recognition sequence and restriction site: TGGVCCA. See, for example, Roberts, R.

J. Nucl. Acids Res. 19: 2077 (1991).

DETAILED DESCRIPTION OF THE INVENTION Overview and Utility The present invention provides, among other things, methods of characterizing a polynucleotide. Thus, the present invention provides utility in such exemplary applications as the physical mapping of a gene, constructing a physical map of a genome by alignment of sub-genomic DNA fragments, determining phylogenetic relationships between organisms of the same species, and identifying genotype-specific markers.

The restriction site profiling method of the present invention allows the physical mapping of more complex genomes which could not be mapped using conventional restriction mapping. Restriction site profiling does not require any preliminary linkage map construction based on any kind of molecular markers nor does it require nucleic acid markers and hybridization or PCR amplification of any probes and thus, the method of the present invention circumvents the obstacle presented by repetitive elements in a genome.

Further, the present invention has an advantage over current methods in that it does not require that the physical distance between restriction sites be obtained. Instead, restriction site profiling utilizes the order of the restriction sites. Consequently, the invention may be practiced without the optional step of approximating the physical distance between restriction sites.

Obtaining a Partia ! Digest of a First Sample of a First Polynucleotide In the methods of the present invention, a partial digest of a polynucleotide is obtained using a restriction enzyme. The polynucleotide sample can be from any eukaryotic or prokaryotic organism. Eukaryotic organisms to which this invention applies include, for example, humans, other mammals, reptiles, birds, arthropods, fungi, and plants. Prokaryotic organisms to which the methods of this invention can be applied include, for example, bacteria. The methods of the present invention can also be applied to viruses. In a preferred embodiment, the organism used in the present invention is a plant.

The plant used in the present invention can be, for example, either a monocot or dicot. For example, the invention can be used in species from the genera: Hordeum, Secale, Tritium, Sorghum (e. g., S. bicolor), Zea (e. g., Z. mays), Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, and Avena.

The polynucleotide isolated from these organisms is duplex DNA (e. g., genomic or cDNA) or heteroduplex DNA. Methods of isolating polynucleotides from organisms are well known to those in the art. See, for example, Sambrook, et al., Molecular Cloning : A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Vols. 1-3 (1989); Plant MolecularBiology : A LaboratoryManual, Clark, Ed., Springer-Verlag, Berlin (1997).

For use in this invention, the polynucleotide molecules are optionally in a cloning vector such as but not limited to YAC clones, BAC clones, PAC clones, cosmid clones, lambda clones, and phagemid clones. Those in the art will recognize that the invention may be modified to be practiced using polynucleotide molecules from any cloning system currently in use or later developed. Preferably, the polynucleotide molecule is at least 20Kb to 1Mb in length. For example, the polynucleotide may be at least 20 Kb, 30 Kb, 40

Kb, 50 Kb, 60 Kb, 70 Kb, 80 Kb, 90 Kb, 100 Kb, 200 Kb, 300 Kb, 400 Kb, 500 Kb, 600 Kb, 700 Kb, 900 Kb, or 1 Mb in length.

The restriction enzyme used in the partial digest can be any restriction enzyme which has a specific cleavage site within or adjacent to a recognition site. Suitable restriction enzymes include but are not limited to Types II and III restriction enzymes, lambda terminase, Cre recombinase, FLP recombinase, and certain cleaving peptide nucleic acids such as those described in US Patent No. 5,641,625. In preferred embodiments, the restriction enzymes used in the present invention have hexanucleotide recognition sequences.

Conditions for obtaining a partial digest can be determined for a particular restriction enzyme in use with a particular polynucleotide using methods known to those in the art. Such methods include but are not limited to: decreasing the units of restriction enzyme per microgram of DNA from the concentration required for complete digestion of the polynucleotide; limiting reaction time to a time less than that required for complete digestion of the polynucleotide with that restriction enzyme; and conducting the digestion in temperatures below the optimum temperature for obtaining complete digests with that restriction enzyme.

Obtaining a Partial Digest of a Second Sample of a First Polvnucleotide Using the methods explained supra, a partial digest of a second sample of the same polynucleotide is obtained using a second restriction enzyme. This restriction enzyme should not be an isoschizomer of the restriction enzyme used to obtain the partial digest of the first sample.

One of skill in the art will recognize that this process can be repeated any number of times with different restriction enzymes. The uniqueness of a restriction site profile depends upon the number and type of restriction enzymes used. More complex and potentially more specific sequences can be generated when more restriction enzymes are used for the restriction site profiles.

For a given genome, it is often possible to predict the theoretical minimum number of restriction sites in a polynucleotide segment for a given set of restriction enzymes that is sufficient for the unique identification of that segment. This statement is based on the assumption that those recognition sites are dispersed randomly and evenly along a polynucleotide sequence. For example, for the maize genome, calculations indicate that a string of greater than eleven restriction sites (e. g., 12,13, or 14) generated by four different

hexanucleotide enzymes is sufficient to uniquely identify a DNA segment and its position within the maize genome.

In one embodiment of the present invention, at least four non-isoschizomeric restriction enzymes are used. The total number of non-isoschizomeric restriction enzymes can be at least 2 to 10. For example, the total number of non-isoschizomeric restriction enzymes may be at least 2,3,4,5 or 6.

In some embodiments it is necessary that each partial digest be detectable by a different label, for example, when samples will be multiplexed into a single lane for electrophoresis. In other embodiments, is not critical that each partial digest be detectable by a different label, for example, when samples will be run in separate lanes for electrophoresis. Those of skill in the art will be able determine when different labels must be applied.

Detection of Partial Disert The products of cleavage of a polynucleotide with a restriction enzyme which are detectable represent the partial digest. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.

Energy transfer labels are preferred. Ju et al., PNAS USA 92: 4347-4351 (1995).

Particularly preferred are multiple energy transfer labels for each polynucleotide. Energy transfer labels are commercially available from Amersham (Arlington Heights, IL).

One common method of detection is the use of autoradiography using radioactive probes. The choice of radioactive isotope depends on research preferences due to ease of synthesis, stability, and half lives of the selected isotopes. The polynucleotide can be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. Labeling of the polynucleotide of the present invention is readily achieved such as by the use of labeled oligonucleotides (e. g., PCR primers). Alternatively, oligonucleotides or other probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents or enzymes. The polynucleotides may also be

labeled before or after cleavage with a restriction enzymes with, for example, labeled triplex-forming oligonucleotides or a labeled oligonucleotide which anneals to a polynucleotide's cohesive end. Labeling of the polynucleotide with a duplex or triplex forming oligonucleotide can occur before or after cleavage with a restriction enzyme. The means by which nucleic acids of the present invention are labeled is not a critical aspect of the present invention and the labels can be attached directly or indirectly such as via a linker molecule. In preferred embodiments, labels are attached covalently.

Polynucleotides can be labeled at their 5'end, 3'end, or internally. Internal labels can be located between the 5'end and the restriction site nearest the 5'end or between the 3'end and the restriction site nearest the 3'end. The polynucleotide can be labeled at more than one location using labels which can be distinguished from each other.

Obtaining a First Restriction Site Profile The partial digests are used to obtain a restriction site profile, alternatively referred to as"RSP". An RSP is the linear order of restriction sites of restriction enzymes along the length of the polynucleotide molecule.

There are several ways to obtain restriction site profiles of the partial digests. Any method typically known and used in the art for fractionation of fragments is appropriate for use in this invention, including but not limited to, electrophoresis, capillary electrophoresis or optical mapping. In one embodiment, the restriction site profiles are obtained by electrophoresis.

Electrophoresis fractionates the fragments according to length. Electrophoretic methods are well known to those in the art. See, for example, Maniatis, T. et al., "Molecular Cloning: A Laboratory Manual,"Cold Spring Harbor Labs (1982). The samples may each be run in a separate lane or they may be multiplexed into a single lane.

To multiplex, the cleaved polynucleotide samples, each labeled with a different label for the restriction enzyme with which it was cleaved, are mixed together and loaded in the same lane for electrophoresis.

The electrophoresis is coupled with a method of detection appropriate to the detectable label used. The restriction site profile is determined by the order of fractionation of the detectable fragments typically determined from smallest to largest detectable fragment. The smallest detectable fragment represents the restriction site closest to the label. For example, if the label was on the 5'end, then the smallest detectable fragment represents the first restriction site and the largest detectable fragment

represents the last restriction site. By reading from the end of a lane back towards where the lane was loaded, the restriction site order from the label can be read. The restriction site order can also be read from the loading site of a lane towards the end of the lane, recognizing that the fragment at the end of the lane represents the restriction site closest to the label.

In embodiments where the samples have been multiplexed, each partial digest has a different detectable label. By differentiating between the labels after fractionation, one of skill in the art can determine which restriction enzyme is responsible for each fragment, and thus determine the order of restriction sites.

In embodiments where the samples are in separate lanes, each lane represents fragments generated by one restriction enzyme. The order of restriction sites is then a matter of comparing position both within and between lanes. The smallest fragment from one lane represents the first restriction site from the label only if it is smallest fragment compared across the lanes for all the partial digests of the polynucleotide.

A device to detect the labels of the detectable fragments can be coupled with a fractionating apparatus such as electrophoresis such that the device reads the labels of the fragments as they are fractionated. For example, if fluorescent labels are used, a scanning device with fluorescence excitation and detection capabilities can be coupled with the gel and the restriction fragment profile detected.

Such scanning devices can be, for example, horizontal, agarose-based electrophoresis systems with fluorescence excitation and detection capabilities. Upon fractionation, DNA fragments go through the read region of the scanning device where they migrate past a laser beam. Fluorescence signal data are collected in real time by a computer. Fragment peak extraction and mobility conversion are automatically performed at the end of each run. The restriction site profile is the order in which the fluorescence signal data are read. Devices to detect labels are well known and available to those of skill in the art.

Optionally, the approximate physical distance between at least one pair, for example at least 1 to 6 pairs, of restriction sites is obtained using techniques known to those of skill in the art. For example, the approximate distance between at least 1,2,3,4, 5, or 6 pairs of restriction sites in a partial digest is obtained. The approximate physical distance can be used in cases of conflict on the alignment of RSPs when the same overlapping sequence links different DNA segments having different RSP for non- overlapping segments. However, it should be noted that the distance between restriction

sites, that is, the size of the fragments generated by the restriction digest, is not necessary for determining the restriction site profile. The present invention utilizes the order of the restriction sites, not the size of the various fragments. Consequently, the present invention can be practiced without determining the distance between any pair (s) of restriction sites.

A Second Polvnucleotide Molecule The methods of the present invention as described for a first polynucleotide molecule can be applied to a second polynucleotide molecule to generate a second restriction site profile. This second restriction site profile can then be used in combination with the first restriction site profile for physical mapping, identifying genotype-specific markers and phylogenetic analysis of two organisms of the same species.

Obtaining a Second Restriction Site Profile of a Second Polvnucleotide Molecule A partial digest of a second polynucleotide molecule is obtained using a set of restriction enzymes in the same manner set forth above for the first polynucleotide molecule. At least two of the restriction enzymes used to obtain the first RSP, or their isoschizomers, are used to obtain the RSP of the second polynucleotide molecule. The second RSP is obtained in the same manner as discussed for the first RSP.

Determining the Presence of a Contiguous Nucleic Acid Segment Restriction site profiles of different polynucleotides which overlap are identified.

The number of restriction sites of the first and second RSPs which must overlap in order to consider the first and second polynucleotides a contiguous nucleic acid segment may be based on any number of factors, for example, the level of statistical certainty desired for a determination that two polynucleotides are a contiguous nucleic acid segment, the size of the genome from which the polynucleotides were isolated, and the complexity of that genome. Those of skill in the art will recognize that the greater the number of overlapping restriction sites, the higher the probability that two polynucleotides are a contiguous nucleic acid segment. Any number of overlapping restriction sites may be determined to be the number required to consider two polynucleotides a contiguous nucleic acid segment at a desired level of statistical certainty, for example, from 2 to 50. For example, 2,6,10, 15,20, or 50 may be determined to be the number of restriction sites required to overlap between two polynucleotides in order for the polynucleotides to represent a contiguous region. Statistical methods to determine the number of overlapping restriction sites

required for a contiguous region at a desired level of significance for a given genome are known in the art.

The first polynucleotide molecule can be physically mapped to the second polynucleotide molecule if the first and second polynucleotide molecules are determined to represent a contiguous nucleic acid segment.

Physical Mapping Because a restriction enzyme has a specific recognition sequence and restriction site, the order of restriction sites in a polynucleotide segment for two and more restriction enzymes may be considered as reduced polynucleotide sequence information on a given polynucleotide segment. Therefore, the order of restriction sites may be treated as polynucleotide sequence information for the purpose of sequence comparison.

Alignment of the RSPs can be performed with the aid of computer software programs which perform polynucleotide sequence alignment or protein alignment.

Alignment software is available from a number of commercial suppliers such as the Genetics Computer Group's (Madison, WI) PILEUP software, Vector NTI's (North Bethesda, MD) ALIGNX, or Genecode's (Ann Arbor, MI) SEQUENCHER.

Physical distances between adjacent restriction sites are ignored at the primary round of alignment. They can be retrieved in case of conflicts on the alignment of RSPs when the same overlapping sequence links multiple polynucleotide segments having different RSPs for non-overlapping segments. However, they are not essential to the current invention.

The aligned RSPs provide an ordered list of the restriction sites for the contiguous nucleic segment. By aligning the RSPs, the first polynucleotide has been physically mapped to the second polynucleotide. If one of the polynucleotides has already been placed on a physical map of the genome, the other polynucleotide can readily be added.

Phylogenetic Analysis of Two Organisms of the Same Species Methods of determining the phylogenetic relationship of organisms are of interest because they can facilitate a determination of the probability that analogous stretches of polynucleotide in two organisms are identical by descent. They can be used alone or in conjunction with other methods to determine the genetic relatedness of organisms. Such information is useful, for example, in determining whether two organisms of unknown origin are derived from a common ancestral line or in determining whether two varieties or

lines of the same species were derived from a single variety or line. Therefore, the methods of the present invention are useful for monitoring germplasm security. Current methods relying on polymorphisms between restriction maps are subject to the limitations of traditional restriction mapping.

When the first polynucleotide molecule and the second polynucleotide molecule of the present invention are from two organisms of different genotypes of the same species, the phylogenetic relationship of the two individuals can be determined if the polynucleotide samples contain overlapping segments of polynucleotide from the organisms, and if at least two of the same restriction enzymes, or isoschizomers of those restriction enzymes, used to create the RSP of the first polynucleotide molecule are used to create the RSP of the second polynucleotide molecule. RSPs of each polynucleotide molecule are obtained using the procedures set forth by the methods of this invention.

The overlapping segments of the RSPs of the first organism and the second organism are compared. Restriction sites present in one organism and absent in the other are identified. The resulting information is analyzed using methods commonly used for DNA fingerprinting by DNA restriction mapping. Phylogenetic trees based on this information can be constructed using methods known and used in the art for distance- based construction. The degree of divergence of the two organisms can also be calculated.

Such methods are well known in the art and a variety of modeling and analytical tools are available from commercial vendors such as the Genetics Computer Group's PAUPSEARCH and GROWTREE programs of the WISCONSIN PACKAGE (Version 10.0, Madison, WI).

Identifving Genotvpe-Specific Markers When the first polynucleotide molecule and the second polynucleotide molecule of the present invention are from two different genotypes of the same species, genotype- specific markers can be developed if the polynucleotide samples are overlapping segments of polynucleotide. RSPs of each polynucleotide molecule are obtained using the procedures set forth by the methods of this invention.

The RSPs for the genotypes are aligned and any sites present in one genotype and not present in the other genotype in overlapping regions are identified. Sites associated with one genotype and not the other are genotype-specific markers. Those of skill in the art will recognize that this method could be applied to any number of genotypes within a species to create a profile of genotype-specific markers for each of the genotypes.

Identifving Tandem Repeated Elements The present invention provides methods of identifying tandem repeat elements in a polynucleotide. Exemplary tandem repeat elements are 5S rDNA, 18-26S rDNA, 180- knob tandem repeats, centromeric CentC repeats, and TR1 knob repeats. A monomeric unit within a tandem repeat element will often have at least one recognition site for a restriction enzyme. The RSP of each monmeric unit within a tandem repeating element will be repeated as many times as the number of repeats in the tandem repeating element.

For those tandem repeats with known nucleotide sequence it is possible to select an appropriate restriction enzyme with a recognition site within the unit.

The correspondence of the tandem repeat discovered via RSP can optionally be verified by the comparison of the sizes of adjacent restriction fragments. Those of skill in the art will recognize that this method could be applied to any number of tandem repeats within a species to identify their presence in a polynucleotide molecule as well as for the discovery new tandem repeats.

Identifvinç Disperse Repeated Elements Genomes of eukaryotic organisms contain a large number of dispersed repetitive elements like retrotransposable elements, LINE-elements, transposons. The elements of the same family will typicaly have a specific RSP which is repeated many times throughout a genome. These dispersed repeated elements can be identified by their unique RSP. Such dispersed repeated elements can be mapped across a genome. For those tandem repeats with known nucleotide sequence it is possible to select a set of restriction enzymes, which will create a specific RSP for a dispersed repeating unit. Those of skill in the art will recognize that this method can be applied to any number of dispersed repeats within a species to identify their presence as well as for the discovery of new dispersed repeats.

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Example I This example provides an outline of one embodiment of the restriction site profiling method.

Design: A DNA sample of a cosmid clone is digested to completion with lambda terminase.

To generate an RSP for four restriction enzymes, four aliquots of the DNA sample are cleaved under partial digest conditions with each restriction enzyme.

The CosL oligo, which is complementary to the left protruding end of the cos site (12 nucleotides in length) is labeled with four different fluorochromes, each of which could be used in combination with a certain enzyme. Each of the four different fluorochrome labeled oligos are mixed with a different one of the four DNA aliquots to allow the annealing of the oligo to a cos site.

After annealing, the DNA samples are loaded into separate lanes of an agarose gel for electrophoresis or mixed together and loaded on the same lane. The resulting restriction site profile is detected with the aid of, for example, a scanning device with fluorescence excitation and detection capabilities. The order of the restriction sites can then be determined in any given DNA segment. The restriction site profile can be determined a second time for the same DNA.

Steps: 1. Prepare oligonucleotides complementary to the CosL (SEQ ID NO: 1) and CosR (SEQ ID NO: 2) overhangs labeled at 3'end or at 5'end or internally with a fluorochrome, see Table 1.

Table 1 NameSequenceWavelength CosL-FAM 5-AGGTCGCCGCCC-3'-FAM Blue 490-520 5- (SEQ ID NO:1)-3'-FAM CosL-JOE 5-AGGTCGCCGCCC-3'-JOE Green 521-550 5- (SEQ ID NO:1)-3'-JOE CosL-TAMRA 5-AGGTCGCCGCCC-3'-TAMRA Yellow552-575 5- (SEQIDNO:1)-3'-TAMRA CosL-ROX5-AGGTCGCCGCCC-3'-ROXRed580-605 5- (SEQ ID NO:1)-3'-ROX CosR-FAM 5-GGGCGGCGACCT-3'-FAM Blue 5- (SEQ ID NO:2)-3'-FAM CosR-JOE 5-GGGCGGCGACCT-3'-JOE Green 5- (SEQ ID NO:2)-3'-JOE CosR-TAMRA 5-GGGCGGCGACCT-3'-TAMRA Yellow 5- (SEQ ID NO:2)-3'-TAMRA A CosR-ROX 5-GGGCGGCGACCT-3'-ROX Red 5- (SEQ ID NO:2)-3'-ROX

2. Cosmid DNA with insert is cut with lambda terminase to linearize a cosmid and to expose the cos overhangs.

3. DNA sample is divided into 4 aliquots and each aliquot is cleaved under partial digest conditions with one of the four mapping enzymes. Enzymes are inactivated.

4. Each aliquot is mixed with CosL oligos labeled with different fluorochromes assigned for a specific enzyme. Altogether there are 4 samples with CosL.

5. Four samples annealed to CosL primers labeled with different fluorochromes are mixed together and loaded on the same lane. Final amount of DNA in a sample is about 500 ng.

(12 fmol).

6. Fragments are fractionated in agarose gel with the help of inverse field gel electrophoresis system to ensure good resolution in the area of long DNA fragments (20 to 50kb). The gel is coupled with a scanning device and the restriction fragment profile is detected. The laser scanning device is a vertical agarose-based electrophoresis system with fluorescence excitation and detection capabilities (Perkin-Elmer/Applied Biosystems 370). Upon fractionation DNA fragments go through the read region (scanning region) where they migrate past a laser beam. Fluorescence signal data are collected in real time by a computer. Fragment peak extraction and mobility conversion are automatically performed at the end of each run. An internal size standard is used to enable accurate size determination from mobility data.

7. A restriction site profile is determined for four restriction enzymes. The order of restriction sites is the result necessary for DNA fragment alignment. However, information on sizes of restriction fragments can also be saved.

8. Two RSPs are obtained for the same cloned DNA fragment from both ends. This increases the accuracy of the map construction.

9. The resulting RSP is aligned using software generally used for nucleotide sequence alignment.

The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications cited herein are hereby incorporated by reference.