WO2001034840A2 - Genetic compositions and methods - Google Patents

Abstract

This invention provides nucleic acid segments derived from the human genome, including polymorphic sites. Allele-specific primers and probes hybridizing to these sites and to regions flanking these sites are also provided. This invention further provides methods of analyzing a nucleic acid from an individual or a group of individuals. The nucleic acids, primers, and probes are useful tools for applications including forensics, paternity testing, medicine, e.g., the corelation of polymorphisms with phenotypic traits, and genetic analysis, e.g., genetic mapping of such phenotypic traits.

Description

GENETIC COMPOSITIONS AND METHODS

BACKGROUND OF THE INVENTION

The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution generating variant forms of progenitor sequences

(See, e.g., Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). A variant form may be neutral or confer an evolutionary advantage or disadvantage relative to a progenitor sequence. In some instances, a variant form confers a lethal disadvantage. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the deoxyribonucleic acid (DNA) of many or most members of the species and effectively becomes the progenitor sequence. In many instances, both the progenitor and the variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms.

Several different types of polymorphism have been reported. A restriction fragment length polymorphism (RFLP) is a variation in a DNA sequence that alters the length of a restriction fragment (see, e.g., Botstein et al., Am. J. Hum. Genet. 32, 314-331 (1980). The RFLP may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses (See, e.g, WO 90/13668; WO 90/11369; Donis-Keller, Cell 51, 319-337 (1987); and, Lander et al, Genetics 121, 85-99 (1989)). Where a heritable trait can be linked to a particular RFLP, the presence of such RFLP in a human can be used to predict the likelihood that the individual will also exhibit the trait.

Other polymorphisms take the form of short tandem repeats (STRs), for example, tandem di-, tri- and tetra-nucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analyses as well as in a large number of genetic mapping studies. (See, e.g, US 5,075,217; Armour et al, FEBS Lett. 307, 113-115 (1992); Horn et al, WO 91/14003; Jeffreys, EP 370,719).

Other polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than

RFLPs, STRs and VNTRs. Some single nucleotide polymorphisms (SNPs) occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective, or other variant, protein and, potentially, a genetic disease.

Examples of genes, in which polymorphisms within coding sequences give rise to genetic disease include β - globin (sickle cell anemia) and CFTR (cystic fibrosis). SNPs also occur in noncoding regions and these SNPs may result in defective protein expression (e.g, as a result of defective splicing). Further, some SNPs have no phenotypic effects. SNPs can be used in the same manner as RFLPs and VNTRs but offer several advantages. SNPs occur with greater frequency and are spaced more uniformly throughout the genome than other polymorphisms. The greater frequency and uniformity of SNPs mean that there is a greater probability that such a SNP will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms.

Also, the different forms of characterized SNPs are often easier to distinguish than other polymorphisms (e.g, by use of assays employing allele-specific hybridization probes or primers).

Despite the increased amount of nucleotide sequence data being generated in recent years, only a minute proportion of the total repository of polymorphisms in humans and other organisms has been identified. The paucity of polymorphisms hitherto identified is due to, in substantial part, the large amount of work required for their detection by conventional methods. A conventional approach to identifying polymorphisms, for example, sequences the same stretch of oligonucleotides in a population of individuals by dideoxy sequencing. In this type of approach, the amount of work increases in proportion to both the length of the sequence and the number of individuals in a population and, as those skilled in the art will understand, becomes impractical for large stretches of DNA or large numbers of subjects.

All documents, i.e., publications and patent applications, cited in this disclosure, including the foregoing, are incorporated by reference herein in their entireties for all purposes to the same extent as if each of the individual documents were specifically and individually indicated to be so incorporated by reference herein in its entirety. SUMMARY OF THE INVENTION

This invention provides nucleic acid segments of between 10 and 100 bases from a fragment shown in TABLE 1, column 6 including a polymorphic site. Complements of these segments are also provided in this invention. The segments can be DNA or ribonucleic acid (RNA), and can be double- or single-stranded. Some segments are 10-20 or 10-50 bases length. Preferred segments include a diallelic polymorphic site. The base occupying the polymorphic site in the segments can be the reference (TABLE 1, column 4) or an alternative base (TABLE 1, column 5). The invention further provides allele-specific oligonucleotides that hybridizes to a segment of a fragment shown in TABLE 1 , column 6 or its complement. These oligonucleotides can be probes or primers. Also provided are isolated nucleic acids comprising a sequence shown in TABLE 1 , column 6, or the complement thereto, in which the polymorphic site within the sequence is occupied by a base other than the reference base shown in TABLE 1 , column 4.

The invention further provides methods of analyzing a nucleic acid from a subject, which may be, but is not limited to a human being. The novel methods determine which base is present at any one of the polymorphic sites shown in TABLE 1. Optionally, a set of bases occupying a set of the polymorphic sites shown in TABLE 1 is determined. These types of analyses can be performed on a plurality of subjects who are tested for the presence of a disease phenotype. The presence or absence of disease phenotype can then be correlated with a base or set of bases present at the polymorphic sites in the subjects tested.

DEFINITIONS An oligonucleotide, as the case may be, can be DNA or RNA, and single- or double-stranded. Oligonucleotides can be composed of naturally occurring or synthetic bases, but in either case are generally prepared by synthetic means. Preferred oligonucleotides of the invention include segments of DNA, or their complements including any one of the polymorphic sites shown in TABLE 1. The segments are usually between 5 and 100 bases in length, and often between 5-10, 5- 20, 10-20, 10-50, 20-50 or 20-100 bases in length. The polymorphic site can occur within any position of the segment. The segments can be from any of the allelic forms of DNA shown in TABLE 1.

Hybridization probes are oligonucleotides capable of binding in a base- specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids. (See, e.g, Nielsen et a , Science 254, 1497-1500 (1991)). A primer is a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions e.g, buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase. The length of the primer, in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with such template. The primer site is the area of the template to which a primer hybridizes. The primer pair is a set of primers including a 5' upstream primer that hybridizes with the 5' end of the sequence to be amplified and a 3' downstream primer that hybridizes with the complement of the 3' end of the sequence to be amplified.

Linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome, and can be measured by percent recombination between the two genes, alleles, loci or genetic markers.

Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than about 1 percent (%), and more preferably greater than about 10% or 20% of a selected population. A polymorphic locus may be as small as one base pair. Polymorphic markers include RFLPs, variable number of tandem repeats VNTRs, hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as, for example, Alu. The first identified allelic form is arbitrarily designated the reference form while subsequently identified allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is often referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic polymorphism has two forms. A triallelic polymorphism has three forms. A SNP occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. This site of variation is usually both preceded by and followed by highly conserved sequences e.g, sequences that vary in less than 1/100 or 1/1000 members of the populations of the given allele. A SNP usually arises due to the substitution of one nucleotide for another at the polymorphic site. These substitutions include both transitions (i.e. the replacement of one purine by another purine or one pyrimidine by another pyrimidine) and transversions (i.e. the replacement of a purine by a pyrimidine or vice versa). SNPs can also arise from either a deletion of a nucleotide or from an insertion of a nucleotide relative to a reference allele. Hybridizations, e.g, allele-specific probe hybridizations, are generally performed under stringent conditions. For example, conditions where the salt concentration is no more than about 1 Molar (M) and a temperature of at least 25 degrees-Celcius (°C), e.g, 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4 (5X SSPE)and a temperature of from about 25 to about 30°C. An isolated nucleic acid is an object species invention that is the predominant species present ( . e., on a molar basis it is more abundant than any other individual species in the composition). Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.

Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods). DESCRIPTION OF THIS INVENTION

I. NOVEL POLYMORPHISMS OF THIS INVENTION The novel polymorphisms of this invention are listed in TABLE 1.

The first column lists the sequence identification number (SEQ ID NO). The second column of TABLE 1 lists the unique names assigned to the fragments in which the polymorphism(s) occur. These names are from the GenBank database (release 114) and can be accessed by querying the database with a program such as Blast. All information related to these sequences available through GenBank is hereby incorporated by reference for all purposes. The fragments are all human genomic fragments. Also included within the scope of this invention are all analogous fragments of other species to such human genomic fragments of TABLE 1.

The third column of TABLE 1 lists the position of a polymorphic site within the fragment. These positions are numbered consecutively with the first base of the fragment sequence assigned the number one. The fourth column of TABLE 1 lists the base occupying the polymorphic site in the sequence in the data base. This base is arbitrarily designated the reference, or prototypical form, but is not necessarily the most frequently occurring form. The fifth column of TABLE 1 lists other base(s) found at the polymorphic site. The seventh column of TABLE 1 lists about 10 bases of sequence on either side of the polymorphic site in the fragment. The sequences can be either DNA or RNA. In the latter, the T's shown in TABLE 1 are replaced by U's. The base occupying the polymorphic site is indicated by an asterisk (*).

Table 1

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Table 1

Table 1

Table 1

Table 1

ANALYSIS of POLYMORPHISMS

A. PREPARATION of SAMPLES

Polymorphisms are detected in a target nucleic acid from the subject(s) being analyzed. Any suitable biological sample can be used for assay of genomic DNA. Convenient suitable tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. Pure red blood cells are not suitable. As those skilled in the art will appreciate, for assays of cDNA or mRNA, the tissue sample must be obtained from an organ in which the target nucleic acid is expressed, e.g., the liver for a target nucleic acid of a cytochrom P450. Many known methods such as, for example those described below require amplification of the DNA from target samples. This can be accomplished by e.g., PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Patent 4,683,202.

Other suitable amplification methods include the ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4,560 (1989) and Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)) and nucleic acid based sequence amplification (NABS A). The latter two amplification methods include isothermal reactions based on isothermal transcription, which produce both single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.

B. DETECTION of NOVEL POLYMORPHISMS in TARGET DNA There are two distinct types of analyses depending upon whether a given polymorphism has already been characterized. The first type of analysis is sometimes referred to as de novo characterization and compares target sequences in different subjects to identify points of variation, i.e., polymorphic sites. By analyzing a group of subjects representing the greatest ethnic diversity among human beings and the greatest breed and species variety of plants and animals, patterns characteristic of the most common alleles/haplotypes of a given locus can be identified, and the frequencies of such patterns in the population can be determined. Additional allelic frequencies can be determined for subpopulations characterized by criteria such as, for example, geography, race, or gender. This de novo characterization of the polymorphisms of this invention is described in the EXAMPLES section of this description. The second type of analysis determines which form(s) of a characterized polymoφhism are present in the subjects under test. Many suitable procedures exist and these are discussed immediately below.

1. ALLELE-SPECIFIC PROBES

The design and use of allele-specific probes for analyzing polymorphisms is known in the art, e.g., Saiki et al., Nature 324, 163-166 (1986); Dattagupta, EP 235,726 and Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one subject but not to the corresponding segment from another subject due to the presence of different polymoφhic forms in their respective segments. Hybridization conditions should be suitably stringent so that there is a significant difference in hybridization intensity between alleles, preferably an essentially binary response, whereby the chosen probe hybridizes to only one of the alleles. Some probes are designed to hybridize to a segment of target DNA such that the polymoφhic site aligns with a central position (e.g., in a 15 mer at the 7 position; in a 16 mer, at either the 8 or 9 position) of the probe. This particular design of probe achieves useful discrimination in hybridization between different allelic forms.

Allele-specific probes are often used in pairs, with one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analyses of multiple polymoφhisms within the same target sequence. 2. TILING ARRAYS

Polymoφhisms can also be identified by hybridization to nucleic acid arrays, (See, e.g., WO 95/11995). One form of such arrays is described in the EXAMPLES section of this description in connection with de novo identification of polymoφhisms. The same array or a different array can be used for analysis of characterized polymoφhisms. The aforementioned WO 95/11995 also discloses subarrays that are optimized for the detection of variant forms of a pre-characterized polymoφhism. Such subarrays contain probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence. The second group of probes is designed by the same principles as described in the EXAMPLES section of this description except that the probes exhibit complementarity to the second reference sequence. The inclusion of a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are predicted to occur within a short distance commensurate with the length of the probes (i.e., two or more mutations within 9 to 21 bases).

3. ALLELE-SPECIFIC PRIMERS

An allele-specific primer hybridizes to a site on a target DNA overlapping a polymoφhism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. (See, ej*., Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989)). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers leading to a detecTABLE product which signifies that the particular allelic form is present. A control is generally performed with a second pair of primers, one of which shows a single base mismatch at the polymoφhic site and the other of which exhibits perfect complementarity the distal site. This single-base mismatch prevents amplification and, as such, no detectable product is formed. Most preferably, the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymoφhism, i.e., the 3'-most position is the position most destabilizing to elongation from the primer. (See, e.g., WO 93/22456.) 4. DIRECT SEQUENCING

The direct analysis of the sequence of polymoφhisms of this present invention can be accomplished by using either the dideoxy chain termination method or the Maxam Gilbert method (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989) and Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

5. DENATURING GRADIENT GEL ELECTROPHORESIS Amplification products generated using PCR can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. (See, e.g., Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7.)

6. SINGLE STRAND CONFORMATION POLYMORPHISM ANALYSIS

Alleles of target sequences can be differentiated using single-strand conformation polymoφhism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products. (See, e.g., Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989)). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence difference between alleles of the target sequences.

II. METHODS of USE

The presence of polymoφhic form(s) in a subject at one or more polymoφhic sites is useful for, e.g., forensics, paternity testing, correlation of polymoφhisms with phenotypic traits, and genetic mapping of phenotypic traits. A. FORENSICS

Determination of which polymoφhic forms occupy a set of polymoφhic sites in an individual identifies a set of polymoφhic forms that distinguishes the individual. (See, e.g., National Research Council, The Evaluation of Forensic DNA Evidence (Eds. Pollard et al., National Academy Press, DC, 1996)). Increasing the number of sites analyzed the probability that the set of polymoφhic forms in one individual is the same as that in an unrelated individual. Preferably, where multiple sites are analyzed, the sites are unlinked. Thus, polymoφhisms of this invention are often used in conjunction with polymoφhisms in distal genes. Preferred polymoφhisms for use in forensics are diallelic because the population frequencies of two polymoφhic forms can usually be determined with greater accuracy than those of multiple polymoφhic forms at multi-allelic loci.

As discussed above, the capacity to identify a distinguishing or unique set of forensic markers in an individual is useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining whether the set of polymoφhic forms occupying selected polymoφhic sites is the same in the samples from suspect and the sample taken from the crime scene. Where the set of polymoφhic markers taken from the crime scene does not the sample from the suspect, it can be concluded (barring experimental error) that the suspect is not the source of the sample taken from the crime scene. Where the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene. Where frequencies of the polymoφhic forms at the loci tested have been determined (e.g., by analysis of a suitable population of individuals), a statistical analysis can be performed to determine the probability that such a match of suspect and crime scene would occur merely by chance. p(ID) is the probability that two random individuals have the same polymoφhic or allelic form at a given polymoφhic site. In diallelic loci, four genotypes are possible: AA, AB, BA, and BB. Where alleles A and B occur in a haploid genome of the organism with frequencies x and y, the probability of each genotype in a diploid organism, as disclosed in WO 95/12607: Homozygote: p(AA)= x².

Homozygote: p(BB)= y² = (1-x)²

Single Heterozygote: p(AB)= p(BA)= xy = x(l-x).

Both Heterozygotes: p(AB+BA)= 2xy = 2x(l-x).

The probability of identity at one locus (i.e, the probability that two individuals, picked at random from a population will have identical polymoφhic forms at a given locus) is given by the equation: p(ID) = (x²)² + (2xy)² + (y²)².

These calculations can be extended for any number of polymoφhic forms at a given locus. For example, the probability of identity p(ID) for a 3 -allele system where the alleles have the frequencies in the population of x, y and z, respectively, is equal to the sum of the squares of the genotype frequencies:

p(ID) = x⁴ + (2xy)² + (2yz)² + (2xz)² + z⁴ + y⁴

In a locus of n alleles, the appropriate binomial expansion is used to calculate p(ID) and p(exc).

The cumulative probability of identity (cum p(ID)) for each of multiple unlinked loci is determined by multiplying the probabilities provided by each locus.:

cum p(ID) = p(IDl)p(ID2)p(ID3).... p(IDn).

The cumulative probability of non-identity for w loci (i.e., the probability that two random individuals will be different at 1 or more loci) is given by the equation: cum p(nonΙD) = 1-cum p(ID).

If several polymoφhic loci are tested, the cumulative probability of non-identity for random individuals becomes very high (e.g., one in a billion). Such probabilities can be taken into account together with other evidence in determining the guilt or innocence of the suspect.

B. PATERNITY TESTING

The object of paternity testing is usually to determine whether a particular male is the father of a given child. In most cases, the mother of the child is known and thus, the mother's contribution to the child's genotype can be traced. Hence, paternity testing investigates whether the part of the child's genotype not attributable to the mother is consistent with that of the alleged father. Paternity testing can be performed by analyzing the sets of polymoφhisms in both the alleged father and in his alleged child.

Where the set of polymoφhisms in the child not attributable to the mother does not match the set of the alleged father, it can be concluded (barring experimental error) that the alleged father is not the child's father. Where the set of polymoφhisms in the child not attributable to the mother does match the set of polymoφhisms of the alleged father, a statistical calculation can be performed to determine the probability of a coincidental match.

The probability of parentage exclusion (representing the probability that a random male will have a polymoφhic form at a given polymoφhic site that makes him incompatible as the father) is given by the equation disclosed WO 95/12607: p(exc) = xy(l-xy) where x and y are the population frequencies of alleles A and B of a diallelic polymoφhic site.

At a triallelic site p(exc) = xy(l-xy) + yz(l- yz) + xz(l-xz)+ 3xyz(l- xyz), where x, y and z are the respective population frequencies of alleles A, B, and C. The probability of non-exclusion is: p(non-exc) = l-p(exc).

The cumulative probability of non-exclusion (representing the value obtained when n loci are used) is thus: cum p(non-exc) = p(non-excl)p(non-exc2)p(non-exc3).... p(non-excn). The cumulative probability of exclusion for z loci (representing the probability that a random male will be excluded) cum p(exc) = 1 - cum p(non-exc).

Where several polymoφhic loci are included in the analysis, the cumulative probability of exclusion of a random male is very high. This probability can be taken into account in assessing the liability of an alleged father whose polymoφhic marker set matches the child's polymoφhic marker set attributable to the child's father.

C. CORRELATION of POLYMORPHISMS with PHENOTYPIC TRAITS The polymoφhisms of this invention may contribute to the phenotype of an organism in different ways. As discussed above, some polymoφhisms occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect of such a change to the structure of a protein may be neutral, beneficial, or detrimental, or both beneficial and detrimental. For instance, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. Other polymoφhisms occur in noncoding regions and exert phenotypic effects indirectly via influence on, e.g., replication, transcription, and translation. Moreover, a single polymoφhism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymoφhisms in different genes. Further, some polymoφhisms predispose an individual to a distinct mutation that is causally related to a certain phenotype. Phenotypic traits include diseases that have known (but hitherto unmapped) genetic components (e.g., agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, poly cystic kidney disease, hereditary spherocytosis, von Willebrand's disease, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, osteogenesis imperfecta, and acute intermittent poφhyria and the like). Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is, or may be, genetic, such as autoimmune diseases, inflammation, cancer, diseases of the nervous system, and infection by pathogenic microorganisms. Some examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependent and non-independent), systemic lupus erythematosus, and Graves disease. Some examples of cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, skin, stomach and uterus. Phenotypic traits also include characteristics such as longevity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments.

Correlation of polymoφhisms with phenotypic traits is performed for a population of subjects who have been tested for the presence or absence of a phenotypic trait of interest and for polymoφhic markers sets. To perform such analysis, the presence or absence of a set of polymoφhisms (i.e. a polymoφhic set) is determined for a set of the subjects, some of whom exhibit a particular trait, and some of whom do not. The alleles of each polymoφhism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the phenotypic trait of interest. Correlation can be performed by using standard statistical methods such as a κ-squared test and by noting any statistically significant correlations between the polymoφhic form(s) and the phenotypic characteristics. For example, it might be found that the presence of allele Al at polymoφhism A correlates with heart disease. As a further example, it might be found that the combined presence of allele Al at polymoφhism A and allele Bl at polymoφhism B correlates with increased milk production of a farm animal.

These correlations are useful in several ways. For example, where a strong correlation exists between a set of one or more polymoφhic forms and a disease for which treatment is available, detection of the polymoφhic form set in a subject, i.e., a human being or an animal may justify immediate administration of treatment, or at least the institution of regular monitoring of the subject. Detection of a polymoφhic form correlated with a serious disease can assist in various types of decisions, for example, reproductive decision making. For instance, a female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymoφhism from her male partner to her offspring.

Where a weaker yet statistically significant correlation exists between a polymoφhic set and a human disease, immediate therapeutic intervention or monitoring may not be justified. Nevertheless, the patient can be motivated to begin simple life-style changes (e.g., diet, exercise) that can be accomplished at little cost to the patient but confer potential benefits in reducing the risk of conditions to which the patient may have increased susceptibility by virtue of variant alleles. Identification of a polymoφhic set in a patient correlated with enhanced receptiveness to one of several treatment regimes for a disease indicates that this treatment regime should be followed. For animals and plants, correlations between characteristics and phenotype are useful for e.g., breeding for desired characteristics. For example, Beitz et al., in US 5,292,639, disclose use of bovine mitochondrial polymoφhisms in a breeding program to improve milk production in cows. To evaluate the effect of mitochondrial DNA (mtDNA) displacement loop (D-loop) sequence polymoφhism on milk production, each cow was assigned a value of 1 , where variant, or 0, where wildtype, with respect to a prototypical mtDNA sequence at each of the 17 locations considered. Each production trait was analyzed individually with the following animal model:

Y,_jkpn= μ + YS, + P. + X_k + β, + ... β„ + PE_n + a, +e_p wherein: Y_ljknp is the milk, fat, fat percentage, solids-not-fat (SNF), SNF percentage, energy concentration, or lactation energy record; μ is an overall mean; YS, is the effect common to all cows calving in year-season; X is the effect common to cows in either the high or average selection line; β, to β₁₇ are the binomial regressions of production record on mtDNA D-loop sequence polymoφhisms; PE_n is permanent environmental effect common to all records of cow n; a_ is the effect of animal n and is composed of the additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect; and e_p is a random residual. It was found that eleven of the seventeen polymoφhisms tested influenced at least one production trait. Bovines having the best polymoφhic forms for milk production at these eleven loci are selected to breed the next generation of the herd. D. GENETIC MAPPING of PHENOTYPIC TRAITS

The previous section described the identification of correlations between phenotypic traits and polymoφhisms that directly or indirectly contribute to those traits. The present section describes the identification of a physical linkage between a genetic locus associated with a trait of interest and polymoφhic markers that are not associated with that trait, but rather are in physical proximity with the genetic locus responsible for the trait and co-segregate with it. Such analysis is useful for, e.g., mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning the gene(s) responsible for the trait. (See, e.g., Lander et ah, Proc. Natl. Acad. Sci. (USA) 83, 7353-7357 (1986); Lander et aL, Proc. Natl. Acad. Sci. (USA) 84, 2363-2367 (1987); Donis-Keller et al., Cell 51, 319-337 (1987); Lander et al., Genetics 121, 185-199 (1989)). Genes localized by linkage can be cloned by a process known as directional cloning. (See, e.g., Wainwright, Med. J. Australia 159, 170-174 (1993); and Collins, Nature Genetics 1, 3-6 (1992)).

Linkage studies are generally performed on members of a family. Available members of the family are characterized for the presence or absence of a phenotypic trait and for a set of polymoφhic markers. The distribution of polymoφhic markers in an informative meiosis is then analyzed to determine which polymoφhic markers co-segregate with a phenotypic trait. (See, e.g., Kerem et al., Science 245, 1073-1080 (1989); Monaco et al, Nature 316, 842 (1985); Yamoka et ah, Neurology 40, 222-226 (1990); and Rossiter rt ak, FASEB Journal 5, 21-27 (1991)).

Linkage is analyzed by calculation of log of the odds (LOD) values. A LOD value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction θ, versus the situation in which the two are not linked, and thus segregating independently (See, e.g., Thompson & Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company, Philadelphia, 1991); and Strachan, "Mapping the human genome" in The Human Genome (BIOS Scientific Publishers Ltd, Oxford), Chapter 4). A series of likelihood ratios are calculated at various θ, ranging from θ = 0.0 (coincident loci) to θ = 0.50 (unlinked). Thus, the likelihood at a given value of θ is the probability of data where loci are linked at θ to the probability of data where loci are unlinked. The computed likelihoods are usually expressed as the log_I0 of this ratio (i.e., a LOD value). For example, a LOD value of 3 indicates 1000:1 odds against an apparent observed linkage being a coincidence. The use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of LOD scores for differing values of θ

(e.g., LIPED, MLINK (See e.g., Lathrop, Proc. Nat. Acad. Sci. (USA) 81, 3443-3446 (1984)). For any particular LOD score, a recombination fraction may be determined from mathematical tables. (See, e.g., Smith et al., Mathematical TABLEsfor research workers in human genetics (Churchill, London, 1961); and Smith, Ann. Hum. Genet. 32, 127-150 (1968)). The value of θ at which the LOD score is the highest is considered to be the best estimate of the recombination fraction.

Positive LOD score values suggest that the two loci are linked, whereas negative LOD values suggest that linkage is less likely (at that value of θ) than the possibility that the two loci are unlinked. By convention, a combined LOD value of +3 or greater (equivalent to greater than 1000:1 odds in favor of linkage) is considered definitive evidence that the two loci are linked. Similarly, by convention, a negative LOD value of -2 or less is taken as definitive evidence against linkage of the two loci being compared. Negative linkage data are useful in excluding a chromosome or a segment thereof from consideration. The search then focuses on the remaining non- excluded chromosomal locations.

E. DISEQUILIBRIUM MAPPING OF THE ENTIRE GENOME

Linkage disequilibrium or allelic association is the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. For example, if locus X has alleles a and b, which occur equally frequently, and linked locus Y has alleles c and d, which occur equally frequently, one would expect the combination ac to occur with a frequency of 0.25. If ac occurs more frequently, then alleles a and c are in linkage disequilibrium. Linkage disequilibrium may result from natural selection of certain combination of alleles or because an allele has been introduced into a population too recently to have reached equilibrium with linked alleles.

A marker in linkage disequilibrium can be particularly useful in detecting susceptibility to disease (or other phenotype) notwithstanding that the marker does not cause the disease. For example, a marker (X) that is not itself a causative element of a disease, but which is in linkage disequilibrium with a gene (including regulatory sequences) (Y) that is a causative element of a phenotype, can be detected to indicate susceptibility to the disease in circumstances in which the gene Y may not have been identified or may not be readily detectable.

F. MARKER ASSISTED BREEDING

Genetic markers can decipher the genomes in animals and crop plants. Genetic markers can aid a breeder in the understanding, selecting and managing of the genetic complexity of an agronomic or desirable trait. The agriculture world, for example, has a great deal of incentive to try to produce food with a rising number of desirable traits (high yield, disease resistance, taste, smell, color, texture, etc.) as consumer demand and expectations increase. However, many traits, even when the molecular mechanisms are known, are too difficult or costly to monitor during production. Readibly detectable polymoφhisms which are in close physical proximity to the desired genes can be used as a proxy to determine whether the desired trait is present or not in a particular organism. This provides for an efficient screening tool which can accelerate the selective breeding process.

G. PHARMACOGENOMICS Genetic information can provide a powerful tool for doctors to determine what course of medicine is best for a particular patient. A recent Science paper entitled "Molecular Classification of Cancer: Class Discovery and Class Prediction by Gene Expression Monitoring," (to be published 10/15/99 hereby incoφorated by reference in its entirety for all puφoses) discusses the use of genetic information discovered through the use of arrays to determine the specific type of cancer a particular patient has. The paper goes on to discuss the ways in which particular treatment options can then be tailored for each patient's particular type of cancer. Similar uses of genetic information for treatment plans have been disclosed for patients with HIV. (See US Patent Application 5,861,242).

The pharmaceutical industry is likewise interested in the area of pharmacogenomics. Every year pharmaceutical companies suffer large losses from drugs which fail clinical trials for one reason or another. Some of the most difficult are those drugs which, while being highly effective for a large percentage of the population, prove dangerous or even lethal for a very small percentage of the population. Pharmacogenomics can be used to correlate a specific genotype with specific responses to a drug. The basic idea is to get the right drug to the right patient. If pharmaceutical companies (and later, physicians) can accurately remove from the potential recipient pool those patients who would suffer adverse responses to a particular drug, many research efforts which are currently being dropped by pharmaceutical companies could be resurrected saving hundreds of thousands of dollars for the companies and providing many currently unavailable medications to patients.

Similarly, some medications may be highly effective for only a very small percentage of the population while proving only slightly effective or even ineffective to a large percentage of patients. Pharmacogenomics allows pharamaceutical companies to predict which patients would be the ideal candidate for a particular drug, thereby dramatically reducing failure rates and providing greater incentive to companies to continue to conduct research into those drugs.

IV. MODIFIED POLYPEPTIDES and GENE SEQUENCES This invention further provides variant forms of nucleic acids and corresponding proteins. The nucleic acids comprise one of the sequences described in TABLE 1, column 8, in which the polymoφhic position is occupied by one of the alternative bases for that position. Some nucleic acid encode full-length variant forms of proteins. Similarly, variant proteins have the prototypical amino acid sequences encoded by the nucleic acid sequence shown in TABLE 1, column 8, (read so as to be in-frame with the full-length coding sequence of which it is a component) except at an amino acid encoded by a codon including one of the polymoφhic positions shown in TABLE 1. That position is occupied by the amino acid coded by the corresponding codon in any of the alternative forms shown in TABLE 1.

Variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter. Usually, the promoter is a eukaryotic promoter for expression in a mammalian cell. The transcription regulation sequences typically include a heterologous promoter and optionally an enhancer that is recognized by the host. The selection of a promoter, for example tφ, lac, phage, glycolytic enzyme and tRNA promoters, depends, in part, on the host selected. Commercially available expression vectors can be used in this invention. Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.

The means of introducing the expression construct into a host cell varies depending upon, for example, the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described, for example, in Sambrook, supra. A wide variety of prokaryotic and eukaryotic host cells can be employed for expression of the variant gene. Suitable host cells include, for example, bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines, as well as derivatives thereof. Preferred host cells are able to process the variant gene product to produce the mature polypeptide, where processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like.

The protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i. e. , 80, 95 or 99% free of cell component contaminants. (See, e.g., Jacoby, Methods in Enzymology Volume 104, Academic Press, New York (1984); Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer- Verlag, New York (1987); and Deutscher (ed), Guide to Protein Purification, Methods in Enzymology, Vol. 182 (1990)). Where the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. Where the protein is not secreted, the protein can be isolated from a lysate of the host cells.

This invention further provides transgenic nonhuman animals capable of expressing an exogenous variant gene and/or having one or both alleles of an endogenous variant gene inactivated. Expression of an exogenous variant gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinjecting the construct into a zygote. (See, e.g., Hogan et al., "Manipulating the Mouse Embryo, A Laboratory Manual," Cold Spring Harbor Laboratory.) Inactivation of endogenous variant genes can be achieved by forming a transgene in which a cloned variant gene is inactivated by insertion of a positive selection marker. (See, e.g., Capecchi, Science 244, 1288-1292 (1989)). The transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous variant gene. Mice and other rodents are preferred animals. Such animals provide useful drug screening systems.

In addition to substantially full-length polypeptides expressed by variant genes, this present invention also includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides. Biologically active fragments include any portion of the full-length polypeptide that confers a biological function on the variant gene product, including ligand and antibody binding. Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.

Polyclonal and/or monoclonal antibodies that specifically bind to variant gene products but not to corresponding prototypical gene products are also provided by this invention. Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof.

Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); and Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.

V. KITS This invention further provides kits comprising at least one allele- specific oligonucleotide as described above. Preferably, the kits contain one or more pairs of allele-specific oligonucleotides hybridizing to different forms of a polymoφhism. In some kits, the allele-specific oligonucleotides are provided immobilized to a substrate. For example, the same substrate can comprise allele- specific oligonucleotide probes for detecting at least 10, 100 or all of the polymoφhisms shown in TABLE 1. Optional additional components of the kit include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin- enzyme conjugate and enzyme substrate and chromogen where the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions. Generally, the kit also contains instructions for carrying out the methods.

EXAMPLES The polymoφhisms shown in TABLE 1 were identified by resequencing of target sequences from eight unrelated individuals of diverse ethnic and geographic backgrounds by hybridization to probes immobilized to microfabricated arrays. The strategy and principles for design and use of such arrays are generally described in WO 95/11995. The strategy provides arrays of probes for analysis of target sequences showing a high degree of sequence identity to the reference sequences of the fragments shown in TABLE 1, column 1. The reference sequences were sequence-tagged sites (STSs) developed in the course of the Human Genome Project (see, e.g., Science 270, 1945-1954 (1995); Nature 380, 152-154 (1996)). Most STS's ranged from 100 base pair (bp) to 300 bp in size.

A typical probe array used in this analysis has two groups of four sets of probes that respectively tile both strands of a reference sequence. A first probe set comprises a plurality of probes exhibiting perfect complementarily with one of the reference sequences. Each probe in the first probe set has an interrogation position that corresponds to a nucleotide in the reference sequence. That is, the interrogation position is aligned with the corresponding nucleotide in the reference sequence, where the probe and reference sequence are aligned to maximize complementarity between the two. For each probe in the first set, there are three corresponding probes from three additional probe sets. Thus, there are four probes corresponding to each nucleotide in the reference sequence. The probes from these three additional probe sets are identical to the corresponding probe from the first probe set except at the interrogation position, which occurs in the same position in each of the four corresponding probes from the four probe sets, and is occupied by a different nucleotide in the four probe sets. In the present analysis, probes were 25 nucleotides long. Arrays tiled for multiple different reference sequences were included on the same substrate.

Multiple target sequences from an individual were amplified from human genomic DNA using primers for the fragments. The amplified target sequences were fluorescently labeled during or after PCR. The labeled target sequences were hybridized with a substrate bearing immobilized arrays of probes. The amount of label bound to the probes was measured. Analysis of the pattern of label revealed the nature and position of differences between the target and reference sequence. For example, comparison of the intensities of four corresponding probes reveals the identity of a corresponding nucleotide in the target sequences aligned with the interrogation position of the probes. The corresponding nucleotide is the complement of the nucleotide occupying the interrogation position of the probe showing the highest intensity. (See, e.g., WO 95/11995). The existence of a polymoφhism is also manifested by differences in normalized hybridization intensities of probes flanking the polymoφhism where the probes hybridized to corresponding targets from different individuals. For example, relative loss of hybridization intensity in a "footprint" of probes flanking a polymoφhism signals a difference between the target and the reference sequence (i.e., a polymoφhism) (See, e.g., EP 717,113). Additionally, hybridization intensities for corresponding targets from different individuals can be classified into groups or clusters suggested by the data, not defined a priori, such that isolates in a give cluster tend to be similar and isolates in different clusters tend to be dissimilar. See US Patent Application No. 08/797,812 filed February 7, 1997. Hybridizations to samples from different individuals were performed separately. TABLE 1 summarizes the data obtained for target sequences in comparison with a reference sequence for the eight individuals tested.

From the foregoing, it is clear that the invention includes a number of general uses that can be expressed concisely as follows. This invention provides for the use of any of the nucleic acid segments described above in the diagnosis or monitoring of diseases, such as cancer, inflammation, heart disease, diseases of the CNS, and susceptibility to infection by microorganisms. This invention further provides for the use of any of the nucleic acid segments in the manufacture of a medicament for the treatment (includes, inter alia, preventative (e.g., prophylactic), palliative and curative treatment) of such diseases. This invention further provides for the use of any of the DNA segments as a pharmaceutical in any suitable dosage form. The present invention is described in full, clear, concise and exact terms to enable those skilled in the art to make and use this invention. IN addition, EXAMPLES and illustrations are provided albeit solely for the puφoses of even more clarity and understanding and, as such, do not limit this invention which is defined by the appendant claims. It will also be understood that certain changes and modifications that may be practiced are within the scope of the appendant claims.

Claims

WHAT IS CLAIMED IS:

1 A nucleic acid segment of between 10 and 100 bases selected from a fragment shown in TABLE 1 , wherein said segment comprises a polymoφhic site, or the complement of said segment.

2. The segment of claim 1 that is DNA.

3. The segment of claim 1 that is RNA.

4 The segment of claim 1 that is less than 50 bases.

5. The segment of claim 1 that is less than 20 bases.

6. The segment of claim 1 , wherein the fragment is Z95152 and the polymoφhic site is at position 34953.

7. The segment of claim 1, wherein the polymoφhic site is diallelic.

8. The segment of claim 1, wherein the polymoφhic form occupying the polymoφhic site is the reference base for the fragment listed in TABLE 1, column 4.

9. The segment of claim 1 , wherein the polymoφhic form occupying the polymoφhic site is an alternative form for the fragment listed in TABLE 1, column 5.

10. An allele-specific oligonucleotide that hybridizes to a segment of a fragment shown in TABLE 1 , column 7 or its complement.

11. The allele-specific oligonucleotide of claim 10 that is probe.

12. The allele-specific oligonucleotide of claim 10, wherein a central position of the probe aligns with the polymoφhic site of the fragment.

13 The allele-specific oligonucleotide of claim 10 that is a primer.

14. The allele-specific oligonucleotide of claim 13, wherein the 3' end of the primer aligns with the polymoφhic site of the fragment.

15. An isolated nucleic acid comprising a sequence of TABLE 1, column 7 or the complement thereof, wherein the polymoφhic site within the sequence or complement is occupied by a base other than the reference base show in TABLE 1 , column 4.

16. A method of analyzing a nucleic acid, comprising: obtaining the nucleic acid from an individual; and determining a base occupying any one of the polymoφhic sites shown in TABLE 1.

17. The method of claim 16, wherein the determining comprises determining a set of bases occupying a set of the polymoφhic sites shown in TABLE 1.

18. The method of claim 16, wherein the nucleic acid is obtained from a plurality of individuals, and a base occupying one of the polymoφhic positions is determined in each of the individuals, and the method further comprising testing each individual for the presence of a disease phenotype, and correlating the presence of the disease phenotype with the base.

19. The nucleic acid of claim 15 that is DNA.

20. The nucleic acid of claim 15 that is RNA.

21. The nucleic acid of claim 15 that is less than 50 bases.

22. The nucleic acid of claim 15 that is less than 20 bases.

23. The nucleic acid of claim 15, wherein the fragment is Z95152 and the polymoφhic site is at position 34953.

24. The nucleic acid of claim 15, wherein the polymoφhic site is diallelic.

25. The nucleic acid of claim 15, wherein the base occupying the polymoφhic site is the correlating base for the fragment listed in TABLE 1 , column 4.

26. The nucleic acid of claim 15, wherein the base occupying the polymoφhic site is the correlating base for the fragment listed in TABLE 1 , column 5.

27. The segment of claim 1, wherein said fragment is selected from TABLE 1, Seq ID Nos.1-10.

28. The segment of claim 1 , wherein said fragment is selected from TABLE 1, Seq ID Nos. 11-20.

29. The segment of claim 1, wherein said fragment is selected from TABLE 1, Seq ID Nos.21-30.

30. The segment of claim 1 , wherein said fragment is selected from TABLE 1, SEQ ID NOS.31-40.

31. The segment of claim 1 , wherein said fragment is selected from TABLE , SEQ ID NOS:41-50.

32. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:51-60.

33. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:61-70.

34. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:71-80.

35. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:81-90.

36. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:91-100.

37. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:101-110.

38. The segment of claim 1 , wherein said fragment is selected from TABLE , SEQ ID NOS:l 11-120.

39. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:121-130.

40. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:131-140.

41. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:141-150.

42. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS: 151-160.

43. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:161-170.

44. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:171-180.

45. The segment of claim 1, wherein said fragment is selected from TABLE , SEQ ID NOS:181-190.

46. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS: 191-200.

47. The segment of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS: 1-10.

48. The segment of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS: 11-20.

49. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS: 21-30.

50. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:31-40.

51. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS.41-50.

52. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:51-60.

53. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS :61-70.

54. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS :71-80.

55. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:81-90.

56. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:91-100.

57. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:101-110.

58. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:l 11-120.

59. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:121-130.

60. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:131-140.

61. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:141-150.

62. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:151-160.

63. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:161-170.

64. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:171-180.

65. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:181-190.

66. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:191-200.

67. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:201-210.

68. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS :211-220.

69. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:221-230.

70. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS:231-240.

71. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS.241-250.

72. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1, SEQ ID NOS: 151-260.

73. The nucleic acid of claim 15, wherein said fragment is selected from TABLE 1 , SEQ ID NOS : 161 -265.

74. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:201-210.

75. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:211-220.

76. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:221-230.

77. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS.231-240.

78. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:241-250.

79. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS :251-260.

80. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS.261-265.

81. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:266-275.

82. The segment of claim 1, wherein said fragment is selected from TABLE l, SEQ ID NOS:276-285.

83. The segment of claim 1, wherein said fragment is selected from TABLE l, SEQ ID NOS:286-295.

84. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:296-305.

85. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS.306-315.

86. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:316-325.

87. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:326-335.

88. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:336-345.

89. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:346-355.

90. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:356-365.

91. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:366-375.

92. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:376-385.

93. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:386-395.

94. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS:396-405.

95. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS.406-415.

96. The segment of claim 1, wherein said fragment is selected from TABLE 1, SEQ ID NOS :416-423.