MXPA97001549A - Detection of sequences of nucleic acids, hydrochlortable in teji - Google Patents

Abstract

The present invention provides an assay for detection of cellular proliferative disorder in mammals, associated with hypermutable nucleic acid sequences. The identification of particular hypermutable sequences such as microsatellite sites correlates with a particular cancer, thereby allowing the detection of both primary tumors and metastatic sites in a patient.

Description

DETECTION OF SEQUENCES OF NUCLEIC ACIDS H I PERMUTABLE IN TISSUES BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to the detection of an objective nucleic acid sequence, and specifically to the detection of a cell proliferative disorder associated with a hypermutable nucleic acid sequence in a sample. 2. Description of the Related Art Mammalian genomes consist of unique DNA sequences interspersed with moderately and highly repetitive DNA sequences. Traditionally genetic mapping has been performed by meiotic linkage using variations in single sequence DNA, such as restriction fragment length polymorphisms (Botstein et al., Am. J. Hum. Genet., 3.2: 314-331, 1980), as genetic markers It has recently been discovered that variations in repetitive sequence elements, such as variable number row repetition sequences (VNTR) or in minisatellite (Jeffreys et al., Nature, 314: 67-73, 1985;? A amura et al. , Science, 235: 1616-1622, 1987), and single variable sequence motifs (VSSM) or microsatellite motifs (Litt and Luty, Am. J. Hum.

Genet 44: 397-401, 1989; Weber and May, Am. J. Hum. Genet 44: 388-396, 1989), are useful for link studies. An advantage of the use of repetitive sequence variations instead of single sequence variations is the apparently greater number of alleles present in normal populations when compared to restriction fragment length polymorphisms (RFLP). A second advantage is the ability to easily detect variations in sequence length using the polymerase chain reaction to facilitate the rapid and economical analysis of large numbers of DNA samples. Microsatellite elements consist of simple sequences of mono-, di-, or tri-nucleotides, where the alleles differ by one or more repeating units (Luty et al., Am. J. Hum. Genet., 46: 776- 783, 1990; Tautz and collaborators, Nature, 322: 652-656, 1986; Weber and May, Am. J. Hum. Genet., 4_4: 388-396, 1989). The minisatellites, or variable number row repeat sequences, typically have a repeating unit of 20 to several hundred nucleotides, and the alleles differ by as little as one repeating unit. Among the simple sequences, it has recently been found that repeating elements (TG) n or (CA) n are extremely useful for meiotic mapping, because: (1) they are abundant in the genome, (2), they exhibit a large number of different alleles, and (3) can be assayed rapidly using the polymerase chain reaction (Litt and Luty, Am. J. Hum. Genet., 44: 397-401, 1989; Weber and May, Am. J. Hum. Genet , 44: 388-396, 1989). A number of other short sequence motifs have been discovered in mammalian genomes (Hellman et al., Gene, 6_8: 93-100, 1988; Knott et al., Nuc Acids Res., 14: 9215-9216; Litt and Luty, Am. J. Hum. Genet., 44: 397-401, 1989, Milstein et al., Nuc.Acids Res., 12: 6523-6535, 1984, Stoker et al., Nuc Acids Res., 13: 4613-4621. , 1985, Vassart et al, Science, 23_3: 683-684, 1987, and Vernaud, Nuc Acids Res. 17: 7623-7630, 1989), and in bird genomes (Gyllensten et al., Nuc. Acids Res. , 17: 2203-2214, 1989; Longmire et al., Genomics, 2: 14-24, 1988), and are thought to accumulate by the sliding of the AD? during replication (Tautz et al., Nature, 322 .: 652-656, 1986), or by unequal recombination events (Wolff et al., Genomics, 5: 382-384, 1989). Many of these repeating elements exhibit a high degree of genetic variation, and therefore, are also useful for meiotic and mitotic mapping. The row variable repeat sequence isolated by Jeffreys (supra) contains an invariant core sequence GGGCAGGAXG which has some similarities to the chi sequence of the phambu phage (Wolff et al., Genomics, 5: 382-384, 1989), and is detected by a restriction fragment of the bacteriophage M13 (Vassart et al., Science, 231: 683-684, 1987). ? akamura et al. (Science, 235: 1616-1622, 1987) have detected similar repeating elements, and contain a similar, but distinctive, common core unit, GGGGTGGGG. Elements of this type are presented within several known genetic sequences, including the place of ß-globin. Variable row repeat elements of similar variable number have been described within apolipoprotein B (Boerwinkle et al., Proc. Nati, Acad. Sci. USA, 86: 212-216, 1989; Knott et al., Nuc. Acids Res. , 14: 9215-9216, 1986), and in type II collagen genes (Stoker et al., Nuc Acids Res., 13: 4613-4621, 1985), and contain a different AT-rich motif. Although a physiological function has not been defined for repetitive elements of this type, they have been suggested as potential immediate hosts for chromosomal recombination (DeBustros et al., Proc. Nati, Acad. Sci. USA, 85: 5693-5697, 1988) , or as important elements for the control of gene expression (Hellman et al., Gene, 68: 93-100, 1988; Milstein et al., Nuc.Aids Res., 12: 6523-6535, 1984). The microsatellites represent a very common and highly polymorphic class of genetic elements in the human genome. The microsatellite markers containing repeat sequences have been used for primary genetic mapping and for linkage analysis, as described (Weber et al., Am. J. Hum. Genet., 44.:388, 1989). The amplification by polymerase chain reaction of these repeats allows a rapid evaluation of the loss of heterozygosity (LOH), and can greatly simplify the procedures for the mapping of tumor suppressor genes (Ruppert et al., Cancer Res. 53. : 5093, 1993; van der Riet et al., Cancer Res., 54: 1156, 1994). More recently, microsatellites have been used to identify specific mutations in certain inherited disrs, including Huntington's disease (HD), fragile X chromosome (FX), myotonic dystrophy (MD), spinocerebellar ataxia type 1 (SCA1), muscular dystrophy. spinobulbar (SBMA), and hereditary dentatorrubralpalidoluisiana atrophy (DRPLA) (The Huntington 's Disease Collaborative Research Group., Cell. 72: 971, 1993; EJ Kremer et al., Science, 252: 1711, 1991; G. Imbert et al. , Nature Genet., 4:72, 1993; HT Orr et al., Nature Genet., 4: 221, 1993; V. Biancalana et al., Hum. Mol. Genet., 1: 255, 1992; MY Chung et al., Nature Genet., 5: 254, 1993; R. Koide et al., Nature Genet., 6: 9, 1994). Recently, microsatellite instability in human cancers has been described. For example, it has been reported that microsatellite instability is an important feature of tumors of patients with colorectal carcinoma without hereditary polyposis (H? PCC) (Peltomáki et al., Science, 260: 810, 1993; Aaltonen et al. Science, 260: 812, 1993; Thibodeau et al., Science, 260: 816, 1993). Furthermore, microsatellite instability, demonstrated by the expansion or suppression of repeating elements, has been reported in neoplastic colorectal, endometrial, breast, gastric, pancreatic, and bladder tissues (JI Risinger et al., Cancer Res. , 53_: 5100, 1993, HJ Han et al, Cancer Res., 53: 5087, 1993, P. Peltomáki et al, Cancer Res., 53: 5853, 1993, M. Gonzalez-Zulueta et al., Cancer Res. , 53_: 5620, 1993), and recently in (small cell lung carcinoma (SCLC).) In patients with colorectal carcinoma without hereditary polyposis, this genetic instability is due to inherited and somatic mutations of a mismatch repair gene (hMSH-2) Mutations of hMLH-1 and other repair genes of poor critical coupling may also be responsible for the instability detected in patients with colorectal carcinoma without hereditary polyposis. Cer continues to be a major cause of mortality worldwide, and despite advances in diagnosis and treatment, the overall survival rate has not improved significantly in the past twenty years. There continues to be an unmet need for a more sensitive medium for early diagnosis. Typical assays to detect rare infiltration tumor cells in clinical samples use amplification methods that require additional cloning steps and the synthesis of a large number of oligomer-specific probes to detect a wide variety of oncogenic mutations for each type of tumor (Sidransky et al., Science, 252: 706, 1991; Sidransky et al., Science, 256: 102, 1992). The present invention provides an effective assay for detecting a variety of cancers using hypermutable microsatellite markers and an amplification strategy that eliminates the need for additional cloning steps. SUMMARY OF THE INVENTION The present invention provides a rapid, reliable, and effective screening method for the detection of a cell proliferative disorder in different clinical samples. The invention uses amplification and detection of microsatellite nucleic acid (small repeat sequences) to detect a clonal population of cells in a clinical sample. The invention is based on the unexpected discovery that microsatellite alterations are detectable as a clonal population of cells in the DNA of clinical cytological samples. These samples include urine, sputum, and histopathological margins obtained from cancer patients. The invention provides a method for detecting a mammalian cell proliferative disorder (ie, neoplasia) associated with a hypermutable mammalian target nucleic acid in a sample, which comprises isolating the nucleic acid present in the sample, and detecting the presence or the absence (eg, loss of heterozygosity, LOH) of the hypermutable target nucleic acid, typically following amplification of the nucleic acid. In one embodiment, the amplification step in the method of the invention is performed as a multiplex reaction. Therefore, instead of performing multiple amplification reactions to identify each clonal alteration, primers for different markers are combined in a simple amplification reaction, improving the identification of a large proportion of cell proliferative disorders. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a denaturing DNA acrylamide normal (N) and tumoral (T) DNA of lane 1, B17 (TCC) (FGA marker); lane 2, L21 (SCLC) (marker AR); lane 3, B30 (TCC) (marker UT762); and lane 4, L5 (SCLG) (marker D14S50). Figure 2 shows a denaturing gel of acrylamide from panel A), a urine sample (U) of B27 (TCC) analyzed with FGA marker and compared with normal (N) and tumoral (T) tissue DNA; panel B), histological margin (M) of L31 (NSCLC) analyzed with RA and compared with tumor tissue; and panel C), sputum sample (S) of L25 (SCLC) analyzed with CHRNB and compared with normal and tumor tissue DNA. Figure 3 shows lymphocyte (N) DNA from patients, amplified with FGA marker in varying dilutions with tumor DNA in 1 in 5 (20%) to 1 in 1,000 (0.1%). The novel bands are indicated by an arrow. Panel A, B17 tumor DNA (TCC); panel B, B27 tumor DNA (TCC).

Figure 4 shows a multiplex PCR assay using B27 DNA (TCC) and a corresponding urine sample amplified with three different primordial sets (FGA, ACTBP2, AR) in the same PCR reaction (N: normal DNA; T: tumor DNA; U: urine DNA). Figure 5 shows the results of studies in 25 patients having a confirmed diagnosis of cancer through pathology. Blood samples (B), tumor (T) and urine (U) were compared for detection of microsatellite alterations. Detailed Description of the Invention The present invention relates to a method for detecting a cell proliferative disorder associated with an objective nucleic acid having a hypermutable nucleotide sequence. Preferably, the hypermutable nucleotide sequence of the invention is a DNA sequence in microsatellite. The microsatellite alterations in different cancer lesions, for example, can be detected by using known microsatellite repeat markers. A combination of repeating markers may be used in the method of the invention to identify a large percentage of cell proliferative disorders or neoplasms, even when the neoplastic cells comprise only a small fraction of the clinical sample. As used herein, the term "hypermutable" refers to a nucleic acid sequence that is susceptible to instability, thereby resulting in alterations of the nucleic acid. These alterations include the deletion and addition of nucleotides. The hypermutable sequences of the invention are preferably microsatellite DNA sequences which, by definition, are small repeating DNA sequences in a row. DNA markers are used in microsatellite to detect hypermutable sequences, as well as the loss of heterozygosity (LOH) and genomic instability. The hypermutable nucleic acid can be a sequence of neoplastic nucleic acids. The term "neoplastic" nucleic acid refers to a nucleic acid sequence that is directly or indirectly associated with, or causes, a neoplasm. The method of the invention is applicable to the detection of hypermutable nucleotide sequences associated with benign as well as malignant tumors. The method can be used to detect any hypermutable nucleotide sequence, regardless of origin, provided that the sequence is detectably present in a sample. The sample may be blood, urine, sputum, vilis, stool, cervical culture seed, saliva, tears, cerebrospinal fluid, regional lymph node, and histopathological margins, and body fluid draining a cavity or organ from the body. For example, regional lymph node neoplasm associated with a primary breast tumor can be detected using the method of the invention. The term "regional lymph node" refers to the lymphoid tissue that forms the lymphoid organs or nodes that are in close proximity to the primary tumor. For example, regional lymph nodes, in the case of carcinomas of the head and neck, include cervical lymph nodes, prelaryngeal lymph nodes, juxta-phage lung lymph nodes, and submandibular lymph nodes. Regional lymph nodes for mammary tissue carcinomas include axillary and intercostal nodules. The term "external to a primary neoplasm" means that the sample is taken from a different site than directly from the primary neoplasm itself. This sample may be useful to evaluate whether metastasis of the primary neoplasm has occurred. The method can also be used to detect a hypermutable nucleic acid sequence associated with a primary tumor, by assaying the surrounding tumor margin. As used herein, the term "tumor margin" refers to tissue that is around a discernible tumor. In the case of surgical removal of a solid tumor, the tumor margin is the tissue cut with the discernible tumor that normally seems to be normal to the naked eye. In a more particular way, as used herein, "margin" refers to the edge, border, or boundary of a tumor. The margin usually extends from approximately 0.2 centimeters to approximately 3 centimeters from the primary tumor, but may be greater, depending on the size of the primary solid tumor.

In its broadest sense, the present invention allows the detection of any objectively hypermutable nucleic acid sequence of diagnostic or therapeutic relevance, wherein the objective nucleic acid sequence is present in a tissue sample. The objective nucleotide sequence may be, for example, a restriction fragment length polymorphism (RFLP), a nucleotide deletion, a nucleotide addition, or any mammalian nucleic acid sequence of interest in those tissue samples. Preferably, the microsatellite hypermutable nucleic acid of the invention contains deletions or additions of nucleic acids. The most preferred hypermutable microsatellites in the method of the invention comprise the sequence (X) n, wherein X is the number of nucleotides in the repeat sequence, and is greater than, or equal to, 1, preferably greater than, or equal to 2, and more preferably greater than, or equal to 3, and wherein n is the number of repetitions, and is greater than, or equal to 2, and preferably from 4 to 6. Preferably, when X is 2 , the nucleotide sequence is TC. Preferably, when X is 3, the nucleotide sequence is selected from AGC, TCC, CAG, CAA, and CTG. Preferably, when X is 4, the nucleotide sequence is selected from AAAG, AGAT, and TCTT. The hypermutable nucleic acid sequence is preferably associated with a known site. For example, alterations of the hyperchangeable microsatellite can be detected using a marker selected from ARA (chromosome X), D14S50 (chromosome 14), AR (chromosome X), MD (chromosome 19), SAT (chromosome 6), DRPLA (chromosome 12), ACTBP2 (chromosome 6), FGA (chromosome 4), D4S243 (chromosome 4), and UT762 (chromosome 21). Repetition sequences in row have been identified as associated with Huntington's disease (HD), with fragile X syndrome (FX), with myotonic dystrophy (MD), with spinocerebellar ataxia type 1 (SCA1), spinobulbar muscular dystrophy, and hereditary dentatorrubralpalidoluisiana atrophy (DRPLA). When the nucleotide sequence of X is larger, it is more likely that the microsatellite site has alterations, for example, it is more possible that a trinucleotide repeat has deletions or additions than a repeat of dinucleotides. Accordingly, in the present invention, it was found that 8 percent of trinucleotide or tetranucleotide markers exhibited microsatellite alterations per 100 tumor samples examined, whereas only 0.7 percent of 83 dinucleotide microsatellite markers contained alterations. In addition, it was found that a regular repeat, such as AAT AAT AAT is more likely to be hyperchangeable than a sequence containing interruptions in the repeat sequence, eg, AAT GAC AAT AAT. Accordingly, ordinary hypermutable nucleic acid sequences can be readily identified by ordinary experts in the field, by considering the size of the candidate sequence, and if the sequence is uninterrupted, without resorting to undue experimentation. Other microsatellite markers will be known by the criteria described herein, and are accessible to those skilled in the art. The smaller microsatellite markers, which include the dinucleotide and mononucleotide repeats, can be hypermutable and useful for this analysis. The present invention identifies hypermutable target sequences, preferably microsatellite sites, which are unique to a particular cellular proliferative disorder, primary tumor, or metastatic sites derived from the primary tumor. In the tumor cell, the hypermutable nucleotide sequence is evidenced by deletions of nucleic acids, or by the expansion of repeat sequences, compared to a normal cell; therefore, it is possible to design appropriate diagnostic techniques directed to the specific sequence, and design therapeutic strategies once the sequence is identified. The term "cell proliferative disorder" denotes benign as well as malignant cell populations that often appear to differ morphologically from the surrounding tissue. For example, the method of the invention is useful for detecting malignancies of different organ systems, such as, for example, lung, breast, lymphoid, gastrointestinal, and genitourinary tract, as well as epithelial carcinomas that include malignancies, such as such as most cancers of the colon, renal cell carcinoma, prostate cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the head and neck, stomach cancer, cancer of the bladder, kidney cancer , cervical cancer, cancer of the esophagus, and any other type of organ that has a fluid that is drained or tissue accessible for analysis. The method of the invention is also useful for detecting non-malignant cell proliferative diseases such as colon adenomas, hyperplasty, dysplasia, and other "pre-malignant" lesions. Essentially, any disorder that is etiologically linked to a hyperchangeable microsatellite site would be considered susceptible to detection. When it is desired to amplify the objective nucleotide sequence prior to detection, such as a hypermutable nucleotide sequence, this can be done using oligonucleotides that are primers for amplification. Oligonucleotide primers are designed based on the identification of the nucleic acid sequence of the flanking regions contiguous to the hypermutable nucleotide sequence. For example, in the case of hypermutable microsatellite nucleic acid sequences, the oligonucleotide primers comprise sequences that are capable of hybridizing to the nucleotide sequences flanking the sites of the mutations, such as the following nucleotide sequences: a. 5 '-CTT CTG TCC CGG CGT CTG-3' (SEQ ID NO: 1); b. 5'-C AGC CCA GCA GGA CCA GTA-3 '(SEQ ID NO: 2); c. 5 '-TGG TAA CAG TGG AAT ACT GAC-3' (SEQ ID NO: 3); d. 5 '-ACT GAT GCA AAA ATC CTC AAC-3' (SEQ ID NO: 4); e. 5'-GA TGG GCA AAC TGC AGG CCT GGG AAG-3 '(SEQ ID NO: 5); 5 '-GCT ACA AGG ACC CTT CGA GCC CCG TTC-3' (SEQ ID NO: 6); g 5 '-GAT GGT GAT GTG TTG AGA CTG GTG-3' (SEQ ID NO: 7); h 5 '-GAG CAT TTC CCC ACC CAC TGG AGG-3' (SEQ ID NO: 8); 5 '-GTT CTG GAT CAC TTC GCG GA-3' (SEQ ID NO: 9); 3 5 '-TGA GGA TGG TTC TCC CCA AG-3' (SEQ ID NO: 10); k 5 '-AGT GGT GAA TTA GGG GTG TT-3' (SEQ ID NO: 11); 1 5 '-CTG CCA TCT TGT GGA ATC AT-3' (SEQ ID NO: 12); m 5 '-CTG TGA GTT CAA AAC CTA TGG-3' (SEQ ID NO: 13); n 5 '-GTG TCA GAG GAT CTG AGA AG-3' (SEQ ID NO: 14); or 5 '-GCA CGC TCT GGA ACA GAT TCT GGA-3' (SEQ ID NO: 15); P 5 '-ATG AGG AAC AGC AAC CTT CAC AGC-3' (SEQ ID NO: 16); q 5 '-TCA CTC TTG TCG CCC AGA TT-3' (SEQ ID NO: 17); r 5 '-TAT AGC GGT AGG GGA GAT GT-3' (SEQ ID NO: 18); 5 '-TGC AAG GAG AAA GAG AGA CTG A-3' (SEQ ID NO: 19); t 5 '-AAC AGG ACC ACA GGC TCC TA-3' (SEQ ID NO: 20); and u complementary sequences for the sequences a to t. The primers that hybridize in these flanking sequences are, for example, the following: a. 5 '-CAG ACG CCG GGA CAC AAG-3' (SEQ ID NO: 21); b. 5 '-TAC TGG TCC TGC TGG GCT G-3' (SEQ ID NO: 22); c. 5 '-GTC AGT ATT ACC CTG TTA CCA-3' (SEQ ID NO: 23); d. 5 '-GTT GAG GAT TTT TGC ATC AGT-3' (SEQ ID NO: 24); 5 '-CTT CCC AGG CCT GCA GTT TGC CCA TC-3' (SEQ ID NO: 25); 5 '-GAA CGG GGC TCG AAG GGT CCT TGT AGC-3' (SEQ ID NO: 26); 5 '-CAC CAG TCT CAA CAC ATC ACC ATC-3' (SEQ ID NO: 27); 5 '-CCT CCA GTG GGT GGG GAA ATG CTC-3' (SEQ ID NO: 28); 5 '-TCC GCG AAG TGA TCC AGA AC-3' (SEQ ID NO: 29); 5 '-CTT GGG GAG AAC CAT CCT CA-3' (SEQ ID NO: 30); 5 '-AAC ACC CCT AAT TCA CCA CT-3' (SEQ ID NO: 31); 5 '-ATG ATT CCA CAA GAT GGC AG-3' (SEQ ID NO: 32); 5 '-CCA TAG GTT TTG AAC TCA CAG-3' (SEQ ID NO: 33); 5 '-CTT CTC AGA TCC TCT GAC AC-3' (SEQ ID NO: 34); 5 '-TCC AGA ATC TGT TCC AGA GCG TGC-3' (SEQ ID NO: 35); 5 '-GCT GTG AAG GTT GCT GTT CCT CAT-3' (SEQ ID NO: 36); 5 '-AAT CTG GGC GAC AAG AGT GA-3' (SEQ ID NO: 37); 5'ACÁ TCT CCC CTA CCG CTA TA-3 '(SEQ ID NO: 38); 5 '-TCA GTC TCT CTT TCT CCT TGC A-3' (SEQ ID NO: 39); 5 '-TAG GAG CCT GTG GTC CTG TT-3' (SEQ ID NO: 40); and complementary sequences for the sequences a to t. One skilled in the art will be able to generate suitable primers to amplify additional nucleic acid target sequences, such as those flanking the locations of known microsatellite sequences, using the routine skills known in the art, and the teachings of this invention. In general, the primers used according to the method of the invention, comprise oligonucleotides of sufficient length and of an appropriate sequence, which provide a specific initiation of the polymerization of a significant number of nucleic acid molecules containing the nucleic acid. objective, under the conditions of stringency for the reaction using the primers. In this way, it is possible to selectively amplify the specific objective nucleic acid sequence containing the nucleic acid of interest. Specifically, the term "primer", as used herein, refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least eight, whose sequence is capable of initiating the synthesis of a primer extension product that is substantially complementary to an objective nucleic acid chain. The oligonucleotide primer typically contains 15 to 22 or more nucleotides, although it may contain fewer nucleotides, as long as the primer is of sufficient specificity to allow essentially only the amplification of the specifically desired objective nucleotide sequence (i.e., the primer is substantially complementary). The experimental conditions that lead to the synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH. The primer is preferably single chain for maximum amplification efficiency, but can be double stranded. If it is double-stranded, the primer is first treated to separate its chains before being used to prepare the extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of the extension products in the presence of the induction agent for polymerization. The exact length of the primer will depend on many factors, including the temperature, the pH regulator, and the composition of the nucleotide. The primers used according to the method of the invention are designed to be "substantially" complementary to each strand of mutant nucleotide sequence to be amplified. Substantially complementary means that the primers must be sufficiently complementary to hybridize with their respective chains under conditions that allow the agent to polymerize. In other words, the primers must have sufficient complementarity with the flanking sequences to hybridize with, and allow amplification of the nucleotide sequence. Preferably, the 3 'terminus of the extending primer has a perfect complementarity in base pairs with the complementary flanking chain. The oligonucleotide primers used in accordance with the invention are used in any amplification process that produces larger amounts of the target nucleic acid. Typically, one primer is complementary to the negative (-) chain of the mutant nucleotide sequence, and the other is complementary to the positive (+) chain. Tempering the primers with the denatured nucleic acid, followed by extension with an enzyme, such as the large fragment of DNA Polymerase I (Klenow) or Taq DNA polymerase, and the nucleotides or ligases, results in newly synthesized chains (+) and (-), which contain the target nucleic acid. Because these newly synthesized nucleic acids are also templates, repeated cycles of denaturation, priming of the primer, and extension, result in an exponential production of the region (ie, the objective hypermutable nucleotide sequence) defined by the primer. The product of the amplification reaction is a separate nucleic acid duplex, with terms corresponding to the ends of the specific primers employed. Those skilled in the art will know other amplification methodologies that can also be used to increase the number of copies of the target nucleic acid.

Oligonucleotide primers for use in the invention can be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods, or automated embodiments thereof. In an automated mode, diethylphosphoramidites are used as starting materials, and can be synthesized as described by Beaucage et al. (Tetrahedron Letters, 22: 1859-1862, 1981). A method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066. One method of amplification that can be used in accordance with this invention is the polymerase chain reaction (PCR) described in U.S. Patent Nos. 4,683,202 and 4,683,195. The nucleic acid of any sample, in a purified or unpurified form, can be used as the starting acid or nucleic acids, provided that it contains, or is suspected to contain, the specific nucleic acid sequence containing the nucleic acid. objective. Accordingly, the process can employ, for example, DNA or RNA, including messenger RNA (mRNA), wherein the DNA or RNA can be single-stranded or double-stranded. In the case that RNA is going to be used as a template, enzymes and / or optimal conditions would be used for the reverse transcription of the template in the DNA. In addition, a DNA-RNA hybrid containing a chain of each can be used. A mixture of nucleic acids can also be used, or the nucleic acids produced in a previous amplification reaction can be used in the same way, using the same or different primers. The mutant nucleotide sequence to be amplified may be a fraction of a larger molecule, or may be present initially as a separate molecule, such that the specific sequence constitutes all of the nucleic acid. It is not necessary that the sequence to be amplified is initially present in a pure form; it can be a minor fraction of a complex mixture, such as that which is contained in the entire human DNA. Where the objective neoplastic nucleotide sequence of the sample contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as the template. The chain separation can be carried out either as a separate step or simultaneously with the synthesis of the extension primer products. This chain separation can be carried out using different suitable denaturation conditions, including physical, chemical, or enzymatic elements; the word "denaturalization" includes all these elements. A physical method to separate the nucleic acid strands involves heating the nucleic acid until it is denatured. Typical heat denaturation can involve temperatures from about 80 ° C to 105 ° C for times from about 1 to 10 minutes. Chain separation can also be induced by an enzyme from the class of enzymes known as helicases, or by the enzyme RecA, which has helicase activity, and in the presence of riboATP, which is known to denature DNA. The reaction conditions suitable for the separation of nucleic acid chains with helicases are described by Kuhn Hoffmann-Berling (CSH-Quanti tative Biology, 43_: 63, 1978), and the techniques for using ReCA are reviewed in C. Radding (Ann.Rev.Genetics, 16: 405-437, 1982). If the nucleic acid containing the target nucleic acid to be amplified is single stranded, its complement is synthesized by the addition of one or two oligonucleotide primers. If a single primer is used, a primer extension product is synthesized in the presence of the primer, an agent for polymerization, and the four nucleoside triphosphates described below. The product will be complementary to the single-stranded nucleic acid, and will hybridize with a single-stranded nucleic acid to form a duplex of chains of unequal length, which can then be separated into single strands to produce two separate complementary strands. Alternatively, two primers can be added to the single chain nucleic acid, and the reaction is performed as described. When the complementary strands of the acid or nucleic acids are separated, regardless of whether the nucleic acid was originally double-stranded or single-stranded, the separate strands are ready to be used as a template for the synthesis of additional nucleic acid strands . This synthesis is carried out under conditions that allow the hybridization of the primers in the templates. In general, the synthesis is presented in a regulated aqueous solution, preferably at a pH of 7 to 9, more preferably of about 8. Preferably, a molar excess is added (for the genomic nucleic acid, usually about 108: 1 primer template) of the two oligonucleotide primers to the buffer containing the separate template strands. However, it is understood that the amount of complementary strand may not be known if the process of the invention is used for diagnostic applications, such that the amount of primer relative to the amount of complementary strand may not be determined with certainty. However, as a practical matter, the amount of primer added will generally be a molar excess over the amount of complementary strand (template), when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. . A large molar excess is preferred to improve the efficiency of the process. In some embodiments of amplification, substrates, for example, deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP, are added to the synthesis mixture, either separately or together with the primers, and in suitable amounts, and the The resulting solution is heated to about 90 ° C-100 ° C of about 1 to 10 minutes, preferably 1 to 4 minutes. After this heating period, the solution is allowed to cool to room temperature, which is preferable for primer annealing. A suitable agent is added to the cooled mixture to effect the primer extension reaction (referred to herein as "polymerization agent"), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization can also be added together with the other reagents if it is stable to heat. This synthesis reaction (or amplification) can occur at room temperature up to a temperature above which the agent for polymerization no longer functions. Accordingly, for example, if DNA polymerase is used as the agent, the temperature is generally not higher than about 40 ° C. The agent for polymerization can be any compound or system that functions to perform the synthesis of primer extension products, including enzymes. Enzymes suitable for this purpose include, for example, polymerase I of E. coli DNA, Taq polymerase, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse trans-cryptase, ligase, and other enzymes, including heat-stable enzymes (ie, those enzymes that perform primer extension after being subjected to sufficiently high temperatures to cause denaturation). Suitable enzymes will facilitate the combination of the nucleotides in the proper manner to form the primer extension products that are complementary to each mutant nucleotide chain. In general, the synthesis will start at the 3 'end of each primer, and proceed in the 5' direction along the template chain, until the synthesis ends, producing molecules of different lengths. However, there may be agents for polymerization, which initiate synthesis at the 5 'end, and proceed in the other direction, using the same process as described above. In any case, the method of the invention should not be limited to the amplification modalities described herein. The newly synthesized mutant nucleotide chain and its complementary nucleic acid strand will form a double stranded molecule under the hybridization conditions described above, and this hybrid is used in the subsequent steps of the process. The next step, the newly synthesized double stranded molecule is subjected to denaturing conditions using any of the methods described above, to provide single chain molecules. The above process is repeated on single chain molecules. If necessary, additional polymerization agent, nucleotides, and primers may be added for the reaction to proceed under the conditions described above. Again, the synthesis will start at one end of each of the oligonucleotide primers, and proceed along the single strands of the template, to produce additional nucleic acid. After this step, half of the extension product will consist of the specific nucleic acid sequence limited by the two primers. The steps of denaturation and synthesis of the extension product can be repeated as frequently as necessary to amplify the objective hypermutable nucleotide sequence to the extent necessary for detection. The amount of the hypermutable nucleotide sequence produced will accumulate in an exponential manner. In one embodiment of the invention, a combination of hypermutable microsatellite markers are amplified in a single amplification reaction. The markers are "multiplexed" in a single amplification reaction, for example, by combining primers for more than one site. For example, DNA from a urine sample is amplified with three different series of randomly labeled primers such as FGA, ACTBP2, and AR, in the same amplification reaction. The products are finally separated on a denaturing acrylamide gel, and then exposed to film for visualization and analysis.

The amplified product can be detected by Southern blot analysis, without using radioactive probes. In this process, for example, a small sample of DNA containing a very low level of hypermutable nucleotide sequence in microsatellite is amplified and analyzed by means of a Southern blot technique. The use of non-radioactive probes or labels is facilitated by the high level of the amplified signal. In a preferred embodiment of the invention, a triphosphate of a nucleoside triphosphate is radioactively labeled, thereby allowing direct visualization of the amplification product by autoradiography. In another embodiment, the amplification primers are labeled with fluorescent, and passed through an electrophoresis system. The visualization of the amplified products is by laser detection, followed by computer-aided visual display. Nucleic acids having a hyperchangeable microsatellite sequence detected in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after being fixed to a solid support, by any method normally applied. to the detection of a specific DNA sequence, such as polymerase chain reaction, oligomer restriction (Saiki et al., Bio / Technology, 3: 1008-1012, 1985), allele-specific oligonucleotide probe (ASO) analysis (Conner et al., Proc. Nati, Acad. Sci. USA, .50: 278, 1983), oligonucleotide ligation assays (OLA) (Landegren et al., Science 241: 1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren et al., Science 242: 229-237, 1988). In another embodiment of the invention, purified nucleic acid fragments containing oligonucleotide sequences of 10 to 50 bases are radioactively labeled from the microsatellite sites. The labeled preparations are used to probe the nucleic acid by the Southern hybridization technique. The nucleotide fragments from a sample, before or after amplification, are separated into fragments of different molecular masses by gel electrophoresis, and transferred to filters that fix the nucleic acid. After exposure to the labeled probe, which will hybridize to the nucleotide fragments containing the objective nucleic acid sequences, the binding of the radioactive probe to the target nucleic acid fragments by autoradiography is identified (see Genetic Engineering, 1, ed. Robert Williamson, Academic Press. (1981), 72-81). The probes for the microsatellite sites of the present invention can be used to examine the distribution of the specific fragments detected, as well as the quantitative (relative) degree of probe binding to determine the presentation of highly specific binding sequences (and they hybridize), indicating in this way the presence of extensive alterations in a particular place. For the most part, the probe (or the amplification primer) will be labeled with an atom or an inorganic radical, most commonly using radionuclides, but also perhaps heavy metals. Conveniently, a radioactive label can be employed. Radioactive labels include 32P, 125I, 3H, 14C, 35S, or the like. Any radioactive label that provides an adequate signal, and that has a sufficient half-life can be used. Other labels include ligands, which can serve as a specific binding pair member for a labeled ligand, and the like. A wide variety of labels have been used in immunoassays, which can be easily employed in the present assay. The choice of label will be governed by the effect of the label on the rate of hybridization and attachment of the probe to the mutant nucleotide sequence. It will be necessary for the tag to provide sufficient sensitivity to detect the amount of mutant nucleotide sequence available for hybridization. Other considerations will be the ease of synthesis of the probe, easily available instrumentation, the ability to automate, convenience, and the like. The way in which the label is attached to the probe will vary depending on the nature of the label. For a radioactive label, a wide variety of techniques can be employed. Slit translation with a hydrolysis of terminal phosphate or α-32P-dNTP with alkaline phosphatase is usually followed, followed by radioactive 32P labeling, using? -32P-ATP and T4 polynucleotide kinase. Alternatively, nucleotides can be synthesized in which one or more of the present elements are replaced with a radioactive isotope, for example, hydrogen with tritium. If desired, you can / "- use complementary tagged strings as probes for improve the concentration of the hybridized label. Where other radionuclide labels are involved, different linkage groups may be employed. A terminal hydroxyl can be esterified with inorganic acids, for example, 32P phosphate, or 14C organic acids, or another Thus, it can be esterified to provide link groups to the label. Alternatively, the intermediate bases can be replaced with activatable linkage groups, which can then be linked to a label. Enzymes of interest as primary reporter groups will be alkaline phosphatase, hydrolases, particularly esterases and glucosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescents include, for example, luciferin 25, and 2,3-dihydrophthalazineadiones (e.g., luminol).

An oligomer probe can be used to hybridize to a nucleotide sequence attached to a water insoluble porous support. Depending on the source of the nucleic acid, the manner in which the nucleic acid binds to the support can vary. Ordinary experts in the field know, or can readily assert, different supports that can be used in the method of the invention. The nucleic acid in a sample is stained or spread on a filter to provide a plurality of individual portions. The filter is an inert porous solid support, for example, nitrocellulose or nylon membranes. Any mammalian cells present in the sample are treated to release their nucleic acid. The lysis and denaturation of the nucleic acid, as well as the subsequent washings, can be achieved with an appropriate solution for a sufficient time to lyse the cells and denature the nucleic acid. Other denaturing agents include elevated temperatures, organic reactants (e.g., alcohols, amides, amines, ureas, phenols, and sulfoxides), or certain inorganic ions (e.g., thiocyanate and perchlorate). Alternatively, the nucleic acid can be isolated from the blood, using conventional procedures, subsequently applying the DNA to the membrane. After denaturation, the filter is washed in an aqueous regulated solution, such as Tris, generally at a pH of about 6 to 8, usually 7. One or more washes may be involved, conveniently using the same procedure that was used for the treatment. lysis and denaturation. After the lysis, denaturation, and washings have been performed, the filter stained with nucleic acid is dried at an elevated temperature, generally about 50 ° C to 70 ° C, or reticulated with ultraviolet (for the membranes of nylon). Under this procedure, the nucleic acid is fixed in its position, and can be tested with the probe when convenient. Pre-hybridization can be performed by incubating the filter at a slightly elevated temperature, for a sufficient time with the hybridization solution without the probe, to completely wet the filter. Different hybridization solutions can be employed, ranging from about 20 percent to 60 percent by volume, preferably 30 percent, of an inert polar organic solvent or aqueous hybridization solutions. The particular hybridization technique is not essential to the invention. Other hybridization techniques are well known or can be easily asserted by one of ordinary skill in the art. As improvements are made in the hybridization techniques, they can easily be applied in the method of the invention. The amount of labeled probe that is present in the hybridization solution will vary widely, depending on the nature of the label, of the amount of the probe of the labeled probe that can reasonably be fixed to the filter, and of the stringency of the hybridization. In general, a substantial excess over the stequio-metric concentrations of the probe will be employed to improve the rate of fixation of the probe to the fixed target nucleic acid. Different degrees of hybridization astringency can be employed. The more severe the conditions, the greater the complementarity required for hybridization between the probe and the single-stranded objective nucleic acid sequence for duplex formation. Stringency can be controlled by temperature, probe concentration, probe length, ion concentration, time, and the like. Conveniently, the stringency of the hybridization is varied by changing the polarity of the reactive solution, by manipulating the concentration of formamide on the scale of 20 percent to 50 percent. The temperatures used will normally be in the range of approximately 20 ° C to 80 ° C, normally 30 ° C to 75 ° C (see in general Current Protocols in Molecular Biology, Ausubel, ed., Wiley & amp;; Sons, 1989). Alternatively, the stringency can be controlled when the un-tempered probe is washed out. After the filter has been contacted with a hybridization solution at a moderate temperature for a sufficient period of time to allow the hybridization to occur, then the filter is introduced into a second solution having sodium chloride, citrate sodium, and sodium dodecyl sulfate. The time during which the filter is maintained in the second solution can vary from 5 minutes to 3 hours or more. The second solution and the temperature (generally 5 ° C below the melting temperature), determine the stringency, the duplexes in solution, and the short complementary sequences. For short oligonucleotide probes, the melting temperature can be standardized according to the length of the probe, rather than the sequence, by the inclusion of tetramethyl ammonium chloride in the wash solution (DiLella and Woo,? Feth. Enzymol., 152: 447, 1987). Now the filter can be tested for the presence of duplexes according to the nature of the label. Where the label is radioactive, the filter is dried and exposed to X-ray film. The materials for use in the assay of the invention are ideally suited for the preparation of a kit. This case may comprise a carrier element that is divided into compartments to receive in close confinement one or more container elements, such as bottles, tubes, and the like, each of the container elements comprising one of the separate elements to be used in the method . For example, one of the container elements may contain amplification primers for a microsatellite site or a hybridization probe, all of which can be labeled in a detectable manner. If present, a second container may comprise a lysis regulator. The kit may also have containers containing nucleotides for the amplification of the objective nucleic acid sequence, which may or may not be labeled, and / or a container comprising a reporter element, such as a biotin binding protein, such as avidin. or streptavidin, attached to a reporter molecule, such as an enzymatic, fluorescent, or radionuclide label. The foregoing description generally describes the present invention. A more complete understanding can be had by reference to the following specific examples, which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. EXAMPLES During primary mapping studies using 43 dinucleotide markers in more than 300 tumors, including squamous cell carcinoma (SCC), non-small cell lung carcinoma (NSCLC), bladder transition cell carcinoma (TCC), and Skin carcinoma of basal and squamous cells, simple somatic alterations were observed in approximately 0.7 percent of the tumor's DNA. This low index of "background" alterations in the dinucleotide markers is consistent with other studies that show few changes in tumors not associated with colorectal carcinoma without hereditary polyposis (SN Thibodeau et al., Science, 260: 816, 1993; JI Risinger and collaborators, Cancer Res., 5_3: 5100, 1993, HJ Han et al, Cancer Res., 53: 5087, 1993, P. Peltomaki et al, Cancer Res., 53_: 5853, 1993, M. Gonzalez-Zulueta et al. collaborators, Cancer Res., 5_3: 5620, 1993; R. Wooster et al, Nature Gent., 6: 152, 1994), and the AD? of the germline (JL Weber et al., Am. J. Human Genet., 44: 388, 1989; J. Weissenback et al., Nature, 359: 794, 1992; AJ Jeffreys et al., Nature Genet., 6. : 136, 1994). We tested the following pairs of AD? Normal and cancer using repeating markers of tri- and tetra-nucleotides (TABLE 1): 35 of squamous cell carcinoma, 20 of non-small cell lung carcinoma, 10 of small cell lung carcinoma, and 32 of bladder transition cell carcinoma. Of these, approximately 8 percent and 20 percent of the AD? of tumor had somatic alterations using repeat markers of tri- and tetra-nucleotides, respectively. Of the nine markers, four (myot dystrophy, dentatorrubralpalidoluisiana atrophy, RA (spinobulbar muscular dystrophy), and SAT (spinocerebellar ataxia type 1)), were associated with neurological disease, and have revealed germline expansion of their repeat sequences in the affected patients. Due to these alterations of the germline, it was thought that these markers could be more susceptible to expansion or suppression in AD? of the tumor, comparing with the dinucleotide repeat sequences. The other markers were selected from commercially available tri- or tetra-nucleotide repeat markers, with the exception of UT762 (on human chromosome 21), which had previously been reported to have an excess of germline alterations on the human chromosome 21 (CC Talbot, Jr. et al., 43rd American Society of Human Genetics Meeting, Abstract, 1993). EXAMPLE 1 PRIMERS USED FOR AMPLIFICATION OF SAMPLE DNA All tumors were frozen fresh with the exception of small cell lung carcinoma that was embedded in paraffin. The non-neoplastic tissue was microdissected to be used as normal DNA. Alternatively, fresh blood was obtained and the lymphocytes were separated. Tumor and normal tissue was digested with 1% SDS proteinase K, followed by precipitation in ethanol to extract the DNA. Fifty nanograms of DNA were amplified by polymerase chain reaction; the products were passed over denaturing acrylamide gels as described previously (P. van der Riet et al., Cancer Res., 54: 1156, 1994, H. Nawroz, P. Cancer Res., 54: 1152, 1994; Cairns et al., Cancer Res., 54: 1422, 1994). The primers used to amplify each site were obtained from Research Genetics, Inc., with the exception of the following locations: a. 5 '-CAG ACG CCG GGA CAC AAG-3' (SEQ ID NO: 21); b. 5 '-TAC TGG TCC TGC TGG GCT G-3' (SEQ ID NO: 22); c. 5 '-GTC AGT ATT ACC CTG TTA CCA-3' (SEQ ID NO: 23); d. 5 '-GTT GAG GAT TTT TGC ATC AGT-3' (SEQ ID NO: 24); and. 5 '-CTT CCC AGG CCT GCA GTT TGC CCA TC-3' (SEQ ID NO: 25); F. 5 '-GAA CGG GGC TCG AAG GGT CCT TGT AGC-3' (SEQ ID NO: 26); g. 5 '-CAC CAG TCT CAA CAC ATC ACC ATC-3' (SEQ ID NO: 27); h. 5 '-CCT CCA GTC GGT GGG GAA ATG CTC-3' (SEQ ID NO: 28); i. 5 '-TCC GCG AAG TGA TCC AGA AC-3' (SEQ ID NO: 29) j. 5 '-CTT GGG GAG AAC CAT CCT CA-3' (SEQ ID NO: 30) k. 5 '-AAC ACC CCT AAT TCA CCA CT-3' (SEQ ID NO: 31) 1. 5 '-ATG ATT CCA CAA GAT GGC AG-3' (SEQ ID NO: 32) m. 5 '-CCA TAG GTT TTG AAC TCA CAG-3' (SEQ ID NO: 33); n. 5 '-CTT CTC AGA TCC TCT GAC AC-3' (SEQ ID NO: 34); or. 5 '-TCC AGA ATC TGT TCC AGA GCG TGC-3' (SEQ ID NO: 35); p. 5 '-GCT GTG AAG GTT GCT GTT CCT CAT-3' (SEQ ID NO: 36); q. 5 '-AAT CTG GGC GAC AAG AGT GA-3' (SEQ ID NO: 37); r. 5 '-ACÁ TCT CCC CTA CCG CTA TA-3' (SEQ ID NO: 38); s. 5 '-TCA GTC TCT CTT TCT CCT TGC A-3' (SEQ ID NO: 39); t. 5 '-TAG GAG CCT GTG GTC CTG TT-3' (SEQ ID NO: 40); and u. complementary sequences for the sequences a to t. P. Modrich, Annu. Rev. Genet. , 2J5: 229, (1991). Cytological samples were centrifuged at 3000 x g for 5 minutes, and washed with PBS twice. The cell granules were digested with 1% SDS proteinase K, and the DNA was extracted as described above (D. Sidransky et al., Science, 252: 706, 1991; Science, 256: 102, 1992). The DNA of the surgical margin was obtained from plaques that were histopathologically negative. The tissue was scraped and placed in xylene to remove excess paraffin. After centrifugation with a quarter of the volume of 70% ethanol, the granules were digested and the DNA was extracted as described above (D. Sydransky, supra, R.H. Hruban, supra, L. Mao, supra). EXAMPLE 2 DETECTION OF ALTERATIONS IN PLACES OF THE MICROSATELITE Each site of the microsatellite was amplified in normal / tumor DNA in pairs by polymerase chain reaction, and the labeled products were then passed over denaturing acrylamide gels, and exposed to the film. 29 percent of head and neck cancers, 5 percent of non-small cell lung carcinoma, 50 percent of small cell lung carcinoma, and 28 percent of bladder tumors, exhibited abnormalities of the microsatellite in at least one susceptible marker (TABLE 1). These genetic alterations were identified as a novel band (or bands) in the DNA track of the tumor, and were not present in the paired normal DNA track (Figure 1).

Figure 1 shows the microsatellite alterations in the tumor DNA. Normal and tumor DNA was amplified by polymerase chain reaction, and passed over denaturing acrylamide gels as described (P. van der Riet, supra). Novel bands representing the deletion or expansion of row repeat sequences were seen on all four tracks of the tumor, as indicated by the arrows. Lane 1) B17 (bladder transition cell carcinoma) with the FGA marker on chromosome 4; Clue 2) L21 (small cell lung carcinoma) with the AR marker on the chromosome X; Lane 3) B30 (bladder transition cell carcinoma) with marker UT762 on chromosome 21; and Track 4) L5 (small cell lung carcinoma) with marker D14S50 on chromosome 14. (N: normal DNA, T: tumor DNA). For each case, the amplification was repeated and the alterations were reproduced. The frequency of alterations was significantly higher in these repeat markers of tri- or tetra-nucleotides, and also appeared to be specific to the tumor type (TABLE 1). In CBT, for example, AR repeat is altered in 3 percent of tumors, while 18 percent of squamous cell carcinoma exhibited alterations in this location. No significant difference was observed in the frequency of alterations between the repeat sequences of tri- and tetra-nucleotides related to disease and not related to disease, suggesting that a more generalized cellular mechanism could exist for these changes, instead of the differences of sequence inherent in the repetition regions. Small cell lung carcinoma exhibited generalized microsatellite instability similar to that seen in tumors associated with colorectal carcinoma without hereditary polyposis, and altered a high percentage of all markers, including dinucleotides (A. Merlo et al. supra). Although widespread microsatellite instability has been found more frequently in colorectal carcinoma without hereditary polyposis, other tumors that are not colorectal carcinoma without hereditary polyposis contain occasional alterations. These changes typically involve only one site, rather than the multiple sites typically seen in patients with colorectal carcinoma without hereditary polyposis (A. Merlo et al., Supra; R. Wooster et al., Supra). The widespread instability found in small cell lung carcinoma is an exception, and the genetic basis for this is still unknown. In this study, none of the tumors we tested were associated with colorectal carcinoma without hereditary polyposis. The comparison of dinucleotide repeats (29 of 4171 tested) against repeat alterations of tri- or tetra-nucleotides (44 of 874 tested) in these 97 tumors reveals a significant susceptibility to genetic instability in the largest alleles ( p = 0.08 x 10"9 by X2 analysis.) The high frequency of microsatellite alterations found here suggests that certain sites may be inherently more unstable than others.The mechanism that produces the alleles altered in these tumors may differ from that described in colorectal carcinoma without hereditary polyposis (F. Leach et al., Cell, 75: 1215, 1993; R. Fishel et al., Cell, 75: 1027, 1993), or it could reflect more subtle defects in similar or related repair trajectories (P. Modrich, Annu., Rev. Genet., 25_: 229, 1991). From these data, it can be seen that occasional microsatellite alterations can be a relatively general phenomenon in many human cancers with some altered sites in a tumor-specific manner.

TABLE 1 FREQUENCY OF ALTERATIONS OF THE MICRQSATELITE Alterations of the microsatellite for each tested marker; number of alterations detected in each type of tumor. SCC = squamous cell carcinoma. NSCLC = non-small cell lung carcinoma. SCLC = small cell lung carcinoma. CBT • = bladder transition cell carcinoma. 26 out of 100 (26 percent) of the total tumors exhibited alterations in at least one location. * Related disease.

EXAMPLE 3 DETECTION OF CLONIAL POPULATIONS OF DERIVATIVE CELLS TUMOR These clonal microsatellite alterations were studied to see if they could be detected in cytological samples as specific tumor markers. To demonstrate this potential clinical application, several corresponding cytological samples that were considered negative due to the presence of cancer cells by the light microscope were analyzed. When B27 bladder tumor DNA was screened with the FGA tetranucleotide marker, a novel band was identified in the tumor track, compared to normal (Figure 2a). Figure 2 shows the detection of clonal microsatellite alterations in clinical samples. The conditions of the polymerase chain reaction and the gel analysis have already been described (P. van der Riet et al., Supra). The novel bands are indicated with arrows. Panel (A), the corresponding cytological urine sample of B27 (bladder transition cell carcinoma) was analyzed using the FGA marker, and compared with normal and tumor DNA. A novel or "changed" band is seen on the tumor track against the normal track, and to a lesser but significant intensity on the urine track. Panel (B), this patient's lymphocyte DNA was not available, but a clear band is clearly identified in the "negative" histological range of L31 (non-small cell lung carcinoma), which responds to the most intense novel band in the tumor track, when being traced with the AR marker. Panel (C), CHRNB marker amplification; the corresponding sputum sample of L25 (small cell lung carcinoma) revealed two clear bands consistent with the novel bands in the tumor track. These bands are not present in normal DNA. (N: normal DNA, U: urine DNA, T: tumor DNA, M: margin DNA, S: sputum DNA). The DNA obtained from the patient's urine sample before surgery was screened with the same marker, revealing the same novel band at a lower intensity in the urine DNA (D. Sidransky et al., Supra). The intensity of the changed band was approximately 5 percent of the intensity of the corresponding band in the tumor DNA, indicating that only a small population of cells in the urine sample were derived from the tumor. It was found that a patient with small cell lung carcinoma had a novel allele of CHRNB in the tumor DNA, when compared to normal DNA. When DNA was screened from a corresponding prospectively collected sputum sample with the CHRNB marker, the identical genetic change was found in the sputum DNA initially found in the patient's primary small cell lung carcinoma (Figure 2c). Again, the lower intensity of the novel sputum bands suggested that only a small fraction of the cancer cells was present in the sample. Several histopathologically negative surgical margins were examined (D. Sidransky et al., Supra). The L29 tumor DNA demonstrates a smaller novel band in the IFN microsatellite marker, whereas paired DNA from a histologically negative surgical margin presented the same band changed at a lower intensity (Figure 2b). This is consistent with the previous observation that infiltrating tumor cells not detected in the surgical margins can be identified after a "complete" surgical resection., with effective molecular techniques. These examples demonstrate the ability to detect clonal populations of tumor-derived cells in cytological samples and in histopathological tissue. This assay could be easily reproduced using other altered markers to test corresponding samples in these cases, and paired samples from other patients. The clinical samples of patients without a microsatellite alteration detected in the primary tumor were consistently negative. EXAMPLE 4 DETERMINATION OF SENSITIVITY THROUGH DILUTION An obvious problem in the screening of clinical samples is the need to detect an extremely small number of cancer cells among a large pool of normal cells, especially in body fluids such as urine and sputum. To demonstrate the sensitivity of the method of the invention, bladder tumor DNA B17, which has a novel larger allele, and B27, which has a smaller novel allele, was used. These two samples were selected because of our observation that the smaller alleles tend to be amplified better than the larger alleles by the polymerase chain reaction. The tumor DNA was diluted with normal lymphocyte DNA from the same patient, and 50 nanograms of DNA from each dilution was amplified by polymerase chain reaction. Figure 3 shows the determination of sensitivity by simple dilution. The DNA of tumors (T) containing alterations, was diluted with the lymphocyte DNA of the corresponding patients (N) from 1 in 5 (20 percent) to 1 in 1000 (0.1 percent). The samples were then amplified by polymerase chain reaction with the FGA marker, separated by denaturing gel electrophoresis, and visualized by autoradiography, as described (P. van der Riet et al., Supra). The novel bands are indicated by an arrow. Panel (A) shows the novel band seen in the tumor track of B17 (bladder transition cell carcinoma), still visible when diluted with its normal 0.1 percent DNA. In a similar manner, in Panel (B), the novel band is clearly seen in the tumor DNA of B27 (bladder transition cell carcinoma) when diluted 0.5%.

The "changed" band was seen in the dilution mixture containing only 0.1 percent of tumor DNA in B17, and 0.5 percent of tumor DNA in B27. These results suggest that this method can potentially detect a cancer cell between 200 to 1000 normal cells, thus proving its potential utility as a clinical screening assay. EXAMPLE 5 MULTIPLEX PQLIMERASE MULTIPLEX CHAIN REACTION TEST FOR THE DETECTION OF HYPERMUTABLE NUCLEIC ACID Microatellite alterations could be detected in colorectal carcinoma without hereditary polyposis and in small cell lung carcinoma, by using only a few microsatellite repeat markers , since these tumors alter a high percentage of all alleles tested. For tumors other than colorectal carcinoma without hereditary polyposis, a single well-selected repeat tri- or tetra-nucleotide marker could potentially identify more than 15 percent of the tumors of a particular cancer type. In squamous cell carcinoma and in bladder transition cell carcinoma, seven selected markers detected more than 28 percent of the tumors (TABLE 1). Because the genetic mechanism underlying these occasional alterations is not yet known, it is possible that some cancers do not exhibit alterations, regardless of how many markers are tested. However, the human genome contains more than 100,000 repeating regions, and it is very likely that other candidate markers can be identified for this analysis. A combination of repeating markers could be used to identify a high percentage of cancer patients; The ability to potentially test several markers in a single polymerase chain reaction could simplify the final screening approach. The B27 DNA (bladder transition cell carcinoma) and a corresponding urine sample were amplified with three sets of different primers (FGA on chromosome 4, ACTBP2 on chromosome 6, and AR on chromosome X) in the same polymerase chain reaction, were separated on denaturing acrylamide gels, and exposed to film. The concentration of each primer was diluted to 100 nanograms / mi-crogram in the final polymerase chain reaction. A novel band on the tumor track is identical to the corresponding less intense band on the DNA track of urine. (N: normal DNA, T: tumor DNA, U: urine DNA). The feasibility of this approach is shown in Figure 4, where the three markers are multiplexed into a single polymerase chain reaction. The results clearly demonstrate a novel band in the urine DNA identical to the altered allele in the corresponding primary bladder transition cell carcinoma tumor. This test is much simpler to perform than tests based on previous polymerase chain reaction, followed by cloning and specific hybridization of the oligomer, to detect mutations of the oncogene (D. Sidransky et al., Supra). Although the sensitivity of this assay decreases slightly, with the detection of cancer cells limited to a background of approximately 500 normal cells, compared with 10,000 normal cells using the previous approach, evidence from previous studies suggests that this is sufficient to detect cells from cancer in most clinical samples, including sputum (Figure 3). Moreover, the rare oligoclonal events, perhaps secondary to inflammation or hyperplasia, would not be detected, due to their dilution between a background excess of normal cells that do not have these alterations. Although oncogene mutations that give neoplastic cells a distinct growth advantage are not specifically detected, monoclonality is a fundamental characteristic of all neoplasms, and the detection of clonal cell populations in cytological samples remains an ominous signal (PJ Fialkow , Biochem Biophys. Acta., 458: 283, 1976; P.C. Nowell, Science, 94:23, 1976). The accumulation of genetic events in the following daughter cells is well recognized, and a detectable clone would be expected to persist and probably continue along the path of neoplastic progression (ER Fearon et al., Cell, 61: 709, 1990; Sidransky et al., Nature, 355: 846. 1992; D. Sidransky et al., N. Engl. J. Med., 326: 737, 1992). Although many of the genetic events in the progress of colorectal cancer are known (E.R. Fearon et al., Supra), few events have been well characterized in most other tumor types, and not all occur in a given tumor. The ability to detect early clonal cell populations in patients without an accurate knowledge of specific genetic mutations in the primary tumor is an important force in this trial. Actually, these are preferably patients who may be ideal candidates for chemopreventive strategies and / or susceptible to surgical resection with careful follow-up. Furthermore, the detection of rare infiltration cancer cells in the histopathological margins can have a great impact on current surgical practice. Because the primary tumor is already resected in these cases, a rapid screening of the AD? The tumor can provide a single marker for the detection of these infiltration tumor cells. The present invention indicates that microsatellite alterations appear to be a common feature in human cancers, and that larger repeats are probably more susceptible to this type of genetic instability. The high frequency of alterations observed in several markers seems to be specific to the type of tumor; the selection of appropriate markers with a relatively high rate of instability for a given tumor type may allow the use of a multiplex polymerase chain reaction test to identify a high percentage of neoplasms in patients. The identification of these alterations in body fluids and surgical margins, testifies to their potential use as clonal markers in the detection of neoplastic cells. In the present invention, a simple and powerful screening test that can be applied to a variety of cancers and pathological samples is demonstrated. Because cancer is so prevalent in the population, this molecular approach has important implications for cancer detection. EXAMPLE 6 ALTERATIONS OF THE MICROSATELITE AND LOSS OF HETEROCIGQSITY IN PRIMARY BLADDER CANCER To test the approach used in the previous examples for the detection of bladder cancer, 60 markers of tri and tetra-nucleotides were screened in 50 anous primary bladder cancers from a tumor bank of Johns Hopkins University School of Medicine Although many markers did not exhibit alterations, 80 percent (40/50) of the neoplasms contained at least one novel alteration in the tumor, when compared to normal DNA coupled. Furthermore, a panel selected from the 10 most susceptible markers could theoretically detect 52 percent of all primary bladder cancers (TABLE 2).

This panel of 10 selected markers was tested using the urine sediment of a group of 25 patients with a suspicious lesion of bladder cancer in the cystoscopy, and 5 controls without prior knowledge of their pathological diagnosis. Urine samples were collected before cystoscopy, and microsatellite analysis was done blindly. Urine and normal paired DNA was amplified from each patient, and polymorphic alleles were compared at these 10 microsatellite sites. Urine sediment in 7 of 20 (45 percent) patients with bladder cancer, conenía a novel microsatellite alteration (expansion or suppression of repeat units) in close agreement with the expected frequency based on the analysis of primary tumors (TABLE 1) (Figure 5). However, in an unexpected manner, the additional samples showed a marked loss of heterozygosity (LOH) in the urine sediment consistent with the allelic loss. Because the loss of chromosome 9 is a common genetic event in bladder cancer, three dinucleotide markers were examined to expand the analysis in the critical region of loss on chromosome 9p21. These markers confirmed the presence of deletions in tumors that showed loss of chromosome 9 with the marker D9S747. Above all, the analysis of the microsatellite with the 13 markers, demonstrated the presence of neoplastic cells in 19 of 20 patients with pathologically confirmed cancer, by detecting either an alteration of repetition or loss of heterozygosity. In the only case where a neoplastic clone was not identified in the urine, the patient lodged a small tumor that did not contain alterations in any of the tested sites. It is important that none of the 5 control patients demonstrated microsatellite alterations or deletions of chromosomes. Figure 5 shows the results of studies on 25 patients who had a confirmed diagnosis of cancer by pathology. Nineteen of twenty (95 percent) had identical clonal alterations in the urine sample, and at least nine (45 percent) were cytologically negative for cancer or atypia. To further confirm that these deletions were not noisy, but were intimately associated with neoplastic progression, the primary tumor of the initial biopsies was obtained in 18 of the 20 cancer patients that were analyzed in the study (in 2 cases the the biopsy was not enough for another analysis). In each case (Table 2), the same microsatellite alterations were identified in the primary tumor, which were present in the urine sediment. Furthermore, virtually the same pattern of heterozygosity loss was confirmed in each of the primary tumors, which was initially identified in the urine sediment. However, in two patients additional deletions were identified in the urine, which were not identified in the primary biopsies. In both cases, the loss of heterozygosity in at least one place (and the loss of the identical allele) was shared between the urine sediment and the primary tumor, indicating the detection of a more advanced genetic clone in the urine, probably derived from the same progenitor cell. In five cases, a pathological diagnosis of inflammation was established in the biopsy, but in two of these cases atypical cells were identified (suspicious but not to diagnose neoplasm). In both cases, genetic changes were detected in the urine sediment [in one case abundant atypical cells were also identified in the urine sediment by cytological analysis (see below)]. The cells of the urine sediment were then examined by independent morphological analysis (light microscope). Cytological analysis was performed in 18 of the 20 patients with bladder cancer, and in 3 of the 5 patients with apparent inflammation. Neoplastic cells were identified in 9 patients where the molecular analysis was positive, and in one where the molecular analysis failed to identify the neoplasm. In four additional cases, atypical cells were again seen that were not diagnostic for cancer, but clearly clonal by molecular analysis. It is important that many of the cells that were considered atypical or neoplastic really prevailed throughout the sample, and composed most of the epithelial cells, in a manner consistent with the clear loss of heterozygosity demonstrated by molecular analysis. Consistent with this observation, in some patients the small biopsies contained only a small percentage of neoplastic cells, and yet the urine sediment showed a clear loss of heterozygosity, and seemed to be almost entirely composed of neoplastic cells that shared the same genetic alteration. . This is also consistent with the suppressions of recent reports in most cells within the urine sediment in some patients with bladder cancer. Among the cases detected by molecular analysis, three were the so-called "flat" CIS lesions, and five were early IT cancers. These lesions are those with the greatest potential for clinical progress, and those that would be expected to benefit most from early detection. There was only one case (a small IT lesion) that did not contain a clonal genetic alteration in both the primary tumor and the corresponding urine. Interestingly, the cytology was positive in this patient, although it is not clear if an additional molecular analysis would identify an alteration or a suppressed marker if more sites were tested. The above examples have shown that microsatellite analysis can be a powerful tool in the detection of primary bladder cancer. A panel of markers that were particularly susceptible to alterations was selected, and the number of alterations observed in the urine sediment is consistent with the analysis of the present patent application, demonstrating at least one alteration in many primaries (see Figure 5). The unexpected observation that the loss of heterozygosity could actually be determined, significantly improved the detection strategy of the present invention. Moreover, the identification of both the alteration and the loss of heterozygosity seems to be complementary, since three patients were detected by identifying alterations without loss of heterozygosity in any of the places tested. Initially, it was reported that the loss of chromosome 9 is an early and frequent event in the progression of bladder cancer. Furthermore, the molecular analysis of patients with multiple tumors showed that these multiple tumors appeared to be presented by a single progenitor cell that sowed and populated the bladder mucosa, potentially counting for the high risk of recurrence in these patients. The present findings are consistent with the hypothesis that there are large areas of transformed bladder mucosa in patients with only small neoplasms. Additionally, several factors can contribute to the enrichment of the population of tumor cells in the urine: 1) the normal epithelium lodges more slowly than the tumor epithelium, which can increase the proportion of tumor cells in the urine sediment; 2) normal cells can undergo apoptosis, while tumor cells can survive during normal storage, which can also increase the proportion of tumor cells; and 3) the outer portions of the tumors usually represent actively growing populations that can expand from the subclones of a tumor and lodge heavily in the urine, and increase the population of tumor cells. Two interesting cases contained microsatellite alteration or loss of heterozygosity in multiple locations without definite evidence of a primary tumor by pathology. However, the clinical impression suspected cancer, and atypical cells were identified, despite severe inflammation in both cases. These two cases are probably at high risk of cancer, and probably harbor tumor lesions lost in the biopsy, or premalignant lesions that lack significant morphological alterations. Another interesting phenomenon is the presence of additional clonal alterations in the corresponding urine in some cases of cancer. This observation suggests that the cells of a genetically altered clone, potentially more aggressive, detached into the urine. P53 mutant cells may comprise only a small fraction of the cells in the urine, despite their overwhelming prevalence in the primary tumor. Together, these observations suggest that the clinically obvious primary lesion may not contribute to most of the neoplastic cells detected in the urine sediment. Although 95 percent of the primary tumors in this blinded study were detected, the results are probably an underestimate of the usefulness of this approach. First, a large number of susceptible markers can be used to detect primary microsatellite disturbances. Only 60 microsatellite markers have been examined in the present, and potentially many others that are more susceptible to alteration in neoplasms, including larger repeats, could be potentially identified. Second, the ease of detecting the loss of heterozygosity now puts the usefulness of molecular progress models into perspective. There are more than a dozen chromosomes with common suppressions in primary bladder cancer. Additional markers that form the frequently depleted 9p21 region were tested, and additional markers from other common regions of suppression in primary bladder cancer can be tested. For example, loss of the distal portion of 14q is rare in papillary tumors of the bladder but ubiquitous in clinically aggressive CIS lesions. In fact, a patient with CIS lodged loss of heterozygosity on chromosome 14q as the only abnormality in the urine sediment. Someday a complete "alelotype" of neoplastic cells may be integrated into the urine sediment with prognostic information taken from clinical correlations of genetic changes in primary tumors. Allelicities were detected by a comparison of normal DNA with primary tumor DNA, demonstrating the deletion or recombination of a chromosome. In previous allelic studies, more than 95 percent of transitional cell carcinomas of the bladder have shown loss of heterozygosity in at least one location, using a panel of microsatellite markers across the entire genome. The present invention now brings to light the immediate utility of accumulation studies that demonstrate loss of heterozygosity in many places, and development of molecular progress models for clinical detection. In most of the cases of this study, the morphological and cytological analyzes were not diagnostic. The microsatellite analysis is a simple and effective assay for the detection of clonal tumor populations in urine sediment, and seems very susceptible to low-cost automated approaches.

TABLE 2 CHARACTERISTICS OF PATIENTS WITH BLADDER CANCER Age of Patient urine (years) Number / - Primary Tumor Site of Pathology Stage Citolo-gla LOH Alt Sex 1 80 / F Left Medium Rear TCC T3NOMO Positive 2 43 / M TCC Not done 3 75 / M Right side wall TCC T1N0MO Negative 4 60 / TCC in situ TaNOMO Negati o 5 78 / F TCC Positive 6 45 / M Right side wall TCC T1NOMO Atipia 7 72 / M Prostate fossa Mucina + adenoCA Positive 8 84 / TCC T1NOMO Positive 9 75 / M TCC T1NOMO Positive 10 82 / F TCC T4N2MO Atypia 11 80 / TCC in situ TaNOMO Atypia 12 67 / M TCC T1NOMO Not done 13 l 'Right side wall TCC T2NOMO Negative 14 TCC in situ TaNOMO Inflam. 15 sf * 'TCC Negative 16 5 / M TCC T3NOMO Positive 17 51 / M TCC T1NOMO Positive 18 72 / M Prostate fossa Intraductal TCC Positive 19 65 / M TCC T2NOMO Positive 20 TCC Negative 21 65 / F Atypia / inflammation Not done 22 86 / M Chronic Inflammation Not done 23 71 / M Chronic Inflammation Negative 24 79 / M Atypia / inflammation Atypia 25 65 / M Normal Negative 26 70 / M Control patient without cancer Not done 27 70 / M Control patient without cancer Not done 28 54 / M Control patient without cancer Not done 29 35 / M Control patient without cancer Not done 30 68 / M Control patient without cancer Not done Having now fully described the invention, it can be seen by one of ordinary skill in the art that many changes and modifications can be made without departing from the spirit or scope of the invention.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE (ii) TITLE OF THE INVENTION: DETECTION OF SEQUENCES OF HYPERMUTABLE NUCLEIC ACIDS IN TISSUES (iii) SEQUENCE NUMBER: 40 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTTGTGTCCC GGCGTCTG (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CAGCCCAGCA G GACCAGTA (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TGGTAACAGT GGAATACTGA C ( 2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ACTGATGCAA AAATCCTCAA C (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: No (iv) ANTICIPATE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GATGGGCAAA CTGCAGGCCT GGGAAG (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) ) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCTACAAGGA CCCTTCGAGC CCCGTTC (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GATGGTGATG TGTTGAGACT GGTG ( 2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 GAGCATTTCC CCACCCACTG GAGG (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: AD Nc (iii) HYPOTHETICAL: No (iv) ANTICIPATE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 GTTCTGGATC ACTTCGCGGA (2) INFORMATION FOR SEQ ID NO: 10 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: No (iv) ANTICIPATE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TGAGGATGGT TCTCCCCAAG (2) INFORMATION FOR SEQ ID NO: 11: (i) ) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: .11 AGTGGTGAAT TAGGGGTGTT (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SECUE NCIA: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATE : No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 CTGCCATCTT GTGGAATCAT (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) ) LENGTH: 21 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) ) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 CTGTGAGTTC AAAACCTATG G (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENTO: (vi) FUEN ORIGINAL TE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 GTGTCAGAGG ATCTGAGAAG (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: ( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15 GCACGCTCTG GAACAGATTC TGGA (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic Acids ( C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) DESCRIPTION SEQUENCE: SEQ ID NO: 16 ATGAGGAACA GCAACCTTCA CAGC (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) ) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: CDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 TCACTCTTGT CGCCCAGATT (2) INFORMATION FOR SEQ ID NO: 18 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: No (iv) ANTICIPATE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 TATAGCGGTA GGGGAGATGT (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No ( iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19 TGCAAGGAGA AAGAGAGACT GA (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE : No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20 AACAGGACCA CAGGCTCCTA (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) ) LENGTH: 18 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) ) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 CAGACGCCGG GACACAAG (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT : (vi) FUE ORIGINAL NTE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: TACTGGTCCT GCTGGGCTG (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE : Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23 GTCAGTATTA CCCTGTTACC A (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 GTTGAGGATT TTTGCATCAG T (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: Nucleic Acids (C) YOU CHAIN PO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION : SEQ ID NO: 25: CTTCCCAGGC CTGCAGTTTG CCCATC (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GAACGGGGCT CGAAGGGTCC TTGTAGC (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 27 CACCAGTCTC AACACATCAC CATC (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ) ANTICIPATE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28 CCTCCAGTGG GTGGGGAAAT GCTC (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE : (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: TCCGCGAAGT GATCCAGAAC (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) ) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) ) TYPE OF FRAGM ENTO: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CTTGGGGAGA ACCATCCTCA (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 pairs of bases (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: ( vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: ATACACCCCTA ATTCACCACT (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) ) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) SOURCE ORIGINAL: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: ATGATTCCAC AAGATGGCAG (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: Nuc Acids leicos (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CCATAGGTTT TGAACTCACA G (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids ( C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) DESCRIPTION SEQUENCE: SEQ ID NO: 34: CTTCTCAGAT CCTCTGACAC (2) INFORMATION FOR SEQ ID NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic Acids (C) TYPE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35 TCCAGAATCT GTTCCAGAGC GTGC (2) INFORMATION FOR SEQ ID NO: 36: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36 GCTGTGAAGG TTGCTGTTCC TCAT (2) INFORMATION FOR SEQ ID NO: 37: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37 AATCTGGGCG ACAAGAGTGA (2) INFORMATION FOR SEQ ID NO: 38: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) T MOLECULE IPO: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SUIT: No (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38 ACATCTCCCC TACCGCTATA (2) INFORMATION FOR SEQ ID NO: 39: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 pairs of bases (B) TYPE: Nucleic Acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: ( vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39 TCAGTCTCTC TTTCTCCTTG CA (2) INFORMATION FOR SEQ ID NO: 40: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) ) TYPE: Nucleic Acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: No (v) TYPE OF FRAGMENT: (vi) SOURCE ORIGINAL: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40 TAGGAGCCTG TGGTCCTGTT

Claims (21)

1. CLAIMS 1. A method for detecting a mammalian cell proliferative disorder associated with a hyperchangeable target nucleic acid, comprising isolating the nucleic acid present in a mammalian sample and detecting the presence or absence of the hyperchangeable target nucleic acid.
2. 2. The method of claim 1, wherein the hypermutable target nucleic acid is amplified before detection.
3. 3. The method of claim 2, wherein the amplification is by means of oligonucleotides that hybridize to the flanking regions of the hypermutable target nucleic acid.
4. The method of claim 1, wherein the target nucleic acid is selected from the group consisting of nucleic acid suppression and nucleic acid addition.
5. The method of claim 1, wherein the cell proliferative disorder is not due to a gene repair defect.
6. 6. The method of claim 1, wherein the cell proliferative disorder is a neoplasm.
7. The method of claim 5, wherein the neoplasm is selected from the group consisting of head, neck, lung, esophagus, stomach, small intestine, colon, bladder, kidney and nape.
8. 8. The method of claim 6, wherein the neoplasm is benign.
9. The method of claim 6, wherein the neoplasm is malignant.
10. The method of claim 1, wherein the sample is selected from the group consisting of sputum, urine, bile, stool, cervical fluids, saliva, tears, cerebrospinal fluid, regional lymph nodes, and histopathological margins.
11. The method of claim 1, wherein the target nucleic acid comprises the sequence (X) n, wherein X greater than or equal to a nucleotide and wherein n greater than or equal to 2.
12. The method of claim 1, wherein the target nucleic acid comprises the sequence (X) n, where X greater than or equal to two nucleotides and where n greater than or equal to 2.
13. The method of claim 12, wherein the sequence X is selected from the group consisting of TC, AGC, TCC, CAG, CAA, CTG, AAAG, AGAT and TCTT.
14. The method of claim 3, wherein the nucleotide sequence of the flank region to which the oligonucleotide hybridizes is selected from the group of sequences consisting of: a. 5 '-CTT GTG TCC CGG CGT CTG-3' (SEQ ID NO: 1); b. 5'-C AGC CCA GCA GGA CCA GTA-3 '(SEQ ID NO: 2); c. 5 '-TGG TAA CAG TGG AAT ACT GAC-3' (SEQ ID NO: 3); d. 5 '-ACT GAT GCA AAA ATC CTC AAC-3' (SEQ ID NO: 4); and. 5'-GA TGG GCA AAC TGC AGG CCT GGG AAG-3 '(SEQ ID NO: 5' -GCT ACA AGG ACC CTT CGA GCC CCG TTC-3 '(SEQ ID NO: 6); g 5' -GAT GGT GAT GTG TTG AGA CTG GTG-3 '(SEQ ID NO: 7); h 5' -GAG CAT TTC CCC ACC CAC TGG AGG-3 '(SEQ ID NO: 8); 5' -GTT CTG GAT CAC TTC GCG GA- 3 '(SEQ ID NO: 9); JJ 5' -TGA GGA TGG TTC TCC CCA AG-3 '(SEQ ID NO: 10); k 5' -AGT GGT GAA TTA GGG GTG TT-3 '(SEQ ID NO. : 11); 1 5 '-CTG CCA TCT TGT GGA ATC AT-3' (SEQ ID NO: 12); m 5 '-CTG TGA GTT CAA AAC CTA TGG-3' (SEQ ID NO: 13); n 5 '-GTG TCA GAG GAT CTG AGA AG-3' (SEQ ID NO: 14); or 5 '-GCA CGC TCT GGA ACA GAT TCT GGA-3' (SEQ ID NO: 15); P 5 '-ATG AGG AAC AGC AAC CTT CAC AGC-3 '(SEQ ID NO: 16); q 5' -TCA CTC TTG TCG CCC AGA TT-3 '(SEQ ID NO: 17); r 5' -TAT AGC GGT AGG GGA GAT GT- 3 '(SEQ ID NO: 18) s 5' -TGC AAG GAG AAA GAG AGA CTG A-3 '(SEQ ID NO: 19) t 5' -AAC AGG ACC ACA GGC TCC TA-3 '(SEQ ID NO: 20), and its complementary sequences for the sequences aa t 15.
15. The method of claim 14, wherein the or The ligonucleotide is selected from the group consisting of: a. 5 '-CAG ACG CCG GGA CAC AAG-3' (SEQ ID NO: 21); b. 5 '-TAC TGG TCC TGC TGG GCT G-3' (SEQ ID NO: 22); c. 5 '-GTC AGT ATT ACC CTG TTA CCA-3' (SEQ ID NO: 23); d. 5 '-GTT GAG GAT TTT TGC ATC AGT- 3' (SEQ ID NO: 24); and. 5 '-CTT CCC AGG CCT GCA GTT TGC CCA TC-3' (SEQ ID NO: 25); 5 '-GAA CGG GGC TCG AAG GGT CCT TGT AGC-3' (SEQ ID NO: 26); 5 '-CAC CAG TCT CAA CAC ATC ACC ATC-3' (SEQ ID NO: 27); 5 '-CCT CCA GTG GGT GGG GAA ATG CTC-3' (SEQ ID NO: 28); i. 5 '-TCC GCG AAG TGA TCC AFA AC-3' (SEQ ID NO: 29) j. 5 '-CTT GGG GAG AAC CAT CCT CA-3' (SEQ ID NO: 30) k. 5 '-AAC ACC CCT AAT TCA CCA CT-3' (SEQ ID NO: 31) 1. 5 '-ATG ATT CCA CAA GAT GGC AG-3' (SEQ ID NO: 32) m. 5 '-CCA TAG GTT TTG AAC TCA CAG-3' (SEQ ID NO: 33); n. 5 '-CTT CTC AGA TCC TCT GAC AC-3' (SEQ ID NO: 34); or. 5 '-TCC AGA ATC TGT TCC AGA GCG TGC-3' (SEQ ID NO: 35); p. 5 '-GCT GTG AAG GTT GCT GTT CCT CAT-3' (SEQ ID NO: 36); q. 5 '-AAT CTG GGC GAC AAG AGT GA-3' (SEQ ID NO: 37); r. 5 '-ACÁ TCT CCC CTA CCG CTA TA-3' (SEQ ID NO: 38); s. 5 '-TCA GTC TCT CTT TCT CCT TGC A-3' (SEQ ID NO: 39); t. 5 '-TAG GAG CCT GTG GTC CTG TT-3' (SEQ ID NO: 40); and u. complementary sequences for the sequences a to t.
16. The method of claim 1, wherein the hyperchangeable target nucleic acid is selected from the group of microsatellite sites consisting of ARA, D14S50, AR, MD, SAT, DRPLA, ACTBP2, FGA, D4S243 and UT762.
17. 17. A kit useful for the detection of a mammalian cell proliferative disorder associated with a hypermutable target nucleic acid from a tissue sample, the kit comprising carrier means that are arranged in compartments to receive in them in close confinement one or more Containers containing oligonucleotide primers that hybridize to flank nucleic acid sequences of a hypermutable target nucleic acid.
18. 18. The kit of claim 17, wherein the sample is selected from the group consisting of sputum, urine, bile, stool, cervical fluids, saliva, tears, cerebrospinal fluid, regional lymph nodes and histopathological margins.
19. 19. The kit of claim 17, wherein the kit further comprises a deoxynucleotide labeled in detectable form.
20. 20. The kit of claim 17, wherein the target nucleic acid is a site of microsatellite DNA. The kit of claim 20, wherein the microsatellite site is selected from the group of locations consisting of ARA, D14S5CK AR, MD, SAT, DRPLA, ACTBP2, FGA and UT762. 2K The kit of claim 21, wherein the places are multiplexed.