WO2003038125A1 - Modified pcr-sscp method of mutation screening - Google Patents

Abstract

A specific and sensitive method for the identification of mutations in a gene. The method comprises the steps of: obtaining nucleic acids from a sample, preparing from said nucleic acids large segments of said gene of at least about 700bp each. The next step involves independent digestion of each of said segments with at least two different restriction enzymes, each digestion performed in separate reaction. Each of the chosen restriction enzyme/s has a single restriction site in said segment and this site is different from the restriction site/s of the other restriction enzyme/s. Digestion according to the present invention should give for each of said segments two unequal fragments of different length each, for each restriction enzyme. The digestion fragments obtained in the former step are then subjected to SSCP analysis, whereby said two unequal fragments of different length each are separated. Finally, the gel-banding patterns of overlapping fragments, resulting from separate digestion reactions of the same segment, that were separated on different lanes in the SSCP analysis, are compared to a control wild-type sample, and any difference in the resulting gel-banding patterns of overlapping fragments, between the tested sample and the control, indicates the existence of mutation in said segment.

Description

Method of Screening for Mutations

MODIFIED PCR-SSCP METHOD OF MUTATION SCREENING

Field of the Invention

The invention is directed to the field of screening genes for mutations, such as deletions, nonsense, insertion, exon skipping, and missense mutations. The identification of disease-related mutations may serve in genetic counseling, early identification of carriers, and further insight into the genetic disease studied.

Background of the Invention

The demand for tests that allow for the detection of specific nucleic acid sequence changes is rapidly growing in clinical diagnostics. As nucleic acid sequence data for mutations associated with diseases in humans accumulate, the demand for improved tests for as yet unknown mutations within a specific gene sequence is rapidly increasing.

An example for a genetically inherited disorder is von Recklinghausen neurofibromatosis or neurofibromatosis type 1 (NFl), one of the most common autosomal dominant genetic disorders, with an incidence of 1 in 3000 in all ethnic groups. Individuals with NFl are at increased risk of malignant tumors of the nervous system, such as Malignant Peripheral Nerve Sheath Tumor (MPNST- also previously was known as neurofibrosarcoma), optic nerve glioma, and phaeochromoacytoma. More common malignancies may also develop, including certain leukemias and other childhood malignancies (neuroblastoma, rhabdomyoblastoma and Wilms' tumor). Another feature of NFl is the variable phenotypic expression, even in members of the same family, although it is highly penetrant. The mutation rate in the NFl gene is one of the highest reported in any human disease (1 in 10⁴ germ cells) and approximately half of all cases are caused by new mutations. These patients have no family history of the disease.

The disease is caused by mutations of the NFl gene located on chromosome 17qll.2. The gene is one of the largest genes in the genome, it spans 350 kb of genomic DNA, contains 60 exons and the full length transcript is approximately 13 kb [Li et al., Genomics 25:9-187 (1995)]. The NFl gene product neurofibromin is widely expressed in most tissues. One region of neurofibromin (corresponding to exons 21-27b) is structurally and functionally homologous to GTPase-activating protein for p21^ras, accelerating the hydrolysis of p21^ras-GTP to p21^ras-GDP. NFl is considered a tumor suppressor gene because of the observed loss of the wild type NFl allele in different tumors that develop in NFl patients and in sporadic tumors or cell lines prepared from them together with the loss of function of neurofibromin in inactivating the proto-oncogene p21^ras.

The detection of NFl mutations has been particularly arduous due to the gene's large size, the presence of the normal allele and no identified "hot spots" in the gene. Almost every mutation is unique. Thus, relatively few mutations have been identified at the molecular level even though NFl is so common.

Another example is Ataxia telangiectasia (A-T), an autosomal recessive neurological disorder with an incidence of 1 in 40,000 to 1 in 100,000. The major neurological features result from cerebellar degeneration and include progressive cerebellar ataxia presenting in infancy, hypogonadism, growth retardation, the presence of telangiectasia, cellular and humoral immunodeficiency, and high incidence of cancer, particularly lymphoid malignancies. The cellular phenotype of A-T includes a reduced life-span, chromosomal instability, hypersensitivity to ionizing radiation and radiomimetic chemicals and defective checkpoints at the Gl, S and G2 phases of the cell cycle [Taylor A., et al., Int. J. Radiat. Biol. 65:65, (1994), Beamish H., Int. J. Radiat. Biol. 65:175 (1994)].

The causative gene of ataxia telangiectasia gene was identified by positional cloning, termed ATM (A-T mutated) [Savitsky K., et al., Science 268:1749 (1995), Savitsky K., et al., Hum. Moi. Genet. 4:2025, (1995)].

The ATM gene spans approximately 150 kb of genomic DNA, consists of 66 exons with a transcript of 13 kilobases that encodes a 350 kDa protein [Uziel T., et al., Genomics 33:317, (1996)]. The ATM gene product shares a highly conserved carboxy-terminal region of 350 amino acids showing high sequence homology to the catalytic domain of the pllO subunit of phosphatidylinositol 3-kinase (PI-3 kinase) [Savitsky K., et al., Science, ibid (1995), Savitsky K, et al, Hum. Moi. Genet, ibid, (1995)]. This protein is similar to several large proteins in various species that contain PI-3-kinase like domains at their carboxy termini, and are involved in DNA damage processing and cell cycle control. The ATM gene could be referred as a caretaker; since it is involved in maintaining the integrity of the genome by regulating a network of genes (p53, NBS1, BRCAl, c-abl, Chkl) that are involved in the DNA repair of double strand breaks, regulation of cell proliferation and apoptosis [Khanna KK: et al., J. Natl. Cancer Inst. 92: 795, (2000)]. These genes are imp heated in human cancer. The majority of A-T patients carry truncating mutations, resulting in the inactivation of the ATM protein [Gilad S., et al., Hum. Moi. Genet. 5:433, (1996), Gilad S., et al., Hum. Mut. 11:69, (1998), Lavin MF, et al., Cancer Res. 59:3845, (1999)]. One of the most striking features of A-T is the increased predisposition to leukemia and lymphoma. 10%- 15% of all A-T homozygotes develop a malignancy by early adulthood [Taylor A., et al., Br. J. Cancer 66:5, (1992)]. A 70-fold and 250-fold excess for leukemias and lymphomas, respectively, have been reported [Morrell D, et al., J. Natl. Cancer Inst. 77:89, (1986), Swift M, et al, N. Engl. J. Med. 325:1831, (1991)].

The high incidence of lymphoid neoplasms in A-T includes both B- and T-cell subtypes. B-cell lymphoma occurs in older A-T children, while T-cell neoplasms may occur at any age (2-12 years), and could be T-cell acute lymphoblastic leukemia (T-ALL), T-cell prolymphocytic leukemia, T-cell lymphoma or T-cell chronic lymphocytic leukemia (T-CLL). Most strikingly, is the four to fivefold increase in frequency of T-cell compared with that of B-cell neoplasms in these patients which is clearly different from the ratio in the non-A-T population [Taylor A., et al, ibid., (1992), Taylor A., et al, Blood 87:423, (1996)]. Therefore, it is possible that a significant proportion of all T-ALL/T-cell lymphoma in children might be associated with undiagnosed A-T carriers.

A further example is the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disease that belongs to the inherited human chromosomal instability syndromes that include Bloom's syndrome, Fanconi's anemia and ataxia telangiectasia [Shiloh, Annu. Rev. Genet. 31:635-662 (1997)]. All of these disorders are characterized by spontaneous chromosomal instability, immunodeficiency and predisposition to cancer. The majority of families with NBS are from Eastern Europe, with some from Germany, Italy and the United States. Clinical features are low birth weight for days, microcephaly, various degrees of mental retardation, facial erythema, cafe-au-lait spots, immuno- deficiency, chromosomal instability, gonadal dysgenesis and a striking predisposition to lymphoid malignancies. NBS cells are characterized by radiosensitivity and radioresistant DNA synthesis [Shiloh, ibid. (1997)].

The causative gene for NBS is NBS1, a 50 kb long gene, located to chromosome 8q21-24 and encoding a protein of 754 amino acids, termed nibrin [Matsuura et al, Nature Genet 19:179-181 (1998); Naron et al, Cell 93:467-476 (1998)]. Two known domains in the Ν-termini were identified through sequence comparison: a fork head associated domain (FHA) and a breast cancer carboxy-terminal domain (BRCT). Both domains have been found in DΝA damage responsive cell cycle checkpoint proteins.

Carney et al have shown that nibrin can form complexes with at least 2 proteins, hRAD50 and hMREll, that play a role in double strand break repair [Carney et al, Cell 93:477-486 (1999)]. The hMrell/hRad50/Νbsl complex acts as a sensor of DΝA damage. Upon exposure to ionizing radiation the complex becomes rapidly associated with DΝA double strand breaks and remains at these sites until damage is repaired [Νelms et al, Science 280:590-592 (1998)]. The repair of chromosomal double-strand breaks is crucial for genomic integrity. If not repaired, they can result in chromosomal rearrangements, including translocations which are associated with various tumors [Richardson et al, Nature 405:697-700 (2000)].

There exists a great need for efficient and low-cost methods for detecting gene mutations. The NFl, ATM and NBS1 genes are but three examples of such genes of interest.

The Prior Art

A number of methods have been devised to scan nucleic acid segments for mutations. One option is to determine the entire gene sequence of each test sample e.g., by PCR amplification and sequencing of the product. This method is time-consuming, error-prone, and restricted to rather short fragments, though. In view of that, a given segment of nucleic acid may be characterized on other levels. A detailed picture of a DNA molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. However, this method rarely catches small mutations, such as single nucleic acid changes. However, such changes are of importance in a large number of genetic disorders. For detection of single-base differences between like sequences, mutations may be detected and localized by the presence and size of the RNA fragments generated by cleavage at the mismatches of RNA-RNA heteroduplexes. Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage" (MCC) [Gogos et al, Nucl. Acid. Res. 18:6807-6817 (1990)]. However, this method, although exemplified with large genes, requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory. Moreover, the method is time-consuming and requires highly skilled personnel.

Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes. One of these methods, termed "Denaturing Gradient Gel Electrophoresis" (DGGE) is based on the observation that slightly different sequences will display different patterns of local melting when electrophoretically resolved on a gradient gel. In this manner, variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of mutations in the target sequences because of the corresponding changes in their electrophoretic mobilities. The fragments to be analyzed, usually PCR products, are "clamped" at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands. The attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE [Abrams et al, Genomics 7:463-475, (1990)].

Another common method, called "Single-Strand Conformation Polymorphism" (SSCP) was developed by Hayashi, Sekya and colleagues [see Hayashi, PCR Meth Appl 1:34-38 (1991), and references therein, incorporated herein in its entirety by reference] and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations [Orita et al, Genomics 5:874-879 (1989)]. The SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel, so that intra-molecular interactions can form. The SSCP method has the disadvantage that only fragments up to 350 bp can be screened, and the sensitivity is only 70%-80% [Orita et al, Genomics 5: 874-879 (1989)]. SSCP analysis of longer fragments has been developed, enabling the analysis of fragments ranging from 300 to 800 bp by optimizing different assay conditions such as temperature (room temperature vs. 4°C) and lowering the pH by addition of glycerol to gels or by using low pH buffer (about 6.8). The sensitivity of that method for the detection of mutations is higher [87%, see e.g., Kukita et al, Hum Mut 10:400-407 (1997)] Another approach to overcome the length restriction of the SSCP method, was described by Iwahana et al. [Iwahana et al, Biotechniques, 12(l):64-66 (1992)]. In this method, large PCR fragments of about 900bp were digested by variety of endonucleases creating smaller fragments that were end labeled and then subjected to SSCP analysis. Interpretation of results of variety of fragments created by different endonucleases of this method might be very complex.

Another method for the screening of genes for mutations is restriction endonuclease fingerprinting (REF). In this method, long fragments of up to 1000 base pairs can be screened. However, this method is very complex and comprises a large number of steps including amplification of DNA fragments, purification of said DNA fragments, the use of a large number of restriction enzymes for creating small fragments of about 150bp in length and end-labeling. Loss or gain of restriction sites indicates the existence of mutations and therefore, more endonucleases are required to increase the sensitivity. In addition to loss and gain of restriction enzymes, mutations can be detected by the this method by SSCP of the small fragments. However, the possibility that a mutated fragment will overlap with a normal fragment and vice versa, is very high. Nevertheless, the main disadvantage of the REF is that more steps are required, thereby increasing the opportunity for technical error. In addition, interpretation of the REF gels is very complex, since each lane contains about 68 different fragments [Liu et al, BioTechniques i8:470-477 (1995)].

In contrast, the mutation detecting method provided by the present invention is a simple, rapid, sensitive and specific method for detecting and localizing mutations. More specifically, the method of the invention is based on preparation of large segments of a specific gene and digesting each of said segments with two different restriction enzymes. The essence of the invention is that each restriction enzyme chosen is having a single restriction site in the segment, which is different from the restriction site/s of the other restriction enzyme/s. Thus, digestion with each of said restriction enzymes resulting in two unequal fragments of different length each for each of said enzymes. Most preferably, the two restriction enzymes are chosen such that the smaller^' fragment produced by each digest is not derived from the same end of the undigested fragment as the smaller fragment produced by the other restriction enzyme. The specific choice of endonucleases creating the specific overlapping fragments, is mostly preferred for the present invention and enables the detection of mutations which otherwise could not be detected, as demonstrated in the Examples. Moreover, the use of overlapping fragments enables the detection of the precise location of the mutation.

The above-described methods have been used in a number reports describing mutation analysis. In the above-mentioned NFl gene, the detection of mutations has been particularly arduous due to the gene's large size, the presence of the normal allele and the fact that almost every mutation is unique. Thus, relatively few mutations have been identified at the molecular level, even though NFl is so common. Many studies have tended to focus on one portion of the gene (or one exon a time) using the prior art methods mentioned above for mutation screening. NFl mutations were identified in 5% and in two studies 2% using the heteroduplex analysis and screening in each study 9 exons, 1 exon and 3 exons, respectively [Colman et al, J. Med. Genet. 34:579-581 (1997); Maynard et al, Hum. Genet. 99:674-676 (1997); Upadhyaya et al, Hum. Mut. 10:248-250 (1997)]. Heteroduplex combined with SSCP analysis identified 19% for all exons studied, and 5% for only 6 exons screened [Abe nathy et al, Hum. Mut. 9:548-554 (1997); Hudson et al, Hum. Mut. 9:366-367 (1997)]. DGGE analysis of only 4 exons identified 1% mutations [Klose et al, Hum. Genet. 102:367-371 (1998)]. Methods for screening the entire gene region have also been used. The first could identify insertions or deletions by electrophoresis of RT-PCR long products in agarose gels that altered the size of the product. They identified 30% of the mutations [Martinez et al, Genome Res. 6:58-66 (1996)]. The second used the PTT (protein truncation test) and identified mutations in 73% of patients [Park et al, J. Med. Genet. 35:813-820 (1998)]. This is the highest mutation rate detected in a study of NFl patients using a single detection method. The limitations of PTT are that missense mutations are not detected. In a recent report on the evaluation of PTT in fifteen NFl patients with known mutations, only 67% (10/15) could be detected [Osborn and Upadhyaya, Hum. Genet. 105:327-332 (1999)]. This could be attributed to the inabihty of the PTT to detect small or large truncated proteins products and/or transcript instability.

The instability of the transcript results in varying amounts of mutant mRNA. As will be shown hereafter, and particularly in Figs. 7A and 7B, the method of detecting mutations in accordance with the present invention, also referred to as modified SSCP, is much more sensitive than PTT. Mutations have been identified even where the amount of mutant mRNA was very low, undetectable on agarose gels. In Fig. 7A, independent RT-PCR reactions of the same sample yielded twice only the normal fragment (Fig. 7A, lanes 1 and 2), and only the third time, an additional abnormal band could be detected. Fig. 7B shows the modified SSCP analysis of the RT-PCR product from lane 2, with no detectable abnormal transcript, which definitely identified an abnormal electrophoresis pattern (sample is labeled with an arrow). Such low amount of mRNA would not have yielded a mutant protein by PTT, only the normal protein could have been detected.

Combined analysis allowing identification of about 95% of NF-1 mutations was recently reported [Messian L.M. et al, Human Mutation 15: 541-555 (2000)]. This report describes analysis of NF-1 mutations using combination of variety of methods, a protein truncation assay was followed by heteroduplex analysis, FISH, Southern blot and cytogenetic analysis. This combined approach is complicated, time consuming and costly.

In contrast, the present invention provides a method, exhibiting the highest mutation rate detected in a study of NFl patients using a single mutation identification method.

Thus, the art is in need of methods that allow the detection of all or most of the relevant mutations, including missense, nonsense, insertion and deletion mutations. It is an objective of the invention to provide a fast, sensitive method that allows the rapid identification of mutations along the entire mRNA sequence of the gene. The present invention provides for a single and simple method for the screening of genes, particularly large genes, accurately, specifically and more rapidly than any known method. Further advantages and objectives of the invention will become clear as the description proceeds.

Summary of the Invention

The present invention relates to a method for the identification of mutations in a gene comprising the steps of:

(a) obtaining nucleic acids from a sample;

(b) preparing from said nucleic acid large segments of said gene of at least about 700bp each;

(c) independently digesting each of said segments with at least two different restriction enzymes, each digestion in separate reaction, each chosen restriction enzyme having a single restriction site in said segment, which is different from the restriction site/s of the other restriction enzyme/s, digestion with each of said restriction enzyme resulting in two unequal fragments of different length each for each of said enzymes;

(d) subjecting the digestion fragments obtained in step (c) by each of said restriction enzymes to SSCP analysis, whereby said two unequal fragments are separated; and

(e) comparing the gel-banding patterns of overlapping fragments, resulting from separate digestion reactions of the same segment, in step (c), that were separated on different lanes in the SSCP analysis of step (d), to a control sample, whereby any difference in the resulting gel-band pattern between the sample and the control, indicates the existence of mutation in said segment.

In specific embodiments the nucleic acid is mRNA, which undergoes a reverse transcription to form cDNA. The resultant cDNA is then subjected to preparation of large segments of at least about 700 to 1200bp, preferably by primer extension reaction. The segments are then subjected to endonuclease digestion and SSCP analysis as specified in steps (b) to (e) of the method of the present invention.

In another specific embodiment the method of the invention employs genomic DNA (gDNA) as the nucleic acid. The preparation of the large segments is preferably by primer extension reaction. These reactions are performed on the genomic DNA with specific primers and the resulting segments are then analyzed according to the subsequent steps of the method of the invention.

In a specifically preferred embodiment, the method of the invention employs, for each segment, two different restriction enzymes, each of these restriction enzymes having a single restriction site in said segment, for digesting said segment into two unequal fragments of different length each. Each of the enzymes chosen has restriction site that is different from the site of the other restriction enzyme/s.

Further preferably, the two restriction enzymes are chosen such that the smaller fragment produced by each digest is not derived from the same end of the undigested fragment as the smaller fragment produced by the other restriction enzyme. More preferably, the smaller fragment of said unequal fragments of different length produced in one of the digestion reactions, overlaps with the larger fragment of said unequal fragments of different length produced in the other digestion reaction.

According to a specifically preferred embodiment, the sample used by the method of the invention may be obtained from any one of eukaryotic and prokaryotic organisms selected from the group consisting of vertebrates, invertebrates, plants, bacteria, yeast and fungi. More preferably, the sample may be derived from mammalian vertebrates such as humans and bovine, equine, canine, murine and feline animals. Most preferably, the sample is obtained from a human subject.

A control sample, according to another embodiment of the' invention, is a sample of same type obtained from a healthy subject of the same species. Preferably, the subject may be a mammalian subject. More preferably, the subject is a human.

According to another preferred embodiment, the sample used in the method of the invention may be selected from the group consisting of peripheral blood, bone marrow, tumors and embryonic cells. Preferably, the sample is a peripheral blood sample.

An alternative embodiment relates to the use of a sample obtained from a plant. The sample may obtained from any part of a plant including a seed. The modified SSCP method of the invention enables the analysis of large genes. The method of the invention is preferably intended at identifying nonsense, missense, insertion, and deletion mutations. Also preferably, the method of the invention enables identifying substantially all mutations within the mRNA sequence of said gene, all mutations within the coding region of said gene. Further, the method of the invention is intended for identifying mutations in at least one intron of said gene.

In a specifically preferred embodiment the method of the invention is intended at identifying polymorphism in the gene examined.

In a preferred embodiment the primer extension product is preferably a long fragment, more preferably about 1,000 base pairs (bp) to about l,200bp in length.

The method of the invention involves a primer extension step as preferred method for preparation of said large segments. The primer extension comprises nucleic acid amplification, preferably, the primer extension and amplification step may be PCR.

The primer extension or PCR product is not labeled according to one embodiment of the invention. In this case, the gel-bending pattern of the digested fragments may be visualized by silver staining. Alternatively, the primer extension or PCR product may be body-labeled, most preferably by using labeled nucleotide during the PCR reaction. A preferred-labeled nucleotide may be ³³P-dCTP.

Preferably, samples in which a mutation has been identified, may be sequenced in a further step, for verifying the existence of a mutation revealed in the former step (e) or (f) of the method of the invention, by comparing it to the corresponding wild type sequence.

In an embodiment thereof, the invention provides a method for identifying mutations in large genes, for example in the NFl, NBS1 or ATM genes. Preferably, the mutations are identified in patients and/or in asymptomatic individuals.

Most preferably, the method of the invention is intended for identifying patients at risk to develop malignancy. Such malignancy may be selected from the group consisting of carcinoma, melanoma, lymphoma, sarcoma and leukemia.

In a specific example, the method of the invention is intended for identifying a specific mutation in the NF-1 gene. A further example relates to the identification of different mutations in the ATM and the NBS1 genes in patients diagnosed with lymphoid malignancies such as T cell ALL, T cell lymphoma and Hodgkin's lymphoma.

In a further embodiment, the invention provides a method for screening for homozygous or heterozygous carriers of mutations in said genes. This is specifically useful in genetic counseling.

In yet another embodiment, the invention provides a method of prenatal diagnosis of a fetus, comprising the steps of obtaining nucleic acid from a sample comprising fetal cells; subjecting the nucleic acid obtained to the method of the invention, whereby the presence of a mutation in said nucleic acid indicates that said fetus carries the mutation. The sample used may be amniotic fluid, or is chorionic villi. The present invention further provides a method for screening for any one of mutations and polymorphism associated with a desired trait in a plant.

In another preferred embodiment, the method of the invention is intended for screening plants for the existence of a specific mutation leading to a desired phenotype in said mutated plant.

Another aspect of the present invention relates to a kit for the detection of mutations in a gene. The kit provided by the invention comprises: a. means for producing segments of at least about 700bp each from the gene examined, DNA polymerase and buffers for primer extension reaction, which reaction, or preferably PCR, results in the creation of the segments; b. at least two specific endonucleases for each said segment of said gene; each of the endonucleases chosen having a single restriction site in a given segment, to give two unequal fragments of different length each, the smaller fragment in one of the digestion reactions overlapping with the larger fragment in the other digestion reaction; and the single restriction site of a certain endonuclease being different for each of said endonucleases; c. optionally, SSCP gel and suitable buffers; and d. instructions for carrying out the detection of mutations in a gene according to the method of the invention.

In a preferred embodiment, said means for producing segments of at least about 700bp each from said gene are specific primers, DNA polymerase and buffers for primer extension reaction.

In a specifically preferred embodiment, the kit provided by the invention is intended for detection of mutations in a gene such as NF-1, ATM and NBS-1. In yet another preferred embodiment the kit is intended for detection of mutations in plant genes.

Brief Description of the Figures

Figure 1 Results of analysis of the NFl gene

The left-hand side gel (lanes 1 to) shows analysis of the NF1-6 fragment digested with EcoRI, the right-hand side gel (lanes 7 to 12) shows digestion of the NF1-6 fragment with Mspl. The mutation appearing in the 552 bp and the 356 bp fragments is indicated by an arrow.

Lanes 1, 4-6 and 7, 10-12 are NFl patients samples; 2 and 8 are non- NFl control samples, and lanes 3 and 9 are samples from NFl patient with

Optic Glioma. The suspected sample is in lanes 3 and 9.

Figure 2A-2B Analysis of the NFl gene

Fig. 2A shows gel analysis of the NFl-3 fragment. The left-hand side of the gel (lanes 1 to 7) shows fragments digested with Bmpl, while the right-hand side (lanes 8 to 14) shows the same fragments digested with Msp Al. The mutations appear in the 528 bp Bmpl fragment (left hand side, upper arrows) and in the 489 bp Mspl fragment (right hand side, lower arrows). Lanes 1 4, 6, 7 and 8, 11, 13, 14 are samples from non-NFl controls, lanes 2, 3 and 9, 10 are samples from NFl sibhng patients, lanes 5 and 12 are samples from non-NFl control with optic glioma. The suspected samples are in lanes 2, 3 and 9, 10.

Fig. 2B shows a sequencing gel identifying the mutation as a TGT (non-mutated (N), left-hand side) to CGT (mutated (M), right-hand side) substitution. Figure 3A-B SSCP analysis of NF1-5 fragment

Fig. 3A - SSCP analysis was performed on samples obtained from the

NF-1 patient parent (lane 12) as well as from different NF-1 patients

(lanes 1-11 and 13-16, 17). Samples were digested using BamHI and

MspAI as indicated.

Fig. 3B - shows sequence analysis of exon 26 sense (S) and antisense (A) of the parent suffering from NFl (P) and a healthy control (N).

Figure 4 Sequence analysis of exon 26 of a fetus

DNA obtained from amniotic fluid of a fetus was subjected to DNA sequence analysis of EXON 26 of the NFl gene. Sequence of the fetus (F) was compared to sequence obtained from his NFl parent (P) and a normal subject (N).

Figure 5 Analysis of the ATM gene

Figure 5 shows the location of the restriction sites Apol and Bsp 12861 within the ATM6 fragment. Gels of the two digests of the ATM6 fragment are shown. The Apol (lanes 1, 2) and the Bsp 12861 (lanes 3,4) are shown. The mutated sample (arrowed) is shown in the 593 bp of the Apol and 649 bp of the Bsp 12681 digests. The suspected sample is in lanes 1 and 3.

Figure 6 Analysis of the NBSl gene

Figure 6 shows identification of the 553 G/C polymorphism in the 696 bp fragment of Aat II (lanes 1 to 4) and the identification of the polymorphism of the 102 G/A , in the 640 bp fragment of Pflm I (lanes 5 to 8).

In the left-hand gel, lanes 1 and 4 are samples from Homozygotes to 553/C, lane 2 is a sample from Homozygote to 553/A, and lane 3 is a sample from heterozygote 553 G/C. In the right-hand gel lanes 5 and 8 are samples of homozygous to 102/A, lane 6 is a sample of homozygous to 102/G and lane 3 is a sample from heterozygous 102 G/A.

Figure 7A-7B Comparative Results of RT-PCR and modified SSCP analysis of the ATM gene Fig. 7A shows the RT-PCR products of an ATM sample, of independent RT-PCR analyses, are shown in lanes 1 to 3. Only in lane 3, an additional band is visible. This band is the consequence of a mutated allele. Fig. 7B shows a modified SSCP analysis of the RT-PCR product of lane 2 of Fig. 7A. A suspected pattern is observed (sample is labeled by an arrow).

Detailed Description of the Invention

For purposes of clarity and as an aid in the understanding of the invention, as disclosed and claimed herein, the following terms and abbreviations are defined below:

Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a "nucleotide sequence", and then grammatical equivalents, and is represented herein by a formula whose left to right orientation is in the conventional direction of 5'-terminus to 3' -terminus. Polynucleotide: a polymer of single or double stranded nucleotides. As used herein "polynucleotide" and its grammatical equivalents will include the full range of nucleic acids. A polynucleotide will typically refer to a nucleic acid molecule comprised of a linear strand of two or more deoxyribonucleotides and/or ribonucleotides. The exact size will depend on many factors, which in turn depends on the ultimate conditions of use, as is well known in the art. The polynucleotides of the present invention include primers, probes, RNA/DNA segments, oligonucleotides (relatively short polynucleotides), genes, and the like.

Base Pair (bp): a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA, uracil (U) is substituted for thymine. Base pairs are said to be "complementary" when their component bases pair up normally when a DNA or RNA molecule adopts a double stranded configuration.

Complementary Nucleotide Sequence: a sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to another single strand to specifically (non-randomly) hybridize to it with consequent hydrogen bonding. The term complementary, as used herein, means that two homologous nucleic acids, e.g., DNA or RNA, contain a series of consecutive nucleotides which are capable of forming base pairs to produce a region of double-strandedness. This region is referred to as a duplex.

Unequal fragments: The term "unequal" means that the sizes of the fragments obtained after restriction digest are sufficiently different so that the restricted fragments can be clearly separated on an SSCP gel. An example of unequal fragments can be found in Fig. 1, where the 552 bp and the 494 bp fragments are clearly separated. A number of methods of the art of molecular biology are not detailed herein, as they are well known to the person of skill in the art. Such methods include general molecular biology methods, RT-PCR reactions, sequencing, SSCP, and the like. Textbooks describing such methods are e.g., Sambrook et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology ,by F. M. Ausubel, ISBN: 047150338X, 1988, Short Protocols in Molecular Biology, by F. M. Ausubel et al (eds.) 3^rd ed. John Wiley & Sons; ISBN: 0471137812, 1995, and Current Protocols in Human Genetics, John Wiley & Sons, Inc., Dracopoli et al, (eds), 1994, ISBN: 0471034207. These publications are incorporated herein in their entirety by reference. The RT-PCR technique is well known to the skilled person and is described in a number of textbooks, see, e.g., The PCR Technique: RT-PCR, The BioTechniques Update Series, by Paul D. Siebert (Ed.), Eaton Pub Co, ISBN:1881299139, 1998, Quantitation of mRNA by PCR, Kohler et al, Springer, Berlin, 1995. A method for the designing of PCR primers is described in Griffais et al, Nucleic Acids Research 19, 3887-3891, (1991), and references therein, incorporated herein by reference.

The method provided by the invention is based on PCR or RT-PCR and electrophoresis on SSCP gels special for long fragments, and is sometimes referred to a "modified SSCP". Using the modified SSCP method of the invention for screening the NF-1 gene, 10 overlapping fragments that cover the entire coding region of the NFl gene can be screened at once for mutations. The method of the invention is capable of detecting NFl mutations in 29 out of 31 screened; i.e., 93.5% of sensitivity. The mutations identified generally fall into five classes: deletions, nonsense, insertion, exon skipping and missense. All of these mutations can be identified with the method of the invention. As a first aspect the present invention relates to a method for the identification of mutations in a gene comprising the steps of:

(a) obtaining nucleic acid from a sample;

(b) preparing from said nucleic acid large segments of said gene of at least about 700bp each;

(c) independently digesting each of said segments with at least two different restriction enzymes, each digestion in separate reaction, each restriction enzyme having a single restriction site in said segment, which is different from the restriction site/s of the other restriction enzyme/s, where digestion with each restriction enzyme gives, for each of said segments, two unequal fragments of different length each;

(d) subjecting the digestion fragments obtained by each of said restriction enzymes in step (c) to SSCP analysis, whereby said two unequal fragments of different length each are separated; and

(e) comparing the gel-banding patterns of overlapping fragments, resulting from separate digestion reactions of the same in step (c), that were separated on different lanes in the SSCP analysis of step (d), to a control sample, whereby any difference in the resulting gel-banding patterns of overlapping fragments, between the tested sample and the control, indicates the existence of mutation in said segment.

In a preferred embodiment, preparation of large segments may be performed by a primer extension reaction.

As used herein, the term "nucleic acid" refers to polymer of nucleotides, which may be either single- or double-stranded, which is a polynucleotide such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded p oly nucle otide s .

The term DNA used herein also encompasses cDNA, i.e. complementary or copy DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).

As used herein, the term "gene" refers to a nucleic acid comprising an open reading frame, coding for an RNA, DNA or polypeptide molecule. Genes may be uninterrupted sequences of nucleotides or they may include such intervening segments as introns, promoter regions, splicing sites and repetitive sequences. The term "intron" refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.

By primer is meant a polynucleotide, whether purified from a nucleic acid restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a template nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization, such as DNA polymerase, reverse transcriptase and the like, under suitable temperature and pH reaction conditions.

The primer is preferably single-stranded for maximum efficiency, but may alternatively be in double-stranded form. If double-stranded, the primer is first treated to separate it from its complementary strand before being used to prepare extension products. Preferably, the primer is a polydeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agents for polymerization. The exact lengths of the primers will depend on many factors, including temperature and the source of primer. For example, depending on the complexity of the target sequence, a polynucleotide primer typically contains 15 to 25 or more nucleotides, although it can contain fewer nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with template.

The choice of a primer's nucleotide sequence depends on factors such as the distance on the nucleic acid from the hybridization point to the region coding for the mutation to be detected, its hybridization site on the nucleic acid relative to any second primer to be used, and the like.

A primer is selected to be "substantially" complementary to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.

In a specific embodiment of the method of the invention, the nucleic acid is mRNA. In this embodiment, a reverse transcription of the mRNA, to form cDNA, is performed using a reverse transcriptase and random hexamers as primers. The resultant cDNA is then subjected to preparation of large segments, preferably by primer extension reaction. The segments preparation is then followed by endonuclease digestion and SSCP analysis as specified in steps (b) to (e) of the method of the invention.

Alternatively, the method of the invention employs genomic DNA (gDNA) as nucleic acid. Large segments of at least about 700bp, are then prepared, preferably by primer extension reaction. The primer extension reaction is performed on the gDNA and the resulting segments are then analyzed according to subsequent steps of the method of the invention.

In a specifically preferred embodiment, the method of the invention employs for each segment, two different restriction enzymes, each of which has a single restriction site in said segment, so that said segment is digested into two unequal fragments of different length each. Each of the enzymes chosen has restriction site that is different from the site of the other restriction enzyme/s.

In a particularly preferred embodiment, the two restriction enzymes are chosen such that the smaller fragment produced by each digest is not derived from the same end of the undigested fragment as the smaller fragment produced by the other restriction enzyme. This is demonstrated in Fig. 1, where it can be seen that the site where EcoRI digests is located left of the center of the undigested segment, while the restriction site for Mspl is located right of the center. The resulting small fragment of the EcoRI digest overlaps the large, but not the small, fragment of the Mspl digest. More preferably, the smaller fragment of said unequal fragments of different length produced in one of the digestion reactions, overlaps with the larger fragment of said unequal fragments of different length produced in the other digestion reaction. The specific choice of overlapping fragments enables the detection of mutations which otherwise could not be detected. Example for such case can be found in Fig. 2, in Example 1 of the present application. In this analysis, separation of the 528 fragment could hardly indicate the existence of a mutation. Only comparison to the gel-bending pattern of the overlapping fragment (356bp) enabled the detection of this point mutation. Thus, the specific choice of endonucleases creating the specific overlapping fragments, is essential for the present invention. Moreover, the use of overlapping fragments enables precise location of the mutation.

According to a specifically preferred embodiment, the sample used by the method of the invention may be obtained from any one of eukaryotic and prokaryotic organisms selected from the group consisting of vertebrates, invertebrates, plants, bacteria, yeast and fungi. More preferably, the sample may derived from mammahan vertebrates such as humans and bovine, equine, canine, murine and feline animales. Most preferably, the sample is obtained from a human subject.

A control sample, according to another embodiment of the invention, is a sample of same type obtained from a healthy subject of the same species. Preferably, the subject may be a mammahan subject. The term mammal includes both human and non-human mammals. More preferably, the subject is a human.

The sample that can be used for the method of the invention may be a peripheral blood, bone marrow, tumor, and embryonic cells sample. Preferably, the sample is a peripheral blood sample. Methods for obtaining such samples are well known to the skilled workers in the fields of oncology and surgery. They include sampling blood in well-known ways, or obtaining biopsies from the bone marrow or other tissue or organ.

The inventors have established a new mutation screening method, particularly efficient for large genes. A large gene would be a gene comprising over 8 exons, usually over 10 exons and/or 2 kb or more.

The method of the invention is preferably intended at identifying nonsense, missense, insertion, frame shift, transition, transversion, re-arrangement, deletion mutations and mutations in splicing sites that may cause exon-skipping, insertions or deletions.

A mutation, as used herein, refers to a nucleotide sequence change (i.e., a nucleotide substitution, deletion, or insertion) in an isolated nucleic acid. An isolated nucleic acid which bears a mutation has a nucleic acid sequence that is statistically different in sequence from a homologous nucleic acid isolated from a corresponding wild-type population.

Occasionally, an incorrect base pairing does occur during replication, which, after further replication of the new strand, results in a double-stranded DNA offspring with a sequence containing a heritable single base difference from that of the parent DNA molecule. Such heritable changes are called genetic mutations, or more particularly in the present case, "single base pair" or "point" mutations. The consequences of a point mutation may range from negligible to lethal, depending on the location and effect of the sequence change in relation to the genetic information encoded by the DNA.

Whereas the normal base pairings in DNA (A with T, G with C) involve one purine (A and G) and one pyrimidine (T and C), the most common single base mutations involve substitution of one purine or pyrimidine for the other (e.g., A for G or C for T), a type of mutation referred to as a "transition". Mutations in which a purine is substituted for a pyrimidine, or vice versa, are less frequently occurring and are called "trans versions". Still less common are point mutations comprising the addition or loss of a single base arising in one strand of a DNA duplex at some stage of the replication process. Such mutations are called single base "insertions" or "deletions", respectively, and are also known as "frame-shift" mutations, due to their effects on translation of the genetic code into proteins. Larger mutations affecting multiple base pairs also do occur and can be important in medical genetics, but their occurrences are relatively rare compared to point mutations.

Mapping of genetic mutations involves both the detection of sequence differences between DNA molecules comprising substantially identical (i.e., homologous) base sequences, and also the physical locahzation of those differences within some subset of the sequences in the molecules being compared.

A mutant nucleic acid which includes a single nucleotide change or multiple nucleotide changes will form one or more base pair mismatches after denaturation and subsequent annealing with the corresponding wild type and complementary nucleic acid. For example, G:A, C:T, C:C, G:G, A:A, T:T, C:A, and G:T represent the eight possible single base pair mismatches which can be found in a nucleic acid heteroduplex, wherein U is substituted for T when a nucleic acid strand is RNA. Nucleic acid loops can form when at least one strand of a heteroduplex includes a deletion, substitution, insertion, transposition, inversion of DNA or RNA and mutations in splicing sites that may cause exon-skipping, insertions or deletions. The method of the invention enables identifying substantially all mutations within the mRNA sequence of said gene, all mutations within the coding region of said gene. Further, the method of the invention is intended for identifying mutations in at least one intron of said gene. As mentioned above, the method of the invention can be applied not only to mRNA, but also to genomic DNA. Today, introns are becoming more widely recognized as having important gene -regulatory roles, such as containing enhancer or silencing elements. It has been predicted that some of the intronic polymorphisms may predispose toward coding-region mutations [Malkinson You, M. Moi. Carcinogenesis 10:61-65 (1994)]. For example, in the 37 bp repeat in the second intron of Ki-ras, a single copy of the repeat is associated with lung tumor development in mice, while two copies are present in tumor resistant strains [You et al, Proc. Nat. Acad. Sci. USA 89:5804-5808 (1992)]. Another example is a minisatelhte upstream from the polyadenylation signal of Ha-rasl. The frequency of the various alleles has shown a rare allele twice as often in cancer patients. Furthermore, it has been shown that these alleles possess two-fold greater enhancer activity. It has been concluded that these variations in the sequence may lead to the disruption of transcriptional controls [Green and Krontiris, Genomics 17:429-434 (1993)]. In the inventors laboratory, a novel germ line p53 mutation has been shown in intron 6 in diverse childhood malignancies. This mutation stabilized the p53 protein, resulting in its abnormal accumulation [Avigad et al, Oncogene 14:1541-1545 (1997)].

It is therefore advantageous to provide a method for screening mutations in introns. The method of the invention, which is particularly suitable for screening large nucleic acid sequences, may thus be advantageous in screening genomic DNA. Thus, in a specifically preferred embodiment the method of the invention is intended at identifying polymorphism in the gene examined.

The term "polymorphism" is often used to denote a sequence variation in DNA which is benign. In the present invention, it is to be understood that the term "mutation" refers to any DNA fragment which has a base sequence which varies from the wild type and includes "polymorphisms".

Single nucleotide polymorphisms are the most common type of DNA sequence variations and occur once every 100-300 bases. Researchers looking for associations between a disease and specific differences in a population use these SNPs (single nucleotide polymorphisms). SNPs present a potentially vast arena for the detection of genetic alterations that seem to relate to medically important differences in disease susceptibility and drug response.

The information on DNA sequence variation could distinguish those individuals who are likely to benefit from a new medication from those who could suffer adverse side-effects, or to determine the optimal dosage.

As described in the Examples, the method of the invention could easily detect SNPs, as single base polymorphisms have been detected in all the genes screened up until now.

In a preferred embodiment the segment is prepared by primer extension reaction. This primer extension product is preferably a long fragment, more preferably about 2000 base pairs (bp) to about 1,500 bp, most preferably, about 1,000 bp to about l,200bp in length.

The method of the invention involves preparation of a large segment preferably by a primer extension step, which comprises nucleic acid amplification. Preferably, the primer extension and amplification step may be PCR.

As used herein "PCR" (polymerase chain reaction) refers to a process of amplifying one or more specific nucleic acid sequences by repeated rounds of synthesis and denaturing under appropriate "amplification conditions".

PCR requires two primers that are capable of hybridization with a single-strand of a double-stranded target nucleic acid sequence which is to be amplified under appropriate "hybridization conditions". In PCR, this double-stranded target sequence is denatured and one primer is annealed to each single-strand of the denatured target. The primers anneal to the target nucleic acid at sites removed (downstream or upstream) from one another and in orientations such that the extension product of one primer, when separated from its complement, can hybridize to the extension product generated from the other primer and target strand. Once a given primer hybridizes to the target sequence, the primer is extended by the action of a DNA polymerase. DNA polymerase which is heat stable is generally utilized so that new polymerase need not be added after each denaturation step. Such thermostable DNA polymerase would be known to one of ordinary skill in the art, such as Taq polymerase. The extension product is then denatured from the target sequence, and the process is repeated.

The primer extension or PCR product may be un-labeled. In this case, the gel-banding pattern of the digested fragments may be visualized by silver staining. Alternatively, the primer extension or PCR product may be body-labeled, most preferably by using labeled nucleotide during the PCR reaction. The term "label" as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. A preferred labeled nucleotide according to the invention may be ssp-dCTP.

The digestion of segments produced by the PCR is carried out for a time suitable to achieve complete digestion, preferably between one hour and 48 hours, more preferably between 6-8 hours. The preferred amount of DNA digested is between about lng and about lμg, more preferably about lOOng. The preferred amount of restriction enzyme used is between about 1 unit and about 20 units, more preferably about 4 units. The person of skill in the art will recognize that the amount of enzyme used and the amount of DNA to be digested, as well as the reaction buffer conditions and digestion time and temperature conditions, are dependent upon each other, with respect to the desired result of complete digestion, but lack of over-digestion.

The modified SSCP analysis of the invention is preferably carried out using a polyacrylamide gel, more preferably 5% acrylamide, with l/30^th bis-acrylamide added (1:29 bis-acrylamide:acrylamide). The acrylamide gel preferably contains glycerol, more preferably about 5% glycerol. The gel is run preferably for between about 3 hours and about 14 hours, more preferably about 7 hours. The power used to drive the DNA through the gel is preferably about 40 Watts. The gel is preferably run at room temperature. More preferably, the gel is run at room temperature with cooling using a fan, so that the gel is not heated. Preferably, samples in which a mutation has been identified, may be sequenced in a further step, for verifying the existence of a mutation revealed in the former step (e) or (f) of the method of the invention, by comparing to the corresponding wild type sequence.

In an embodiment thereof, the invention provides a method for identifying mutations in large genes, for example in the NFl, NBSl or ATM genes. Preferably, the mutations are identified in patients and/or in asymptomatic individuals.

Most preferably, the method of the invention is used in the diagnosis of genetic diseases and the diagnosis and prognosis of human cancer. More particularly, the method of the invention is intended for identifying patients at risk to develop malignancy. Such malignancy may be selected from the group consisting of carcinoma, melanoma, lymphoma, sarcoma and leukemia, but is not h ited thereto. Genetic material from patients can be screened for mutations in a gene of interest, for example the NFl, NBSl or ATM genes.

As used herein to describe the present invention, "cancer", "tumor" and "malignancy" all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the method of the present invention as well as kit of the present invention may be used in the diagnosis of non- solid and solid tumors.

In a further embodiment, the invention provides a method for screening for homozygous or heterozygous carriers of mutations in said genes. This is specifically useful in genetic counseling. When alleles at any one locus are identical, the individual is said to be "homozygous" for that locus, when they different the individual is said to be "heterozygous" for that locus. Allele is a variant of DNA sequence of a specific gene. In diploid cells a maximum of two alleles will be present; each in the same relative position or locus on homologous chromosomes of the chromosome set. Since different alleles of any one gene may vary by only a single base, the possible number of alleles for any one gene is very large. When alleles differ, one is often dominant to the other, which is said to be recessive. Dominance is a property of the phenotype and does not imply inactivation of the recessive allele by the dominant. In numerous examples the normally functioning (wild-type) allele is dominant to all mutant alleles of more or less defective function. In such cases the general explanation is that one functional allele out of two is sufficient to produce enough active gene product to support normal development of the organism (i.e., there is normally a two-fold safety margin in quantity of gene product).

Still further, the invention provides a method for prenatal diagnosis in the fetus, comprising the steps of: (a) obtaining nucleic acid from a sample comprising fetal cells; subjecting the nucleic acid obtained in step (a) to the method of the invention, whereby the presence of a mutation in said nucleic acid sequence indicates that said fetus is a carrier of said mutation. The sample used may be amniotic fluid, or is comprised of chorionic villi.

The method of the invention may be adapted for automated use, for example with fluorescent labeling and/or radioactive labeling, which are techniques known to the man skilled in the art.

The present invention further provides a method for screening of any one of mutations and polymorphism associated with a desired trait in a plant. Analyzing candidate genes will enable the development of new products for improved crops having improved quahty, safety and lower cost. Such improved products may have for example, increased nutritional value or decreased need for pesticides.

In yet a further embodiment, the invention provides a method for screening plants for the existence of a specific mutation leading to a desired phenotype in said mutated plant.

Another aspect of the present invention relates to a kit for detection of mutations in a gene. The kit provided by the invention comprises:

(a) means for producing segments of at least about 700bp each from the gene examined;

(b) at least two specific endonucleases for each said segment of said gene, each of the endonucleases chosen having a single restriction site in the segment, to give two unequal fragments of different length each, the smaller fragment in one of the digestion reactions overlapping with the larger fragment in the other digestion reaction, the single restriction site of a certain endonuclease being different for each of said endonucleases;

(c) SSCP gel and suitable buffers; and

(d) instructions for carrying out the detection of mutations in a gene according to the method of the invention.

According to a preferred embodiment, wherein preparation of the segments is by a primer extension reaction, the kit of the invention comprises as means for producing said segments; DNA polymerase and buffers for primer extension reaction, the primer extension reaction, or preferably PCR. Optionally, the kit according to the present invention may further comprise reverse transcriptase, random hexamers primers and buffers suitable for reverse transcription of mRNA into cDNA, that otherwise are commercially available.

The PCR mixture included in the kit provided by the present invention, may contain the control target DNA, the DNA primer pairs, four deoxyribonucleoside triphosphates (A, T, C, G), MgCl₂, DNA polymerase (thermo-stable), and conventional buffers.

In a preferred embodiment, the kit provided by the invention is intended for detection of mutations in a gene such as NF-1, ATM and NBS-1.

In yet another preferred embodiment, the kit of the invention is intended for detection of mutations in a plant genes.

A number of studies have focused on one portion of the gene (or one exon a time) using the traditional methods for mutation screening. However, none of the prior art methods used is capable of detecting all mutations. The PTT (protein truncation test) was shown to be able to identify mutations in 73% of patients [Park et al, J Med Genet 35:813-820 (1998)]. This is the highest mutation rate detected in a study of NFl patients. The limitations of PTT are that missense mutations are not detected (reference is also made to Osborn and Upadhyaya (1999) supra).

To date, because of technical limitations, despite the very high prevalence of NFl in the populations all over the world, it was impossible to identify the whole scope of the mutations. With the introduction of the method of the invention, it will be possible to identify most of the NFl mutations. This will extend insight into this disease and also other diseases where the screening method of the invention is applied, such as in screening the ATM gene. The method of the invention will thus help clarifying the unknown facts in the genetic background of carriers and their families, namely, distinguish between familial and sporadic cases, and define those who are prone to the more severe phenotype of the disease. Moreover, it may help identifying those carriers with predisposition to develop cancer. Knowing that, it will be possible to provide the carriers/patients with the appropriate surveillance, early detection and early prevention. This will be one of the very rare cases in medicine and agriculture, where the appropriate application of molecular biology will help in providing the optimal care for patients, e.g., children, including genetic counseling, prenatal diagnosis and optimal therapy.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. The following Examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Examples

Experimental procedures

DNA preparation

DNA was prepared according to the procedure described in Miller et al

[Miller et al, Nucl. Acid Res. 3:1215 (1983)].

RNA preparation

RNA was prepared using the RNA/DNA/Protein isolation reagent. Tri Reagent according to the manufacturer's protocol. Reagent was purchased from Molecular Research Center, INC.

RT-PCR

RT-PCR was performed using the Ready To Go, RT-PCR Beads, according to the manufacturer's protocol. Beads werepurchased from Amersham pharmacia biotech.

Radioactive labeling of fragments

Labling of fragments was performed using Amersham pharmacia biotech

P33 α-Dctp, o.3 μCi. per tube according to the manufacturer's protocol. Restriction enzymes

The following restriction - enzymes were obtained from New England Biolabs and from MBI Fermentas: EcoRI, Bglll, Bpml, MspAI, Xbal, Nhwl, BamHI, Mspl, Apol, Aval, Dral, Msll, Avail, BsaJI, Avrll, NdEI, Hindi, Banl, BsaAI, Bsp, Earl, Eael, Earl and BsaBI.

Tlie modified SSCP method

Generally, the screening method of the invention consists of a number of steps. A genomic DNA (gDNA) or mRNA (first converted to cDNA) of the gene which is to be screened for mutations is obtained. The cDNA or gDNA are then amplified in fragments of about 1,000 to about 1,200 bp. Generally, this is done by PCR. At this stage, the fragments may optionally be labeled, e.g., by end-labeling or by body-labeling during the PCR reaction. As the sequence of the gene to be screened is known, primers can be designed specifically for that purpose. In the next step, the amplified fragments are digested with a restriction enzyme. The enzyme is chosen to generate two fragments of unequal size. In a preferred embodiment, two independent restriction digests are carried out for each fragment, each digest resulting in two fragments of unequal length. Preferably, the smaller fragment produced in one of the digests overlaps the larger fragment produced in the other digest. After completion of the reaction, the fragments are then run out on an SSCP gel containing glycerol. The banding pattern on the gel enables quick and easy identification of mutations, by comparison to non-mutated samples run in parallel.

Specifically, the method of the invention is as follows:

(a) extraction of nucleic acids from samples using standard methods. The samples may be taken from a tumor, peripheral blood, bone marrow, prenatal or embryonic cells. In case that the nucleic acids obtained are mRNA, conversion into cDNA should be performed by RT(reverse transcription) using random hexamers.

(b) designing primers for PCR reactions that divide the relevant gene to segments of 1000-1200 base pairs, and choosing 2 restriction enzymes that restrict the fragments to 2 unequal fragments (Tables 1 and 3).

(c) PCR of each segment in the presence of ³³P-dCTP or alternatively, without any radioactive nucleotide.

(d) digesting each segment for between 6-8 hours in two tubes of restriction analysis of 100 ng of cDNA in each tube using about 4U of each enzyme.

(e) electrophoresing the products of the restriction analyses on 5% polyacrylamide gel (1:29 bis:polyacrylacmide) containing 5% glycerol with on for 7 hours 40 Watts, at room temperature using a cooling system, or for over-night (12-17 hours) 12-14 Watts and exposing to film overnight when radioactive material is used, or silver staining and then exposing to film for 2-3 minutes.

(f) analysing the autoradiograph for results.

(g) directly sequencing suspected samples (Tables 2 and 4).

Example 1

Screening the NFl gene for mutations

The study undertaken for screening the NF-1 gene consisted of screening a group of 31 chnically well characterized NFl patients by the Neurological Department, Schneider Children's Medical Center of Israel, according to the NFl criteria according to the NFl consortium. Of these 31 patients, 9 had also developed optic ghoma — a frequent malignancy in NFl patients. Twenty-eight controls were used: 14 normal individuals and 14 patients who developed malignancy, but without NFl. Blood samples were obtained from the patients according to known procedures and in accordance with NIH guidelines. Informed consent was obtained from all patients and healthy volunteers. White blood cells were isolated and RNA was extracted using TRI Reagent (Molecular Research Center, OH, USA) according to the manufacturer's instructions.

The RNA was converted to cDNA using random hexamers, as described above in "methods". PCR fragments of the desired size were then amplified from the cDNA. The fragments and sizes are listed in Table 1. After amplification of the fragments, about 100 ng of each fragment was digested in separate reactions with two restrictions enzymes specifically chosen for each fragment so as to result in two unequal restriction fragments. The restriction enzymes chosen for each fragment, along with the position of the restriction site and the sizes of the resulting restriction fragments are listed in Table 1 on the right-hand side.

Table 1

NFl Fragments

Name Size (bj a) Nucleotides Restriction Size

NF1-1 974 256 - 1230 BbSI - 723 467 507

EcoRI - 616 360 614

NF1-2 1068 1011-2079 Bglll - 1395 384 684

EcoRI - 1411 400 668

NFl-3 1024 1697-2721 Bpml - 2193 496 528

MspAI - 2232 535 489

NF1-4 1236 2559-3795 Xbal - 2977 418 818

Nhwl - 3167 608 628

NF1-5 1263 3437-4700 BamHI- 4114 677 526

MspAI- 4219 782 481

NF1-6 1046 4401-5447 EcoRI - 4895 494 552

Mspl - 5091 690 356

NF1-7 1140 5210-6350 Apol - 5921 714 429

Aval - 5746 536 604

NF1-8 1135 6058-7193 Mspl - 6694 636 499

MspAI- 6665 607 528

NF1-9 1095 6900-7995 Dral - 7332 432 663

Msll - 7587 687 408

NFl- 10 859 7778-8637 Avail - 8294 516 343

BsaJI - 8191 413 446

The modified SSCP analysis of the restriction segments was carried out as described in "methods". It showed that in 29 out of all 31 NFl patients, a mutation was identified. Thus, the method of the invention shows about 93.5% sensitivity in the NFl gene analysis. In contrast, in none of the 28 controls, a mutation was identified. Fig. 1 shows a part of the SSCP gel exposure showing the mutation in the NF-1 6 fragment. The mutation (arrow) can easily be identified by the change in the banding pattern, both when using the EcoRI enzyme and when using the Mspl enzyme. Fig. 2 shows an example of mutations identified in the NFl-3 fragment, and the sequence analysis thereof. Again, the mutations (arrows) are easily identified by the change in banding pattern, both with the Bmpl and with the Msp Al digest. However, the bands resulting from the mutation are running close to the non-mutated bands when using the Bmpl digest (arrow, see the 528 bp fragment). The second enzyme digest assures identification of the mutation, since the mutated band is more clearly distinguished in this digest. SSCP gels may not always resolve bands optimally, so that a mutated band running very close to a non-mutated band, like in the Bmpl digest shown in Fig. 2A, may be missed. However, by running a second restriction enzyme digest in parallel, detection of such mutations can be achieved with great accuracy. Fig. 2B, further shows a portion of a sequencing gel identifying the nucleotide change in the mutation shown in Fig. 2A: The non-mutated sequence (N-left hand side) is TGT, while the mutated (M) sequence is CGT, i.e., a single nucleotide substitution. A complete Hst of all mutations identified in the screening is given in Table 2. From the data in this table, it is clear that majority of the mutations are skipping mutations, but other types of mutations such as nonsense, missense, deletion and insertion mutations could be identified. The method of the invention identifies even mutations that reside outside exons, but affect splicing, such as exon skipping mutations. Table 2

NFl mutations identified

Fragment Exon Change Codon/ Number Nucleotide patients

NF1-1 3 TGT TAT C489Y 1

NF1-2 int 9 (-2 exon 10) GT CT skipping 10a 1

NF1-2 10c delet 2 bp 1752delAG 1

NFl-3 12a delet 1 bp 1948delT 1

NFl-3 13 TGT CGT C709R 1

NFl-3 13 delet 1 bp 2333delT 1

NF1-4 19a TAC^TAG Y1044Term 1

NF1-4 21 delet 2 bp 3736delAA 1

NF1-4 22 delet 2 bp 4030delTC 1

NF1-5 24 insertion 50 bp 1

NF1-6 24 insert 1 bp 4421insA 1

NF1-6 28 skipping 28 1

NF1-6 28 delet 1 bp 5334delC 1

NF1-6 29 skipping 1

NF1-6 29 CGA^TGA R1748Term 1

NF1-7 31 CAA-»TAA Q1972Term 1

NF1-7 32 AGT-^AAT S1232N 1

NF1-8 34 skipping 34 1

NF1-8 36 cryptic splice site 6938AT-»GT 1

NF1-8 36 CGA-»TGA R2237Term 1

NF1-8 36 skipping 36 2

NF1-8 37 delet 2 bp 7000delAA 1

NF1-9 42 GTC A2511V 1 Parental diagnosis of sporadic neurofibromatosis type 1 Currently, mutations in the NFl gene are detected throughout the gene, and the majority of the mutations are unique per patient. The combination of the high mutation rate in the gene and the hmited prenatal diagnosis to familial cases, or to already identified mutations resulted in poor prenatal diagnosis of sporadic neurofibromatosis type 1. The majority of the laboratories offering prenatal diagnosis of NFl, perform linkage analysis according to the familial cases only.

Therefore, one object of the present inventors was to develop a prenatal diagnosis, preferably for NFl, using the mutation detecting method of the invention.

Accordingly, a prenatal diagnosis for NFl was examined in a couple in which the husband had sporadic NFl and the wife was already 14 week pregnant. The husband satisfied the NFl criteria and was the first member of the family affected by the disease. Since this was a case of sporadic NFl, linkage analysis could not be offered.

Peripheral blood sample was obtained from the patient (the husband) and DNA and total RNA were prepared from the sample as described in the experimental procedures. RNA was reverse transcribed into cDNA and segments prepared from this cDNA were digested using BamHI and MspAI and were subjected to the modified SSCP method of the invention. As shown in Fig. 3A, an abnormal pattern was detected within a week, on the fragment NFl-5 containing exon 26 of the NF-1 patient parent (lane 12).

The fragment was next sequence d, revealing a 16 base pairs deletion in exon 26. The genomic DNA of exon 26 of the NFl gene, as well as the flanking introns were sequenced, and a base substitution of AG to AC in the splicing region was identified (Fig. 3B). This sphcing-mutation causes the observed 16 base pairs deletion.

In order to examine whether the fetus of this couple carries the mutation that was identified in a sample obtained from the father, a prenatal diagnosis was next performed.

Amniotic fluid was obtained from the pregnant wife (20 week pregnancy), DNA was extracted and sequence analysis was performed on the same NFl-5 fragment. As shown by the sequence analysis in Fig. 4, the sequence of the fetus DNA was identical to the sequence of the healthy control. These results indicate that the fetus did not carry his father's specific NFl mutation.

Thus, the method of the invention offers rehable and powerful tool for screening of sporadic mutation and implying the results on prenatal diagnosis.

Example 2

Identification of mutations within the ATM gene

The method of the invention was successfully applied in the detection of mutations of the ATM gene. The ATM gene is, like the NF-1 gene, rather large, and therefore detection of mutations therein with prior art methods has been difficult and suffered from lack of sensitivity. Up to date, 44 A-T mutations were identified [Gilad et al, Hum. Moi. Genet.

5:433-439 (1996)]. 89% of these mutations are truncating ones, resulting in inactivation of the ATM protein. Additional five novel mutations have been identified, completing the identification of the Israeli population.

The majority of A-T patients in Israel are North-African Jews, all carry the same mutation, a base substitution in exon 5 [Gilad et al, (1996) ibid.].

In the present study, patients diagnosed with lymphoid malignancies that are not A-T patients were screened for mutations in the ATM gene. The malignancies were T-cell acute lymphoblastic leukemia, T-cell lymphoma and Hodgkin lymphoma in children. Altogether, 72 children with a lymphoid malignancy were screened.

The screening was performed in a manner similar to that described in Example 1. The ATM mRNA extracted from the samples was reverse transcribed into cDNA, and 9 fragments of about 1,000 to about 1,100 bp length were amplified from this cDNA using designed specific primers. The PCR fragments amplified are listed in Table 3. Table 4 lists the mutations found in the screening.

T-cell ALL

35 children were studied: 33 children at diagnosis and 2 in relapse. Mutations were identified in 19/33 (58%) and in both children in relapse. The types of mutations were: missense, inversion, deletion, skipping of exons (Table 4). In addition 3 different polymorphisms in exons 31, and 39 were identified (Table 5).

The most prevalent mutations are: skipping of exons 20 and 34, in 6 and 8 patients, respectively. There was a significant correlation between the presence of a mutation, and worse outcome as revealed by Life table analysis (Kaplan-Meier) for overall survival of 56% in the ATM positive group versus 91% in the negative group (p=0.04). Thus, the identification of an ATM mutation could serve as an adverse prognostic marker in T-cell ALL for high risk patients and appropriate therapy should be given.

Furthermore, in 6 patients that normal tissue was available, the mutation was present in the germ hne of 5 of them (83%). Thus, these patients are carriers of A-T.

Fig. 5 shows an example of the modified SSCP gel-banding pattern in a mutation identified in exon 39 (fragment ATM-6 in Table 3), using Apol and Bsp 12861 digestion.

T-cell lymphoma

15 children were studied. Mutations were identified in 4 (27%) (Table4). In two patients the mutation is in the germ hne, thus both are carriers of A-T. Two different polymorphisms were identified: one unique to this population in exon 38, and a prevalent one in exon 39 (Table 5).

Hodgkin's lymphoma

20 children were studied, in 2 patients a mutation was identified. The mutations were missense: in exons 13 and 14 (Table 4). In both patients the mutation is in the germ hne, thus both are carriers of A-T. Two different polymorphisms have been identified: 1 unique for this malignancy in exon 5, and one very common in exon 39 (Table 5).

Table 3

ATM Fragments

Name Size I Nucleotides Restriction Size

ATM1 1021 137 - 1158 Avrll - 525 388; 648

Bsrl - 655 516; 505

ATM2 1172 1054 - 2226 EcoRI - 1753 699; 473

NdEI - 1546 492; 680

ATM3 1107 2178 - 3285 Hindi - 2606 428; 679

Banl - 2826 648; 459

ATM4 1152 3191 - 4343 Apol - 3626 435; 717 BgUI - 3934 743; 409

ATM5 1121 4212 - 5333 BsaAI - 4651 439; 682

Hindlll- 4868 656; 465

ATM6 1028 5220 - 6248 Apol - 5655 435; 593

Bspl286I-5869 649; 379

ATM7 1229 6181 - 7410 Earl - 6655 474; 755 Eael - 6971 790; 439

ATM8 1017 7304 - 8321 Earl - 7701 397; 620 BsaBI - 7972 668; 349

ATM9 1190 8191 - 9381 BamHI - 8709 518; 672

BglH - 8853 662; 528 Table 4

ATM Mutations Identified

Exon Change Codon Germ hne No. patients

ALL

7-9 inversion 1

11 GTG→GCG V409A + 1

*17 del2bp 762 + 1

19 TTT→CTT F858L + 3

*20 skipping + 6

30+31 skipping 1

*33 skipping 1

34 skipping 8

*34+35 skipping 1

Lymphoma

11 skipping + 2

*20 skipping + 2

Hodgkin's lymphoma

13 V595A +

* identified in A-T patients

These results indicate that the carrier rate of A-T in the population may be much higher than was previously estimated. This result stresses the necessity to study children and adolescents that have developed a lymphoid malignancy. This study may clarify the role of ATM gene in the mechanisms for the development of childhood lymphoid malignancies. It will be possible to estimate the carrier frequency of the ATM gene, and thus to establish whether the ATM gene contributes to the increased risk of cancer, particularly lymphoid malignancies in children.

Table 5

Polymorphisms identified in childhood lymphoid malignancies and their frequency in normal population

Exon Change No. alleles Frequency

/Patients

54 3/44 0.068¹

3/144 0.020

31 F1463C 1/104 0.0096

38 L1826L 1/30 0.033²

1/144 0.0069

39 D1853N 7/108 0.065

(1) frequency only in Hodgkin lymphoma

(2) frequency only in T-cell lymphoma

Example 3

Screening of mutations in the NBSl gene

The cDNA fragments that were obtained during the RT-PCR reaction with the NBSl gene specific primers were divided to 2 overlapping fragments. Table 6 illustrates the fragments and the restriction enzymes used. Thirty-five children diagnosed with T-cell acute lymphoblastic leukemia have been screened for mutations in the NBSl gene. So far, 10 patients are suspected to harbor a mutation by the screening method. In 3, a mutation has already been verified by sequence analysis, 2 in codon 389, and one in codon 355.

In addition, missense mutations that are known polymorphisms in this gene have been identified: G102A, T1197C and A2016G

Fig. 6 shows an example for analysis of the NBSl gene demonstrating identification of the 553 G/C and the 102 G/A polymorphism.

Table 6

NBSl Fragments and Restriction Enzymes

Fragment nucleotides size (bp) Enzyme nucleotide sizes (bp) 1 18-1252 1234 Aat II 555 546+696

Pflrn I 659 640+592

1175-2372 1197 Eae l 1719 544+653 Sty I 1829 654+543

Example 4

Sensitivity of the Modified SSCP method

As shown in Figs. 7A and 7B and described in the legends thereof, the modified SSCP analysis of the invention is very sensitive. Even though the mutated band was not visible after RT-PCR, signifying that a very small amount was present, the modified SSCP analysis clearly shows the mutation.

Claims

CLAIMS:

1. A method for the identification of mutations in a gene comprising the steps of:

(a) obtaining nucleic acids from a sample;

(b) preparing large segments of said gene of at least about 700bp each;

(c) independently digesting each of said segments with at least two different restriction enzymes, each digestion in separate reaction, wherein each restriction enzyme has a single restriction site in said segment, said single restriction site being different from the restriction site/s of the other restriction enzyme/s, to give for each of said segments two unequal fragments of different length each, for each restriction enzyme;

(d) subjecting the digestion fragments obtained in step (c) by each of said restriction enzymes to SSCP analysis, whereby said two unequal fragments of different length each are separated; and

(e) comparing the gel-banding patterns of overlapping fragments, resulting from separate digestion reactions in step (c) of the same segment, that were separated on different lanes in the SSCP analysis of step (d) to a control sample, whereby any difference in the resulting gel-banding patterns of overlapping fragments, between the tested sample and the control, indicates the existence of mutation in said segment.

2. A method for the identification of mutations according to claim 1, wherein said nucleic acids obtained are mRNA, which method comprises the steps of:

(a) obtaining mRNA from a sample;

(b) performing reverse transcription of said mRNA obtained in step (a) to form cDNA; (c) perepering from said cDNA large segments of said gene of at least about 700bp each;

(d) independently digesting each of said segments with at least two different restriction enzymes, each digestion in separate reaction, wherein each restriction enzyme has a single restriction site in said segment, said single restriction site being different from the restriction site/s of the other restriction enzyme/s, to give for each of said segments two unequal fragments of different length each, for each restriction enzyme;

(e) subjecting the digestion fragments obtained in step (d) by each of said restriction enzymes to SSCP analysis, whereby said two unequal fragments of different length each are separated; and

(f) comparing the gel-banding patterns of overlapping fragments, resulting from separate digestion reactions in step (d) of the same segment, that were separated on different lanes in the SSCP analysis of step (e) to a control sample, whereby any difference in the resulting gel-banding patterns of overlapping fragments, between the tested sample and the control, indicates the existence of mutation in said segment.

3. A method for the identification of mutations according to claim 1, wherein said nucleic acids obtained in step (a) are genomic DNA (gDNA) of said gene, which method comprising the steps of:

(a) obtaining genomic DNA (gDNA) of said gene from a sample;

(b) preparing from said gDNA large segments of said gene of at least about 700bp each;

(c) independently digesting each of said segments with at least two different restriction enzymes, each digestion in separate reaction, wherein each restriction enzyme has a single restriction site in said segment, said single restriction site being different from the restriction site/s of the other restriction enzyme/s, to give for each of said segments two unequal fragments of different length each, for each restriction enzyme;

(d) subjecting the digestion fragments obtained in step (c) by each of said restriction enzymes to SSCP analysis, whereby said two unequal fragments of different length each are separated; and

(e) comparing the gel-banding patterns of overlapping fragments, resulting from separate digestion reactions in step (c) of the same segment, that were separated on different lanes in the SSCP analysis of step (d) to a control sample, whereby any difference in the resulting gel-banding patterns of overlapping fragments, between the tested sample and the control, indicates the existence of mutation in said segment..

4. The method according to any one of claims 1 to 3, wherein two different restriction enzymes each having a different single restriction site in said segment, are employed in separate reactions, for digesting said^' segment into two unequal fragments of different length each.

5. The method according to claim 4, wherein the smaller fragment of said two unequal fragments of different length produced in one of the enzyme's digestion reactions, overlaps with the larger fragment of said unequal fragments of different length produced in the digestion reaction of the other enzyme.

6. The method according to claim 5, wherein said sample is obtained from any one of eukaryotic and prokaryotic organisms selected from the group consisting of vertebrates, invertebrates, plants, bacteria, yeast and fungi.

7. The method according to claim 6, wherein said vertebrates are mammalians selected from the group consisting of human, bovine, equine, canine, mice and feline.

8. The method according to claim 7, wherein said mammal is a human.

9. The method according to any one of claims 7 and 8, wherein said sample is selected from the group consisting of peripheral blood, bone marrow, tumors, and embryonic cells.

10. The method according to claim 9, wherein said sample is peripheral blood.

11. The method according to claim 9, wherein said control sample is obtained from a healthy subject of the same species.

12. The method according to claim 6, wherein said sample is obtained from a plant.

13. The method according to any one of claims 6 to 12 for identifying nonsense, missense, insertion, exon skipping, and deletion mutations in said genes.

14. The method according to any one of claims 6 to 12 for identifying polymorphism in said gene.

15. The method according to claim 2 for identifying substantially all mutations within the mRNA sequence encoded by said gene.

16. The method according to claim 1, for identifying substantially all mutations within the coding region of said gene.

17. The method according to claim 3, for identifying mutations in at least one intron of said gene.

18. The method according to any one of claims 1 to 3, wherein the segment is about l,000bp to about l,200bp in length.

19. The method according to any one of claims 1 to 3, wherein the preparation of said segment is by performing a primer extension reaction, which primer extension step comprises nucleic acid amplification.

20. The method according to claim 19, wherein the primer extension and amplification step is PCR.

21. The method according to claim 20, wherein the primer extension or

PCR product is not labeled.

22. The method according to claim 21, wherein the primer extension or

PCR product is body-labeled.

23. The method according to any one of claims 1 to 3, further comprising the step of determining the nucleotide sequence of a fragment for verifying the existence of a mutation revealed in the former step (e) of claims 1 and 3 or (f) of claim 2 by comparing to the corresponding wild type sequence.

24. The method of any one of claims 1 to 11 and 13 to 23, for identifying mutations in the NFl gene.

25. The method of any one of claims 1 to 11 and 13 to 23„ for identifying ^■mutations in the ATM gene.

26. The method of any one of claims 1 to 11 and 13 to 23,, for identifying mutations in the NBSl gene.

27. A method according to any one of claims 24 to 26, for identifying patients at risk to develop malignancy.

28. The method according to claim 27, wherein said malignancy is selected from the group consisting of carcinoma, melanoma, lymphoma, sarcoma and leukemia.

29. A method according to any one of claims 25 or 26, for identifying patients at risk to develop a lymphoid malignancy.

30. The method of any one of claims 24 to 26 for screening for homozygous or heterozygous carriers of mutations in said genes, for use in genetic counseling.

31. A method of prenatal genetic diagnosis of a fetus comprising the steps of:

(a) obtaining nucleic acids from a sample comprising fetal cells;

(b) subjecting the nucleic acids obtained in step (a) to the method of any one of claims 1 or 24 to 26, whereby the presence of a mutation in said nucleic acids indicates that said fetus is a carrier of said mutation.

32. The method of claim 31, wherein the sample is amniotic fluid.

33. The method of claim 31, wherein the sample is chorionic villi.

34. A method of screening a gene for the presence of a mutation, substantially as described and exemplified.

35. A method according to any one of claims 12 to 23, for screening for any one of mutations and polymorphism associated with a desired trait in a plant.

36. A method according to any one of claims 12 to 23, for screening plants for the existence of a specific mutation leading to a desired phenotype in said mutated plant.

37. A kit for detection of mutations in a gene comprising:

(a) means for producing segments of at least about 700bp each from said gene;

(b) at least two specific endonucleases for each said segment obtained by the means defined in (a), wherein each endonuclease has a single restriction site in segment, to give two unequal fragments of different length each, wherein the smaller fragment in one of the digestion reaction, overlaps the larger fragment in the other digestion reaction, said single restriction site being different for each of said endonucleases;

(c) SSCP gel and suitable buffers; and

(d) instructions for carrying out the detection of mutations in a gene according to the method of any one of claims 1 to 3.

38. A kit according to claim 37, wherein said means for producing segments of at least about 700bp each from said gene are specific primers, DNA polymerase and buffers for primer extension reaction.

39. The kit according to any one of claims 37 and 38, wherein said gene is any one of NF-1, ATM and NBS-1.

40. The kit according to any one of claims 37 and 38, wherein said instructions are for carrying out the method of claims 24 to 26.

41. The kit according to any one of claims 37 and 38, wherein said gene is a plant gene.