WO1998005677A1

WO1998005677A1 - Susceptibility mutations for breast and ovarian cancer

Info

Publication number: WO1998005677A1
Application number: PCT/US1997/013654
Authority: WO
Inventors: Patricia D. Murphy; Antonette C. Allen; Marga B. White; Sheri J. Olson; Bin Zeng
Original assignee: Oncormed, Inc.
Priority date: 1996-08-05
Filing date: 1997-08-04
Publication date: 1998-02-12
Also published as: AU4050997A

Abstract

New mutations have been found in the BRCA1 gene. The invention provides a method for diagnosing persons at risk of developing breast or ovarian cancer. The invention also provides a further tool with which to characterize tumors.

Description

TITLE OF THE INVENTION

SUSCEPTIBILITY MUTATIONS FOR BREAST AND

OVARIAN CANCER

FIELD OF THE INVENTION This invention relates to gene mutations that predispose individuals to breast and ovarian cancer. More specifically, this invention relates to specific mutations in the BRCA1 gene. In addition, it also relates to methods for detecting the presence of these mutations.

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. provisional application serial number 60/023,187 (filed August 5, 1996), U.S. provisional application serial number 60/023,223 (filed August 5, 1996), U.S. provisional application serial number 60/023,184 (filed August 5, 1996), and U.S. provisional application serial number 60/022,421 (filed August 6, 1996), all herein incorporated by reference.

BACKGROUND OF THE INVENTION

It has been estimated that about 5-10% of breast cancer is inherited Rowell, S., et al, American Journal of Human Genetics 55:861-865 (1994). Located on chromosome 17, BRCA1 is the first gene identified conferring increased risk for breast and ovarian cancer. Miki et al, Science 266:66-71

(1994). Mutations in this tumor suppressor gene account for roughly 45% of inherited breast cancer and 80-90% of families with increased risk of early onset breast and ovarian cancer. Easton et al, American Journal of Human Genetics 52:678-701 (1993). The location of one or more mutations in the BRCA1 region of chromosome 17 provides a promising approach to reducing the high incidence and mortality associated with breast and ovarian cancer through the early detection of women at high risk. These women, once identified, can be targeted for more aggressive prevention programs. Screening is carried out by a variety of methods which include karyotyping, probe binding and DNA sequencing.

In DNA sequencing technology, genomic DNA is extracted from whole blood and the coding regions of the BRCA1 gene are amplified using the polymerase chain reaction (PCR). Each of the coding regions is sequenced completely and the results are compared to the normal DNA sequence of the gene (GenBank Accession Number U14680). Many mutations have already been reported in the BRCA1 gene. Shattuck- Eidens, D., et al, Journal of the American Medical Association 273: 535-541 (1995).

The BRCA1 gene (GenBank Accession Number U14680) is divided into 24 separate exons. Exons 1 and 4 are noncoding, in that they are not part of the final functional BRCA1 protein product. The BRCA1 coding region spans roughly 5600 base pairs (bp). Each exon consists of 200-400 bp, except for exon 11 which contains about 3600 bp. To sequence the coding region of the BRCA1 gene, each exon is amplified separately and the resulting PCR products are sequenced in the forward and reverse directions. Because exon 11 is so large, we have divided it into twelve overlapping PCR fragments of roughly 350 bp each (segments "A" through "L'Of BRCAl exon ll).

There is a need in the art to identify mutations in the BRCA1 gene. Identification of mutations of the BRCA1 gene and protein would allow more widespread diagnostic screening for hereditary breast and ovarian cancer than is currently possible. SUMMARY OF THE INVENTION

The present invention is based on the discovery of specific mutations of the published BRCAl cDNA sequence which are associated with susceptibility to and development of breast and ovarian cancer. It is an object of the invention to provide a method for determining a predisposition or higher susceptibility to breast and ovarian cancer.

It is another object of the invention to provide a method of characterizing a tumor.

It is another object of the invention to provide primers for detecting and amplifying regions of DNA which contain mutations in the BRCAl gene that predispose individuals to breast or ovarian cancer.

In detail, the invention provides, an allele-specific oligonucleotide (especially, a labeled oligonucleotide) for detecting a mutation in a BRCAl encoding target polynucleotide, wherein the allele-specific oligonucleotide specifically hybridizes to the target polynucleotide, and thereby permits the detection of a mutation in the target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680. The invention also provides a primer oligonucleotide for amplifying a region of a BRCAl-encoding target polynucleotide, wherein the primer oligonucleotide specifically hybridizes to the target polynucleotide, and thereby permits the amplification of a portion of the target polynucleotide that includes nucleotides that flank nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159, or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680. The invention is particularly directed to the embodiment wherein the detected mutation is a 2799delAA, a 4158delAG mutation, a 5053delG mutation and /or a 943insl0 mutation. The invention additionally provides a method for classifying a tumor for diagnostic and prognostic purposes or detecting a predisposition of higher susceptibility to breast and ovarian cancer in an individual, comprising: a) isolating a BRCAl encoding target polynucleotide from an individual; and b) determining the identity of at least one nucleotide in a polymorphic region selected from the group of nucleotides corresponding to nucleotides at positions 943, 944, 2799, 2800, 4158, 4159, and 5053 of a BRCAl cDNA sequence having the nucleotide sequence of Genbank Accession Number U14680; and c) classifying the BRCAl-encoding target polynucleotide for the diagnostic and prognostic purposes or for the predisposition of higher susceptibility to breast and ovarian cancer in the individual by determining the presence or absence of any of the mutations in the isolated polynucleotide. The invention additionally provides a method for classifying a

BRCAl-encoding target polynucleotide for diagnostic and prognostic purposes of detecting a predisposition or higher susceptibility to breast and ovarian cancer in an individual, comprising: a) isolating the BRCAl-encoding target polynucleotide from an individual; b) incubating the target polynucleotide in the presence of an allele- specific oligonucleotide, wherein the incubation is under conditions sufficient to allow hybridization to occur between the target polynucleotide and the allele-specific oligonucleotide; wherein the allele-specific oligonucleotide specifically hybridizes to the target polynucleotide, and thereby permits the detection of a mutation in the target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680; c) allowing the hybridization to occur; d) detecting the hybridization between the target polynucleotide and the allele-specific oligonucleotide; and e) classifying the BRCAl-encoding target polynucleotide for the diagnostic and prognostic purposes or for the predisposition of higher susceptibility to breast and ovarian cancer in the individual by determining the presence or absence of any of the mutations in the isolated polynucleotide.

The invention is particularly directed to the embodiment of such methods wherein the detected mutation is a 2799delAA, a 4158delAG mutation, a 5053delG mutation and/or a 943insl0 mutation.

The invention additionally provides a kit, comprising a carrier means being compartmentalized to receive in close confinement one or more container means, the container means comprising an allele-specific oligonucleotide; wherein the allele-specific oligonucleotide has a nucleotide sequence sufficient to permit the oligonucleotide to specifically hybridize to a BRCAl-encoding polynucleotide, and to detect a mutation in the target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The BRCAl gene is a tumor suppressor gene associated with breast and ovarian cancer. The present invention is based on the discovery of the significance of mutations in the BRCAl cDNA sequence, and in particular, of the significance of certain specific mutations in the BRCAl cDNA sequence. These mutations are referred to as 2799delAA, 4158delAG, 5053delG, and 943insl0, respectively.

The 2799delAA mutation is a two base pair deletion of nucleotides

2799 and 2780, which interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 901. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein.

The 4158delAG mutation is a two base pair deletion of nucleotides

4158 and 4159, which interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TAA at codon position 1354. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein.

The 5053delG mutation is a one base pair deletion of nucleotide

5053, which interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 1657. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein.

The 943insl0 mutation is a 10 base pair tandem repeat insertion between nucleotides 943 and 944, which interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 289. This mutation is also predicted to result in a truncated, and most likely, non-functional protein.

Useful polynucleotides (especially DNA molecules) according to the invention are those which will specifically hybridize to BRCAl sequences in the region of the 2799delAA, the 4158delAG, the 5053delG, or the

943insl0 mutation, such that, by virtue of such hybridization, or by polymerase-mediated extension, or by ligation with other polynucleotides, permit the identification of the nucleotides present (or deleted from) the region of the 2799delAA, the 4158delAG, the 5053delG, or the 943insl0 mutation.

Typically, such polynucleotides will be DNA molecules that are at least about 20 nucleotides in length and have a nucleotide sequence corresponding to a region of the published BRCAl cDNA sequence

(GenBank Accession No. U14680) near mutations at nucleotides 943 and 944, 2799 and 2800, 4158 and 4159, or 5053, respectively. Such molecules can be labeled, according to any technique known in the art, such as with radiolabels, fluorescent labels, enzymatic labels, sequence tags, etc. According to another aspect of the invention, the DNA molecules contain base changes corresponding to the 2799delAA, 4158delAG, 5053delG, or 943insl0 mutations. Such molecules can be used as allele-specific oligonucleotide probes to track a particular mutation through a family.

Body samples can be tested to determine whether the BRCAl gene contains one or more of the mutations 2799delAA, 4158delAG, 5053delG, or 943insl0. Suitable body samples for testing include those comprising

DNA, RNA or protein obtained from biopsies, blood, prenatal, or embryonic tissues, for example.

In one embodiment of the invention, pairs of isolated oligonucleotide primers are provided. Examples of suitable pairs of oligonucleotide primers include:

BRCA1-11G-F: 5'-GGA AGT TAG CAC TCT AGG GA-3' (SEQ ID NO:l), and

BRCA1-11G-R: 5'-GCA GTG ATA TTA ACT GTC TGT A-3' (SEQ ID NO:2).

BRCA1-11L-F: 5'-GTA ATA TTG GCA AAG GCA TCT-3' (SEQ ID NO:3), and

BRCA1-11L-R: 5'-TAA AAT GTC CTC CCC AAA AGC A-3' (SEQ ID NO:4).

BRCA1-16-F: 5'- AAT TCT TAA CAG AGA CCA GAA C-3' (SEQ ID NO:5), and

BRCA1-16-R: 5'-AAA ACT CTT TCC AGA ATG TTG T-3' (SEQ ID NO:6).

BRCA1-11A-F: 5'-CCA CCT CCA AGG TGT ATC A-3' (SEQ ID NO:7), and BRCA1-11A-R: 5'-TGT TAT GTT GGC TCC TTG CT-3' (SEQ ID NO:8).

The designation BRCA1-11G refers to a sequence in the BRCAl gene, Exon 11, section G; BRCA1-11L refers to a sequence in the BRCAl gene, Exon 11, section L; BRCA1-16 refers to a sequence in the BRCAl gene, Exon 16; and BRCA1-11L refers to a sequence in the BRCAl gene, Exon 11, section A. F and R refer to forward and reverse.

The oligonucleotide primers are useful in diagnosis of a subject at risk of having breast or ovarian cancer, and are also useful for characterizing a tumor. The primers direct amplification of a target polynucleotide prior to sequencing. These unique BRCAl oligonucleotide primers were designed and produced at OncorMed based upon identification of the 2799delAA, 4158delAG, 5053delG, and 943insl0 mutations. In another embodiment of the invention, isolated allele specific oligonucleotides are provided. The allele specific oligonucleotides are useful in diagnosis of a subject at risk of having breast or ovarian cancer, and also useful for characterizing a tumor.

5'-CCT TAA AGA CAA AGT CC-3' (SEQ ID NO:9), and 5'-TCC TTA AAG ACA AAG TC-3' (SEQ ID NO:10) and sequences substantially similar thereto.

Allele specific oligonucleotides represented by SEQ ID NO:9 and SEQ ID NO: 10 and sequences substantially similar thereto, hybridize with a target polynucleotide sequence containing the 2799delAA mutation.

5'-ATG AAG AAA GGA ACG GG-3' (SEQ ID NO:ll), and

5'-GAT GAA GAA AGG AAC GG-3' (SEQ ID NO:12) and sequences substantially similar thereto.

Allele specific oligonucleotides represented by SEQ ID NO:ll and SEQ ID NO:12 and sequences substantially similar thereto, hybridize with a target polynucleotide sequence containing the 4158delAG mutation.

5'-AAC AGA AAG GTC AAC AA-3' (SEQ ID NO:13), and 5'-CAA CAG AAA GGT CAA CA-3' (SEQ ID NO: 14) and sequences substantially similar thereto. Allele specific oligonucleotides represented by SEQ ID NO:13 and SEQ ID NO: 14 and sequences substantially similar thereto, hybridize with a target polynucleotide sequence containing the 5053delG mutation.

5'-CCA TGT GGA GCC ATG TG-3' (SEQ ID NO: 15), and 5'-GCC ATG TGG AGC CAT GT-3' (SEQ ID NO:16) and sequences substantially similar thereto.

Allele specific oligonucleotides represented by SEQ ID NO:15 and SEQ ID NO:16 and sequences substantially similar thereto, hybridize with a target polynucleotide sequence containing the 943insl0 mutation. The term "substantially complementary to" or "substantially the sequence" refers to (e.g., SEQ ID NO:9 and SEQ ID NO:10) sequences which hybridize to the sequences provided under stringent conditions and /or sequences having sufficient homology with SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:ll and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, or SEQ ID NO:15 and SEQ ID NO:16 such that the allele specific oligonucleotides of the invention hybridize to the sequence, under conditions in which the non-allele oligonucleotides are substantially less capable or incapable of hybridizing.

The term "isolated" as used herein includes oligonucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which they may be associated. Such association being either in cellular material or in a synthesis medium. A "target polynucleotide" refers to a nucleic acid sequence of interest e.g., the BRCAl encoding polynucleotide. Other primers which can be used for primer hybridization will be known or readily ascertainable to those of skill in the art.

The primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a significant number of nucleic acids in the polymorphic locus. Specifically, the term "primer" as used herein refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and more preferably more than eight and most preferably at least about 20 nucleotides of the BRCAl gene wherein said DNA sequence contains the 2799delAA mutation relative to BRCAl contained in SEQ ID NO's: 9 and 10; the 4158delAG mutation relative to

BRCAl contained in SEQ ID NO's: 11 and 12; the 5053delG mutation relative to BRCAl contained in SEQ ID NO's: 13 and 14; and the 943insl0 mutation relative to BRCAl contained in SEQ ID NO's: 15 and 16. Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be of sufficient length to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition. The oligonucleotide primer typically contains 12- 20 or more nucleotides, although it may contain fewer nucleotides. Primers of the invention are designed to be "substantially" complementary to each strand of the genomic locus to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' sequences flanking the mutation to hybridize therewith and permit amplification of the genomic locus.

Oligonucleotide primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of polymorphic locus relative to the number of reaction steps involved. Typically, one primer is complementary to the "negative" (-) strand of the polymorphic locus and the other is complementary to the "positive" (+) strand. Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA polymerase I (Klenow) and nucleotides, results in newly synthesized + and - strands containing the target polymorphic locus sequence. Because these newly synthesized sequences are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the target polymorphic locus sequence) defined by the primers. The product of the chain reaction is a discreet nucleic acid duplex with termini corresponding to the ends of the specific primers employed.

The oligonucleotide primers of the invention may be preferably prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al., Tetrahedron Letters, 22:1859-1862, 1981. One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066. Any nucleic acid specimen, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, providing it contains, or is suspected of containing, the specific nucleic acid sequence containing the polymorphic locus. Thus, the process may amplify, for example, DNA or RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded. In the event that RNA is to be used as a template, enzymes, and /or conditions optimal for reverse transcribing the template to DNA would be utilized. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of nucleic acids may also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers may be so utilized. The specific nucleic acid sequence to be amplified, i.e., the polymorphic locus, may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as contained in whole human DNA.

DNA utilized herein may be extracted from a body sample, such as blood, tissue material and the like by a variety of techniques such as that described by Maniatis, et. al. In: Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, NY, p 280-281, 1982). If the extracted sample is impure, it may be treated before amplification with an amount of a reagent effective to open the cells, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily. The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to denature the double-stranded molecules (e.g., about 90°C-100°C from about 1 to 10 minutes, preferably from 1 to 4 minutes). After this heating period, the solution is allowed to cool to conditions that are preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein "agent for polymerization"), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization may also be added together with the other reagents if it is heat stable. This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions. Thus, for example, if DNA polymerase is used as the agent, the temperature is generally no greater than about 40°C. Most conveniently the reaction occurs at room temperature. The agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase, polymerase muteins, reverse transcriptase, other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation), such as Taq polymerase. Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each polymorphic locus nucleic acid strand. Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths. The newly synthesized strand and its complementary nucleic acid strand will form a double-stranded molecule under hybridizing conditions described above and this hybrid is used in subsequent steps of the process. In the next step, the newly synthesized double-stranded molecule is subjected to denaturing conditions using any of the procedures described above to provide single-stranded molecules.

The steps of denaturing, annealing, and extension product synthesis can be repeated as often as needed to amplify the target polymorphic locus nucleic acid sequence to the extent necessary for detection. The amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion. PCR. A Practical Approach, ILR Press, Eds. M. J. McPherson, P. Quirke, and G. R. Taylor, (1992).

The amplification products may be detected by analyzing it by Southern blots without using radioactive probes. In such a process, for example, a small sample of DNA containing a very low level of the nucleic acid sequence of the polymorphic locus is amplified, and analyzed via a

Southern blotting technique or similarly, using dot blot analysis. The use of non-radioactive probes or labels is facilitated by the high level of the amplified signal. Alternatively, probes used to detect the amplified products can be directly or indirectly detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.

Sequences amplified by the methods of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al, Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et. al, Proc. Natl. Acad. Sci. (U.S.A.)., 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landgren, et. al, Science, 241:1007, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landgren, et. al, Science, 242:229-237, 1988).

Preferably, the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art. Alternative methods of amplification have been described and can also be employed as long as the BRCAl locus amplified by PCR using primers of the invention is similarly amplified by the alternative means. Such alternative amplification systems include but are not limited to self-sustained sequence replication, which begins with a short sequence of RNA of interest and a T7 promoter. Reverse transcriptase copies the RNA into cDNA and degrades the RNA, followed by reverse transcriptase polymerizing a second strand of DNA. Another nucleic acid amplification technique is nucleic acid sequence-based amplification (NASBA) which uses reverse transcription and T7 RNA polymerase and incorporates two primers to target its cycling scheme. NASBA can begin with either DNA or RNA and finish with either, and amplifies to 10⁸ copies within 60 to 90 minutes. Alternatively, nucleic acid can be amplified by ligation activated transcription (LAT). LAT works from a single-stranded template with a single primer that is partially single-stranded and partially double- stranded. Amplification is initiated by ligating a cDNA to the promoter oligonucleotide and within a few hours, amplification is 10⁸ to 10⁹ fold. The QB replicase system can be utilized by attaching an RNA sequence called MDV-1 to RNA complementary to a DNA sequence of interest. Upon mixing with a sample, the hybrid RNA finds its complement among the specimen's mRNAs and binds, activating the replicase to copy the tag- along sequence of interest. Another nucleic acid amplification technique, ligase chain reaction (LCR), works by using two differently labeled halves of a sequence of interest which are covalently bonded by ligase in the presence of the contiguous sequence in a sample, forming a new target. The repair chain reaction (RCR) nucleic acid amplification technique uses two complementary and target-specific oligonucleotide probe pairs, thermostable polymerase and ligase, and DNA nucleotides to geometrically amplify targeted sequences. A 2-base gap separates the oligonucleotide probe pairs, and the RCR fills and joins the gap, mimicking normal DNA repair. Nucleic acid amplification by strand displacement activation (SDA) utilizes a short primer containing a recognition site for Hindi with short overhang on the 5' end which binds to target DNA. A

DNA polymerase fills in the part of the primer opposite the overhang with sulfur-containing adenine analogs. Hindi is added but only cuts the unmodified DNA strand. A DNA polymerase that lacks 5' exonuclease activity enters at the cite of the nick and begins to polymerize, displacing the initial primer strand downstream and building a new one which serves as more primer. SDA produces greater than 10⁷-fold amplification in 2 hours at 37°C. Unlike PCR and LCR, SDA does not require instrumented temperature cycling. Another amplification system useful in the method of the invention is the QB Replicase System. Although PCR is the preferred method of amplification if the invention, these other methods can also be - lo ¬

used to amplify the BRCAl locus as described in the method of the invention.

In another embodiment of the invention a method is provided for diagnosing a subject having a predisposition or higher susceptibility to (at risk of) breast or ovarian cancer comprising sequencing a target nucleic acid of a sample from a subject, by the dideoxynucleotide sequencing ("dideoxy sequencing") method (Sanger, F., et al, J. Mol Biol. 142:1617 (1980), herein incorporated by reference), following amplification of the target nucleic acid. In another embodiment of the invention a method is provided for diagnosing a subject having a predisposition or higher susceptibility to (at risk of) breast or ovarian cancer comprising contacting a target nucleic acid of a sample from a subject with a reagent that detects the presence of one or more mutations such as 2799delAA, 4158delAG, 5053delG, or 943insl0 and detecting the mutation.

In another embodiment of the invention a method is provided for characterizing a tumor. One method comprises sequencing the target nucleic acid isolated from the tumor to determine if one or more mutations such as 2799delAA, 4158delAG, 5053delG, or 943insl0 has occurred. Another method comprises contacting a target nucleic acid of a sample from a subject with one or more reagents that detect the presence of one or more mutations such as 2799delAA, 4158delAG, 5053delG, or 943insl0 and detecting the mutation. A number of hybridization methods are well known to those skilled in the art. Many of them are useful in carrying out the invention.

The materials for use in the method of the invention are ideally suited for the preparation of a diagnostic kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise means for amplifying BRCAl DNA, said means comprising the necessary enzyme(s) and oligonucleotide primers for amplifying said target DNA from the subject. The oligonucleotide primers include primers having a sequence:

BRCA1-11G-F: 5'-GGA AGT TAG CAC TCT AGG GA-3' (SEQ ID NO:l), and BRCA1-11G-R: 5'-GCA GTG ATA TTA ACT GTC TGT A-3' (SEQ ID NO:2), or

BRCA1-11L-F: 5'-GTA ATA TTG GCA AAG GCA TCT-3' (SEQ ID NO:3), and BRCAl-llL-R: 5'-TAA AAT GTC CTC CCC AAA AGC A-3' (SEQ ID NO:4), or

BRCA1-16-F: 5'-AAT TCT TAA CAG AGA CCA GAA C-3' (SEQ ID NO:5), and BRCA1-16-R: 5'- AAA ACT CTT TCC AGA ATG TTG T-3' (SEQ ID NO:6) or

BRCA1-11A-F: 5'-CCA CCT CCA AGG TGT ATC A-3' (SEQ ID NO:7), and BRCA1-11A-R: 5'-TGT TAT GTT GGC TCC TTG CT-3' (SEQ ID NO:8)

or primer sequences substantially complementary or substantially homologous thereto. The targets flanking 5' and 3' polynucleotide sequences have substantially the sequences selected from the group consisting of:

5'-CCTTAA AGA CAA AGTCC-3' (SEQ ID NO:9), 5'-TCC TTA AAG ACA AAG TC-3' (SEQID NO:10), 5'-ATG AAG AAA GGAACG GG-3' (SEQIDNO:ll), 5'-GATGAA GAA AGG AAC GG-3' (SEQ ID NO:12), 5'-AAC AGA AAG GTC AAC AA-3' (SEQID NO:13), 5'-CAACAG AAAGGTCAACA-3' (SEQIDNO:14), 5'-CCA TGTGGA GCC ATG TG-3' (SEQID NO:15), and 5'-GCC ATG TGG AGC CATGT-3' (SEQ ID NO:16) and sequences substantially complementary or homologous thereto.

Other oligonucleotide primers for amplifying BRCAl will be known or readily ascertainable to those of skill in the art. EXAMPLES

MATERIALS AND METHODS

Genomic DNA was isolated from white blood cells of a subject with a family history of breast cancer. Dideoxy sequence analysis was performed following polymerase chain reaction amplification of segment L of exon 11.

Exon 11 of the BRCAl gene was subjected to direct dideoxy sequence analysis by asymmetric amplification using the polymerase chain reaction (PCR) to generate a single stranded product amplified from this DNA sample. Shuldiner, et al, Handbook of Techniques in Endocrine Research, p. 457-486, DePablo, F., Scanes, C, eds., Academic Press, Inc., 1993. Fluorescent dye was attached for automated sequencing using the Taq Dye Terminator Kit (Perkin-Elmer^® cat# 401628). DNA sequencing was performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) automated sequencer (Model 377). The software used for analysis of the resulting data was "Sequence Navigator" purchased through ABI.

EXAMPLE 1 IDENTIFICATION OF THE 2799DELAA MUTATION 1. Polymerase Chain Reaction (PCR. Amplification

Genomic DNA (100 nanograms) extracted from white blood cells of the subject was amplified in a final volume of 25 microliters containing 1 microliter (100 nanograms) genomic DNA, 2.5 microliters 10X PCR buffer (100 mM Tris, pH 8.3, 500 mM KC1, 1.2 mM MgCl₂), 2.5 microliters 10X dNTP mix (2 mM each nucleotide), 2.5 microliters forward primer (BRCA1- 11G-F, 10 micromolar solution), 2.5 microliters reverse primer (BRCA1- 11G-R, 10 micromolar solution), and 1 microliter Taq polymerase (5 units), and 13 microliters of water. The PCR primers used to amplify segment G of exon 11 (where the mutation was found) are as follows:

BRCA1-11G-F: 5'-GGA AGTTAG CAC TCT AGG GA-3' SEQ ID NO:l BRCA1-11G-R: 5'-GCA GTG ATA TTA ACTGTC TGTA-3' SEQ ID NO:2 The primers were synthesized on a DNA /RNA Synthesizer Model 394®.

Thirty-five cycles were performed, each consisting of denaturing (95 ° C; 30 seconds), annealing (55 ^° C; 1 minute), and extension (72 ° C; 90 seconds), except during the first cycle in which the denaturing time was increased to 5 minutes, and during the last cycle in which the extension time was increased to 5 minutes.

PCR products were purified using Qia-quick^® PCR purification kits (Qiagen^®, cat# 28104; Chatsworth, CA). Yield and purity of the PCR product determined spectrophotometrically at OD₂₆o °^{n a} Beckman DU 650 spectrophotometer.

2. Dideoxy Sequence Analysis

Fluorescent dye was attached to PCR products for automated sequencing using the Taq Dye Terminator Kit (Perkin-Elmer^® cat# 401628).

DNA sequencing was performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) Foster City, CA., automated sequencer (Model 377). The software used for analysis of the resulting data was

"Sequence Navigator^®" purchased through ABI.

3. Detection of the 2799delAA mutation by Allele Specific

Oligonucleotide Hybridization

For rapid detection of the 2799delAA mutation, unlabeled PCR products from segment G of exon 11 were blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization was accomplished with ²P labeled ATP oligonucleotides corresponding to either a normal sequence such as

5'-CTT AAA GAA ACA AAG TC-3', (SEQ. ID. NO. 17), or 5'-CCT TAA AGA AAC AAA GT-3', (SEQ. ID. NO. 18) or with a sequence exhibiting the 2799delAA mutation such as 5'-CCT TAA AGA CAA AGT CC-3' (SEQ ID NO:9), or 5'-TCC TTA AAG ACA AAG TC-3' (SEQ ID NO:10). The membranes were then washed twice in 2X SSPE and 0.05% SDS at

60 ' C for fifteen minutes. Autoradiography was performed at -70 ° C for 4- 12 hours with enhancing screens.

4. Allele Specific Oligonucleotide Analysis

The ³²P labeled ATP oligonucleotides hybridized to PCR products amplified from individuals possessing the 2799delAA mutation, and not to those from "normal" individuals. This hybridization was seen as exposed regions of the autoradiography film. Both positive (2799delAA) and negative (normal) controls were included in this analysis.

5. Result Using the above PCR amplification and standard fluorescent sequencing technology, we have found a previously unidentified mutation in the BRCAl gene. This mutation lies in segment G of exon 11. The DNA sequence results demonstrated a two base pair deletion of nucleotides 2799 and 2800 of the published BRCAl cDNA sequence. This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 901. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein. The formal name of the mutation will be 2799delAA.

EXAMPLE 2

DIAGNOSIS OF INDIVIDUALS CARRYING THE 2799DELAA MUTATION.

Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section G of exon 11 of the BRCA1 gene is amplified from the genomic DNA using the polymerase chain reaction (PCR). The PCR primers used to amplify the suspected mutation are as follows:

BRCA1-11G-F: 5'-CCT TAA AGA CAA AGT CC-3' SEQ ID NO:9 BRCA1-11G-R: 5'-TCC TTA AAG ACA AAG TC-3' SEQ ID NO:10

1. PCR Amplification

The PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 1.

2. Dideoxy Sequence Analysis PCR products are purified using Qia-quick® PCR purification kits

(Qiagen®, cat# 28104). Dideoxy sequence analysis is performed as described in EXAMPLE 1.

3. Analysis

The software used for analysis of the resulting data was "Sequence Navigator®" purchased through ABI.

4. Result

Using the above PCR amplification and standard fluorescent sequencing technology, a two base pair deletion of nucleotides 2799 and 2800 of the published BRCAl cDNA sequence can be found. This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 901. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein. A finding of this mutation may be used either to design a program of gene therapy in a patient having a tumor, to characterize a tumor, or as a prognostic tool for those at risk of developing breast or ovarian cancer EXAMPLE 3

DETECTION OF THE 2799delAA MUTATION BY HYBRIDIZATION

Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section G of exon 11 of the

BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR). The PCR primers used to amplify the suspected mutation are as follows:

BRCA1-11G-F: 5'-GGA AGT TAG CAC TCT AGG GA-3' (SEQ ID NO:l) BRCA1-11G-R: 5'-GCA GTG ATA TTA ACT GTC TGT A-3' (SEQ ID NO:2)

1. PCR Amplification

The PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 1. PCR products are purified using Qia-quick^® PCR purification kits (Qiagen, cat# 28104). DNA sequence analysis is performed as in EXAMPLE 1.

2. Detection of the 2799delAA mutation by Allele Specific Oligonucleotide Hybridization

For rapid detection of the 2799delAA mutation, unlabeled PCR products from segment G of exon 11 can be blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization is accomplished with ³²P labeled ATP oligonucleotides corresponding to either the normal sequence such as

5'-CTT AAA GAA ACA AAG TC-3', (SEQ. ID. NO. 17), or 5'-CCT TAA AGA AAC AAA GT-3', (SEQ. ID. NO. 18) or with a sequence exhibiting the 2799delAA mutation such as

5'-CCT TAA AGA CAA AGT CC-3' (SEQ ID NO:9), or 5'-TCC TTA AAG ACA AAG TC-3' (SEQ ID NO: 10). The membranes are then washed twice in 2X SSPE and 0.05% SDS at 60 ° C for fifteen minutes. Autoradiography is performed at -70 ° C for 4-12 hours with enhancing screens.

EXAMPLE 4 IDENTIFICATION OF THE 4158delAG MUTATION

1. Polymerase Chain Reaction (PCR) Amplification

Segment L of exon 11 (where the mutation was found) was amplified from genomic DNA by PCR as in EXAMPLE 1 using the following primers: BRCA1-11L-F: 5'-GTA ATA TTG GCA AAG GCA TCT-3' SEQ ID NO:3

BRCAl-llL-R: 5'-TAA AATGTC CTC CCC AAA AGC A-3' SEQ ID NO:4

2. Dideoxy Sequence Analysis

Dideoxy sequence analysis was performed as in EXAMPLE 1.

3. Detection of the 4158delAG mutation by Allele Specific Oligonucleotide Hybridization

For rapid detection of the 4158delAG mutation, unlabeled PCR products from segment L of exon 11 were blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization was accomplished with ³²P labeled ATP oligonucleotides corresponding to either a normal sequence such as

5'- ATG AAG AAA GAG GAA CG-3', (SEQ. ID. NO:19), or 5'-TGA AGA AAG AGG AAC GG -3', (SEQ. ID. NO:20) or with a sequence exhibiting the 4158delAG mutation such as 5'-ATG AAG AAA GGA ACG GG-3' (SEQ ID NO.ll), or 5'-GAT GAA GAA AGG AAC GG-3' (SEQ ID NO:12).

The membranes were then washed twice in 2X SSPE and 0.05% SDS at 60°C for fifteen minutes. Autoradiography was performed at -70°C for 4- 12 hours with enhancing screens. 4. Allele Specific Oligonucleotide Analysis

The ³²P labeled ATP oligonucleotides hybridized to PCR products amplified from individuals possessing the 4158delAG mutation, and not to those from "normal" individuals. This hybridization was seen as exposed regions of the autoradiography film. Both positive (4158delAG) and negative (normal) controls were included in this analysis.

5. Result

Using the above PCR amplification and standard fluorescent sequencing technology, we have found a previously unidentified mutation in the BRCAl gene. This mutations lie in segment L of exon 11. The DNA sequence results demonstrated a two base pair deletion of nucleotides 4158 and 4159 of the published BRCAl cDNA sequence. This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TAA at codon position 1354. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein. The formal name of the mutation will be 4158delAG.

EXAMPLE 5 DIAGNOSIS OF INDIVIDUALS CARRYING THE 4158DELAG MUTATION.

Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section L of exon 11 of the

BRCA1-11L-F: 5'-ATG AAG AAA GGA ACG GG-3' SEQ ID NO:ll BRCAl-llL-R: 5'-GAT GAA GAA AGG AAC GG-3' SEQ ID NO:12 1. PCR Amplification

The PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 4.

2. Dideoxy Sequence Analysis PCR products are purified using Qia-quick^® PCR purification kits

(Qiagen^®, cat# 28104). Dideoxy sequence analysis is performed as in EXAMPLE 4.

3. Analysis

The software used for analysis of the resulting data was "Sequence Navigator^®" purchased through ABI.

4. Result

Using the above PCR amplification and standard fluorescent sequencing technology, a two base pair deletion of nucleotides 4158 and 4159 of the published BRCAl cDNA sequence can be found. This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TAA at codon position 1354. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein. A finding of this mutation may be used either to design a program of gene therapy in a patient having a tumor, to characterize a tumor, or as a prognostic tool for those at risk of developing breast or ovarian cancer.

EXAMPLE 6

DETECTION OF THE 4158delAG MUTATION BY HYBRIDIZATION Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section L of exon 11 of the BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR). The PCR primers used to amplify the suspected mutation are as follows:

BRCA1-11L-F: 5'-GTA ATA TTG GCA AAG GCA TCT-3' (SEQ ID NO:3) BRCAl-llL-R: 5'-TAA AAT GTC CTC CCC AAA AGC A-3' (SEQ

ID NO:4)

1. PCR Amplification and Dideoxy Sequence Analysis

The PCR amplification and dideoxy sequence analysis is carried out as described in EXAMPLE 4.

2. Detection of the 4158delAG mutation by Allele Specific

Oligonucleotide Hybridization

For rapid detection of the 4158delAG mutation, unlabeled PCR products from segment L of exon 11 can be blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization is accomplished with ³²P labeled ATP oligonucleotides corresponding to either the normal sequence

5'-ATG AAG AAA GAG GAA CG-3', (SEQ. ID. NO: 19), or 5'-TGA AGA AAG AGG AAC GG -3', (SEQ. ID. NO:20) or with a sequence exhibiting the 4158delAG mutation such as 5'-ATG AAG AAA GGA ACG GG-3' (SEQ ID NO:ll), or

5'-GAT GAA GAA AGG AAC GG-3' (SEQ ID NO:12). The membranes are then washed twice in 2X SSPE and 0.05% SDS at 60°C for fifteen minutes. Autoradiography is performed at -70°C for 4-12 hours with enhancing screens. EXAMPLE 7 IDENTIFICATION OF THE 5053delG MUTATION

1. Polymerase Chain Reaction (PCR) Amplification

Exon 16 (where the mutation was found) was amplified from genomic DNA by PCR as in EXAMPLE 1 using the following primers:

BRCA1-16-F: 5'-AAT TCT TAA CAG AGA CCA GAA C-3' SEQ ID NO:5 BRCA1-16-R: 5'-AAA ACT CTT TCC AGA ATG TTG T-3' SEQ ID NO:6

2. Dideoxy Sequence Analysis

DNA sequencing was performed in both forward and reverse directions on an Applied Biosystems, Inc. (ABI) Foster City, CA., automated sequencer (Model 377). The software used for analysis of the resulting data was "Sequence Navigator^®" purchased through ABI.

3. Detection of the 5053delG mutation by Allele Specific Oligonucleotide Hybridization

For rapid detection of the 5053delG mutation, unlabeled PCR products from exon 16 were blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization was accomplished with P labeled ATP oligonucleotides corresponding to either a normal sequence such as

5'-ACA GAA AGG GTC AAC AA-3', (SEQ. ID. NO. 21), or

5'- AAC AGA AAG GGT CAA CA-3', (SEQ. ID. NO. 22) or with a sequence exhibiting the 5053delG mutation such as 5'-AAC AGA AAG GTC AAC AA-3' (SEQ ID NO:13), or

5'-CAA CAG AAA GGT CAA CA-3' (SEQ ID NO:14).

The ³²P labeled ATP oligonucleotides hybridized to PCR products amplified from individuals possessing the 5053delG mutation, and not to those from "normal" individuals. This hybridization was seen as exposed regions of the autoradiography film. Both positive (5053delG) and negative (normal) controls were included in this analysis.

5. Result

Using the above PCR amplification and standard fluorescent sequencing technology, we have found a previously unidentified mutation in the BRCAl gene. This mutation lies in exon 16. The DNA sequence results demonstrated a one base pair deletion of nucleotide 5053 of the published BRCAl cDNA sequence. This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 1657. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein. The formal name of the mutation will be 5053delG.

EXAMPLE 8

DIAGNOSIS OF INDIVIDUALS CARRYING THE 5053DELG MUTATION. Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Exon 16 of the BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR). The PCR primers used to amplify the suspected mutation are as follows: BRCA1-16-F: 5'-AAC AGA AAG GTC AAC AA-3' SEQ ID NO:13

BRCA1-16-R: 5'-CAA CAG AAA GGT CAA CA-3' SEQ ID NO:14

1. PC Amplification

The PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 1. 2. Dideoxy Sequence Analysis

PCR products are purified using Qia-quick® PCR purification kits (Qiagen®, cat# 28104). Fluorescent dye is attached for automated sequencing using the Taq Dye Terminator® Kit (Perkin-Elmer cat# 401628). DNA sequencing is performed in accordance with the parameters specified in the Taq Dye Terminator® Kit, in both forward and reverse directions, on an Applied Biosystems, Inc ® (ABI) automated sequencer (Model 377).

3. Analysis

4. Result

Using the above PCR amplification and standard fluorescent sequencing technology, a one base pair deletion of nucleotide 5053 of the published BRCAl cDNA sequence can be found. This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 1657. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein. A finding of this mutation may be used either to design a program of gene therapy in a patient having a tumor, to characterize a tumor, or as a prognostic tool for those at risk of developing breast or ovarian cancer

EXAMPLE 9 DETECTION OF THE 5053delG MUTATION BY HYBRIDIZATION

Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Exon 16 of the BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction

(PCR). The PCR primers used to amplify the suspected mutation are as follows: BRCA1-16-F: 5'-AAT TCT TAA CAG AGA CCA GAA C-3' (SEQ ID NO:5) BRCA1-16-R: 5'- AAA ACT CTT TCC AGA ATG TTG T-3' (SEQ ID NO:6)

1. PCR Amplification

The PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 1. PCR products are purified using Qia-quick®

PCR purification kits (Qiagen®, cat# 28104). Fluorescent dye is attached for automated sequencing using the Taq Dye Terminator® Kit (Perkin-Elmer cat# 401628). DNA sequencing is performed in both forward and reverse directions on an Applied Biosystems, Inc.® (ABI) automated sequencer (Model 377). The software used for analysis of the resulting data was

"Sequence Navigator®" purchased through ABI.

2. Detection of the 5053delG mutation by Allele Specific Oligonucleotide Hybridization

For rapid detection of the 5053delG mutation, unlabeled PCR products from exon 16 can be blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization is accomplished with ³²P labeled ATP oligonucleotides corresponding to either the normal sequence: 5'-ACA GAA AGG GTC AAC AA-3', (SEQ. ID. NO. 21), or 5'- AAC AGA AAG GGT CAA CA-3', (SEQ. ID. NO. 22) or with a sequence exhibiting the 5053delG mutation such as :

5'-AAC AGA AAG GTC AAC AA-3' (SEQ ID NO:13), or 5'-CAA CAG AAA GGT CAA CA-3' (SEQ ID NO:14). The membranes are then washed twice in 2X SSPE and 0.05% SDS at 60°C for fifteen minutes. Autoradiography is performed at -70°C for 4-12 hours with enhancing screens. EXAMPLE 10 IDENTIFICATION OF THE 943insl0 MUTATION

1. Polymerase Chain Reaction (PCR) Amplification

Segment A of exon 11 (where the mutation was found) was amplified from genomic DNA by PCR amplification for approximately 35 cycles as described in EXAMPLE 1 using the following primers:

BRCA1-11A-F: 5'-CCA CCT CCA AGG TGT ATC A-3' SEQ ID NO:7 BRCA1-11A-R: 5'-TGT TAT GTT GGC TCC TTG CT-3' SEQ ID NO:8

2. Dideoxy Sequence Analysis Dideoxy sequence analysis was performed as in EXAMPLE 1.

3. Detection of the 943insl0 mutation by Allele Specific Oligonucleotide Hybridization

For rapid detection of the 943insl0 mutation, unlabeled PCR products from segment A of exon 11 were blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization was accomplished with ³²P labeled ATP oligonucleotides corresponding to either a normal sequence such as

5'-CCA TGT GGC ACA AAT AC-3', (SEQ. ID. NO:23), or 5'-GCC ATG TGG CAC AAA TA-3', (SEQ. ID. NO:24) or with a sequence exhibiting the 943insl0 mutation such as

5'-CCA TGT GGA GCC ATG TG-3' (SEQ ID NO:15), or 5'-GCC ATG TGG AGC CAT GT-3' (SEQ ID NO:16). The membranes were then washed twice in 2X SSPE and 0.05% SDS at 60°C for fifteen minutes. Autoradiography was performed at -70°C for 4- 12 hours with enhancing screens.

4. Allele Specific Oligonucleotide Analysis

The ³²P labeled ATP oligonucleotides hybridized to PCR products amplified from individuals possessing the 943insl0 mutation, and not to those from "normal" individuals. This hybridization was seen as exposed regions of the autoradiography film. Both positive (943insl0) and negative (normal) controls were included in this analysis.

5. Result Using the above PCR amplification and standard fluorescent sequencing technology, we have found a previously unidentified mutation in the BRCAl gene. This mutation lies in segment A of exon 11. The DNA sequence results demonstrated a 10 base pair tandem repeat between nucleotides 943 and 944 of the published BRCAl cDNA sequence. This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 289. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein. The formal name of the mutation will be 943insl0.

EXAMPLE 11

DIAGNOSIS OF INDIVIDUALS CARRYING THE 943INS10

MUTATION.

Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section A of exon 11 of the BRCAl gene is amplified from the genomic DNA using the polymerase chain reaction (PCR). The PCR primers used to amplify the suspected mutation are as follows:

BRCA1-11A-F: 5'-CCA TGT GGA GCC ATG TG-3' SEQ ID NO:15 BRCA1-11A-R: 5'-GCC ATG TGG AGC CAT GT-3' SEQ ID NO: 16

1. PCR Amplification

The PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 10. 2. Dideoxy Sequence Analysis

Dideoxy sequence analysis is performed as in EXAMPLE 10.

3. Analysis

4. Result

Using the above PCR amplification and standard fluorescent sequencing technology, a 10 base pair tandem repeat between nucleotides 943 and 944 of the published BRCAl cDNA sequence can be found. This mutation interrupts the normal reading frame of the BRCAl transcript, resulting in the appearance of an in-frame terminator TGA at codon position 289. This mutation is, therefore, predicted to result in a truncated, and most likely, non-functional protein. A finding of this mutation may be used either to design a program of gene therapy in a patient having a tumor, to characterize a tumor, or as a prognostic tool for those at risk of developing breast or ovarian cancer

EXAMPLE 12 DETECTION OF THE 943insl0 MUTATION BY HYBRIDIZATION

Genomic DNA is isolated from white blood cells from a patient with a family history of breast or ovarian cancer. Section A of exon 11 of the

BRCAl-11 A-F: 5'-CCA CCT CCA AGG TGT ATC A-3' (SEQ ID NO:7) BRC Al-11 A-R: 5'-TGT TAT GTT GGC TCC TTG CT-3' (SEQ ID NO:8)

1. PCR Amplification and Dideoxy Sequence Analysis

The PCR amplification is carried out for approximately 35 cycles as described in EXAMPLE 10. PCR products are purified using Qia-quick® PCR purification kits (Qiagen^®, cat# 28104). Dideoxy sequence analysis is performed as described in EXAMPLE 10

2. Detection of the 943insl0 mutation by Allele Specific Oligonucleotide Hybridization For rapid detection of the 943insl0 mutation, unlabeled PCR products from segment A of exon 11 can be blotted in duplicate onto a nylon membrane, air dried, and UV crosslinked. Hybridization is accomplished with ³²P labeled ATP oligonucleotides corresponding to either the normal sequence 5'-CCA TGT GGC ACA AAT AC-3', (SEQ. ID. NO:23), or 5'-GCC ATG TGG CAC AAA TA-3', (SEQ. ID. NO:24) or with a sequence exhibiting the 943insl0 mutation such as 5'-CCA TGT GGA GCC ATG TG-3' (SEQ ID NO:15), or 5'-GCC ATG TGG AGC CAT GT-3' (SEQ ID NO:16). The membranes are then washed twice in 2X SSPE and 0.05% SDS at 60°C for fifteen minutes. Autoradiography is performed at -70°C for 4-12 hours with enhancing screens.

Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims:

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: OncorMed Inc.

(ii) TITLE OF THE INVENTION:

SUSCEPTIBILITY MUTATIONS FOR BREAST AND OVARIAN CANCER

(iii) NUMBER OF SEQUENCES: 24

(iv) CORRESPONDENCE ADDRESS:

(A ADDRESSEE: Howrey & Simon (B STREET: 1299 Pennsylvania Avenue, N.W. (C CITY: Washington (D STATE: DC (E COUNTRY: USA (F ZIP: 20004-2402

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/023,187

(B) FILING DATE: 05-AUG-1996

(A) APPLICATION NUMBER: 60/023,223

(B) FILING DATE: 05-AUG-1996

(A) APPLICATION NUMBER: 60/023,184

(B) FILING DATE: 05-AUG-1996 ^''

(A) APPLICATION NUMBER: 60/022,421

(B) FILING DATE: 06-AUG-1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Auerbach, Jeffrey I

(B) REGISTRATION NUMBER: 32,680

(C) REFERENCE/DOCKET NUMBER: 05371.0017.228

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202 383-7451

(B) TELEFAX: 202 383-6610

(C) TELEX: (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

GGAAGTTAGC ACTCTAGGGA 20

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GCAGTGATAT TAACTGTCTG TA 22

( 2 ) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTAATATTGG CAAAGGCATC T 21 (2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

TAAAATGTCC TCCCCAAAAG CA 22 (2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

AATTCTTAAC AGAGACCAGA AC 22

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

AAAACTCTTT CCAGAATGTT GT 22

( 2 ) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCACCTCCAA GGTGTATCA 19 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

TGTTATGTTG GCTCCTTGCT 20 (2) INFORMATION FOR SEQ ID NO : :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CCTTAAAGAC AAAGTCC 17

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

TCCTTAAAGA CAAAGTC 17

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATGAAGAAAG GAACGGG 17 (2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GATGAAGAAA GGAACGG 17 (2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

AACAGAAAGG TCAACAA 17

(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

CAACAGAAAG GTCAACA 17

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CCATGTGGAG CCATGTG 17 (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GCCATGTGGA GCCATGT 17 (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 17 :

CTTAAAGAAA CAAAGTC 17

(2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

CCTTAAAGAA ACAAAGT 17

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: ATGAAGAAAG AGGAACG 17 (2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

TGAAGAAAGA GGAACGG 17 (2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

ACAGAAAGGG TCAACAA 17

(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

AACAGAAAGG GTCAACA 17

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CCATGTGGCA CAAATAC 17 (2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

GCCATGTGGC ACAAATA 17

Claims

7 - 42 - WHAT IS CLAIMED IS:

1. An allele-specific oligonucleotide for detecting a mutation in a BRCAl encoding target polynucleotide, wherein said allele-specific oligonucleotide specifically hybridizes to said target polynucleotide, and thereby permits the detection of a mutation in said target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680.

2. The allele-specific oligonucleotide of claim 1, wherein the oligonucleotide is an isolated oligonucleotide.

3. The allele-specific oligonucleotide of claim 1, wherein said allele- specific oligonucleotide is labeled with a label selected from the group: radiolabel, fluorescent label, bioluminescent label, chemiluminescent label, or enzyme label.

4. The allele-specific oligonucleotide of claim 1, wherein said detected mutation is a 2799delAA mutation.

5. The allele-specific oligonucleotide of claim 4 having the nucleotide sequence of SEQ ID NO:9.

6. The allele-specific oligonucleotide of claim 4 having the nucleotide sequence of SEQ ID NO:10.

7. The allele-specific oligonucleotide of claim 1, wherein said detected mutation is a 4158delAG mutation.

8. The allele-specific oligonucleotide of claim 7 having the nucleotide sequence of SEQ ID NO:ll.

9. The allele-specific oligonucleotide of claim 7 having the nucleotide sequence of SEQ ID NO:12.

10. The allele-specific oligonucleotide of claim 1, wherein said detected mutation is a 5053delG mutation.

11. The allele-specific oligonucleotide of claim 10 having the nucleotide sequence of SEQ ID NO: 13.

12. The allele-specific oligonucleotide of claim 10 having the nucleotide sequence of SEQ ID NO:14.

13. The allele-specific oligonucleotide of claim 1, wherein said detected mutation is a 943insl0 mutation.

14. The allele-specific oligonucleotide of claim 13 having the nucleotide sequence of SEQ ID NO:15.

15. The allele-specific oligonucleotide of claim 13 having the nucleotide sequence of SEQ ID NO:16.

16. A primer oligonucleotide for amplifying a region of a BRCAl- encoding target polynucleotide, wherein said primer oligonucleotide specifically hybridizes to said target polynucleotide, and thereby permits the amplification of a portion of said target polynucleotide that includes nucleotides that flank nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159, or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680.

17. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:l.

18. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:2.

19. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:3.

20. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:4.

21. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:5.

22. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:6.

23. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:7.

24. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 8.

25. The primer oligonucleotide of claim 16, wherein said primer oligonucleotide is an isolated oligonucleotide.

26. The primer oligonucleotide of claim 16, wherein said primer oligonucleotide is labeled with a label selected from the group: radiolabel, fluorescent label, bioluminescent label, chemiluminescent label, or enzyme label.

27. The primer oligonucleotide of claim 16, wherein said detected mutation is a 2799delAA mutation.

28. The primer oligonucleotide of claim 16, wherein said detected mutation is a 4158delAG mutation.

29. The primer oligonucleotide of claim 16, wherein said detected mutation is a 5053delG mutation.

30. The primer oligonucleotide of claim 16, wherein said detected mutation is a 943insl0 mutation.

31. A method for classifying a tumor for diagnostic and prognostic purposes or detecting a predisposition of higher susceptibility to breast and ovarian cancer in an individual, comprising:

a) isolating a BRCAl encoding target polynucleotide from an individual; and

b) determining the identity of at least one nucleotide in a polymorphic region selected from the group of nucleotides corresponding to nucleotides at positions 943, 944, 2799, 2800, 4158, 4159, and 5053 of a BRCAl cDNA sequence having the nucleotide sequence of Genbank Accession Number U14680; and

c) classifying said BRCAl-encoding target polynucleotide for said diagnostic and prognostic purposes or for said predisposition of higher susceptibility to breast and ovarian cancer in said individual by determining the presence or absence of any of said mutations in said isolated polynucleotide.

32. The method of claim 31, wherein said identity is determined by dideoxy sequencing.

33. The method of claim 31, additionally comprising amplifying said polymorphic region of the BRCAl encoding target polynucleotide.

34. The method of claim 33, wherein said polymorphic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:l.

35. The method of claim 33, wherein said polymorphic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:2.

36. The method of claim 33, wherein said polymorphic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:3.

37. The method of claim 33, wherein said polymorphic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:4.

38. The method of claim 33, wherein said polymorphic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:5.

39. The method of claim 33, wherein said polymorphic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:6.

40. The method of claim 33, wherein said polymorphic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:7.

41. The method of claim 33, wherein said polymorphic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:8.

42. The method of claim 31, wherein said determination identifies a 2799delAA mutation in said target polynucleotide.

43. The method of claim 31, wherein said determination identifies a 4158delAG mutation in said target polynucleotide.

44. The method of claim 31, wherein said determination identifies 5053delG mutation in said target polynucleotide.

45. The method of claim 31, wherein said determination identifies a 943insl0 mutation in said target polynucleotide.

46. A method for classifying a BRCAl-encoding target polynucleotide for diagnostic and prognostic purposes of detecting a predisposition or higher susceptibility to breast and ovarian cancer in an individual, comprising:

a) isolating said BRCAl-encoding target polynucleotide from an individual;

b) incubating the target polynucleotide in the presence of an allele-specific oligonucleotide, wherein said incubation is under conditions sufficient to allow hybridization to occur between the target polynucleotide and said allele-specific oligonucleotide; wherein said allele-specific oligonucleotide specifically hybridizes to said target polynucleotide, and thereby permits the detection of a mutation in said target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680;

c) allowing said hybridization to occur;

d) detecting said hybridization between said target polynucleotide and said allele-specific oligonucleotide; and

e) classifying said BRCAl-encoding target polynucleotide for said diagnostic and prognostic purposes or for said predisposition of higher susceptibility to breast and ovarian cancer in said individual by determining the presence or absence of any of said mutations in said isolated polynucleotide.

47. The method of claim 46, wherein said allele-specific oligonucleotide is labeled with a label selected from the group: radiolabel, fluorescent label, bioluminescent label, chemiluminescent label, or enzyme label.

48. The method of claim 46, further comprising amplifying said polymorphic region of said target polynucleotide prior to said hybridization.

49. The method of claim 46, wherein said hybridization is a Southern blot hybridization.

50. The method of claim 46, wherein said hybridization is a dot blot hybridization.

51. The method of claim 46, wherein said allele-specific oligonucleotide is an isolated allele-specific oligonucleotide.

52. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of

SEQ ID NO:9.

53. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 10.

54. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:ll.

55. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:12.

56. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:13.

57. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of

SEQ ID NO:14.

58. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:15.

59. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:16.

60. The method of claim 46, wherein a 2799delAA mutation is detected.

61. The method of claim 46, wherein a 4158delAG mutation is detected.

62. The method of claim 46, wherein a 5053delG mutation is detected.

63. The method of claim 46, wherein a 943insl0 mutation is detected.

64. A kit, comprising a carrier means being compartmentalized to receive in close confinement one or more container means, said container means comprising an allele-specific oligonucleotide; wherein said allele-specific oligonucleotide has a nucleotide sequence sufficient to permit said oligonucleotide to specifically hybridize to a BRCAl-encoding polynucleotide, and to detect a mutation in said target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 2799, 2800, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession

Number U14680. AMENDED CLAIMS

[received by the International Bureau on 16 January 1998 (16.01.98); original claims 1,16,31,46 and 64 amended; original claims 4-6, 27,42,52,53 and 60 cancelled; remaining claims unchanged (8 pages)]

An allele-specific oligonucleotide for detecting a mutation in a BRCAl encoding target polynucleotide, wherein said allele-specific oligonucleotide specifically hybridizes to said target polynucleotide, and thereby permits the detection of a mutation in said target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U 14680.

3. The allele-specific oligonucleotide of claim 1 , wherein said allele-specific oligonucleotide is labeled with a label selected from the group: radiolabel, fluorescent label, bioluminescent label, chemiluminescent label, or enzyme label.

8. The allele-specific oligonucleotide of claim 7 having the nucleotide sequence of SEQ ID NO:l l .

9. The allele-specific oligonucleotide of claim 7 having the nucleotide sequence of SEQ ID NO: 12.

13. The allele-specific oligonucleotide of claim 1, wherein said detected mutation is a 943ins 10 mutation.

14. The allele-specific oligonucleotide of claim 13 having the nucleotide sequence of SEQ ID NO: 15.

15. The allele-specific oligonucleotide of claim 13 having the nucleotide sequence of SEQ ID NO: 16.

16. A primer oligonucleotide for amplifying a region of a BRCAl-encoding target polynucleotide, wherein said primer oligonucleotide specifically hybridizes to said target polynucleotide and thereby permits the amplification of a portion of said target polynucleotide that includes nucleotides that flank nucleotides corresponding to nucleotides 943, 944, 4158, 4159, or 5053 of a BRCAl CDNA having the nucleotide sequence of Genbank Accession Number Ul 4680.

17. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1.

19. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 3.

22. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:5.

24. The primer oligonucleotide of claim 16 having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:8.

31. A method for classifying a tumor for diagnostic and prognostic purposes or detecting a predisposition of higher susceptibility to breast and ovarian cancer in an individual, comprising: a) isolating a BRCAl encoding target polynucleotide from an individual; and b) determining the identity of at least one nucleotide in a polymorphic region selected from the group of nucleotides corresponding to nucleotides at positions 943, 944, 4158, 4159, and 5053 of a BRCAl cDNA sequence having the nucleotide sequence of Genbank Accession Number U14680; and c) classifying said BRCAl-encoding target polynucleotide for said diagnostic and prognostic purposes or for said predisposition of higher susceptibility to breast and ovarian cancer in said individual by determining the presence or absence of any of said mutations in said isolated polynucleotide.

32. The method of claim 31 , wherein said identity is determined by dideoxy sequencing.

33. The method of claim 31, additionally comprising amplifying said polymoφhic region of the BRCAl encoding target polynucleotide.

34. The method of claim 33, wherein said polymoφhic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1.

35. The method of claim 33, wherein said polymoφhic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:2.

36. The method of claim 33, wherein said polymoφhic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:3.

37. The method of claim 33, wherein said polymoφhic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:4.

38. The method of claim 33, wherein said polymoφhic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:5.

39. The method of claim 33, wherein said polymoφhic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:6.

40. The method of claim 33, wherein said polymoφhic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:7.

41. The method of claim 33, wherein said polymoφhic region is amplified with an oligonucleotide primer having a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:8.

44. The method of claim 31, wherein said determination identifies a 5053delG mutation in said target polynucleotide.

45. The method of claim 31 , wherein said determination identifies a 943ins 10 mutation in said target polynucleotide.

46. A method for classifying a BRCAl-encoding target polynucleotide for diagnostic and prognostic puφoses of detecting a predisposition or higher susceptibility to breast and ovarian cancer in an individual, comprising: a) isolating said BRCAl-encoding target polynucleotide from an individual; b) incubating the target polynucleotide in the presence of an allele-specific oligonucleotide, wherein said incubation is under conditions sufficient to allow hybridization to occur between the target polynucleotide and said allele-specific oligonucleotide; wherein said allele-specific oligonucleotide specifically hybridizes to aid target polynucleotide, and thereby permits the detection of a mutation in said target polynucleotide at nucleotides corresponding to nucleotides 943, 944, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U14680; c) allowing said hybridization to occur; d) detecting said hybridization between said target polynucleotide and said allele- specific oligonucleotide; and e) classifying said BRCAl-encoding target polynucleotide for said diagnostic and prognostic puφoses or for said predisposition of higher susceptibility to breast and ovarian cancer in said individual by determining the presence or absence of any of said mutations ins aid isolated polynucleotide.

48. The method of claim 46, further comprising amplifying said polymoφhic region of said target polynucleotide prior to said hybridization.

54. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 1.

55. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 12.

56. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 13.

57. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 14.

58. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 15.

59. The method of claim 46, wherein said allele-specific oligonucleotide has a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 16.

61. The method of claim 46, wherein a 4158delAG mutation is detected.

62. The method of claim 46, wherein a 5053delG mutation is detected.

63. The method of claim 46, wherein a 943ins 10 mutation is detected.

64. A kit, comprising a carrier means being compartmentalized to receive in close confinement on or more container means, said container means comprising an allele- specific oligonucleotide; wherein said allele-specific oligonucleotide has a nucleotide sequence sufficient to permit said oligonucleotide to specifically hybridize to a BRCAl-encoding polynucleotide, and to detect a mutation in said target polynucleotide at nucleotides coπesponding to nucleotides 943, 944, 4158, 4159 or 5053 of a BRCAl cDNA having the nucleotide sequence of Genbank Accession Number U 14680.

STATEMENT UNDER ARTICLE 19

Responsive to the International Search Report mailed 17 November 1997, Applicant has amended claims 1, 16, 31, 46 and 64 and cancelled claims 4, 5, 6, 27, 42, 52, 53 and 60. All other claims are unchanged.

Applicant's original claim set recited, among others, the BRCAl 1799delAA mutations which forms a 901 stop codon. Upon review of two of the cited references in the International Search Report mailed 17 November 1997, it appears that Friedman et al and Couch et al disclose a BRCAl 1800delAA mutation which forms a 901 stop codon. The actual BRCAl sequence at 1799-1802 is AAA. Accordingly, Applicant has amended the claims to delete reference to the 2799 and 2800 mutations.

To summarize: claims 1, 16, 31, 46 and 64 have been replaced by amended claims bearing the same numbers; claim 4, 5, 6, 27, 42, 52, 53 and 60 have been cancelled; and all other claims remain unchanged.