WO1999015704A1

WO1999015704A1 - Genetic panel assay for susceptibility mutations in breast and ovarian cancer

Info

Publication number: WO1999015704A1
Application number: PCT/US1998/020256
Authority: WO
Inventors: Mark B. Rabin; Jeffrey Farrow
Original assignee: Oncormed, Inc.
Priority date: 1997-09-23
Filing date: 1998-09-23
Publication date: 1999-04-01
Also published as: AU9586598A

Abstract

The invention relates to gene mutations that predispose individuals to breast and ovarian cancer. More specifically, the invention relates to specific mutations in the BRCA1 gene and the BRCA2 gene. In addition, it also relates to methods and molecules for detecting the presence of these mutations and polymorphisms.

Description

GENETIC PANEL ASSAY FOR SUSCEPTIBILITY MUTATIONS IN BREAST AND OVARIAN CANCER

FIELD OF THE INVENTION

The invention relates to gene mutations that predispose individuals to breast and ovarian cancer. More specifically, the invention relates to specific mutations in the BRCAl gene and the BRCA2 gene. In addition, it also relates to methods and molecules for detecting the presence of these mutations and polymorphisms.

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 5^:861-865 (1994). Two genes, BRCAl and BRCA2, have been estimated to account for 60-80% of the predisposition for familial breast cancer. Easton et al, American Journal of Human Genetics 52:678-701 (1993); Wooster et al, Science 265:2088-2090 (1994); Miki et al, Science 266:66-71 (1994); Wooster et al, Nature 378:789-792 (1995); Tavtigian et al, Nature Gent. JJ:333-337 (1996). Located on chromosome 17, BRCAl was 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). More recently, a second tumor suppressor gene (BRCA2) has been identified on chromosome 13 that confers increased risk for breast and ovarian cancer. Knudson, et al, Nature Genet. 5_.J03-104 (1993). In addition to conferring an increased risk for breast and ovarian cancer in women, mutations in BRCA2 also appear to confer an increased risk for breast cancer in male carriers. Wooster et al, Science 265:2088-2090 (1994). The BRCAl gene is divided into 24 separate exons. Exons 1 and 4 are non- coding, in that they are not part of the final functional BRCAl protein product. The BRCAl coding region spans roughly 5600 base pairs (bp) and encodes a protein of approximately 1,863 amino acids.

The BRCA2 gene is divided into approximately 27 exons, spanning as much as 70,000 bp. The BRCA2 gene has recently been sequenced. Tavtigtan et al. Nature Genet. 12:333-337 (1996).

The location of one or more mutations in the BRCAl and the BRCA2 genes 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. It has been estimated that 36% of all women in whom breast cancer appears between the ages of 20 and 29 have a genetic predisposition, as compared with 1% of women with breast cancer diagnosed at the age of 80 or older. Claus et al, Am. J. Hum. Genet. 48:232-242 (1994). These women, once identified, can be targeted for more aggressive prevention programs. Genetic screening of family members can also be a useful approach to assessing risk. For example, genetic screening can confirm a hereditary form of cancer in an affected individual and identify unaffected family members who carry the mutation and are therefore predisposed to cancer. A positive result on such a screen can enable individuals and their physicians to plan a more aggressive monitoring an management strategy. Conversely, a negative result can help to reassure non-carriers that their risk is the same as the general population. Screening is carried out by a variety of methods that include karyotyping, allele- specific probe binding, allele-specific PCR, and DNA sequencing.

Many mutations have already been reported in the BRCAl gene. Shattuck- Eidens, D., et al, Journal of the American Medical Association 273:535-541 (1995). Currently, more than 100 distinct mutations have been identified. Collins et al, New Engl. J. Med. 334:186-189 (1995). A large number of mutations have been identified in the BRCA2 gene as well. Tavtigtan et al, Nature Genet. 12:333-337 (1996). There is a need in the art to develop improved assays to identify individuals carrying these and other mutations in the BRCAl or BRCA2 genes. Such improved assays would allow more widespread diagnostic screening for hereditary breast and ovarian cancer than is currently possible. The present invention addresses this problem by providing an improved assay for mutations in BRCAl and BRCA2. In addition to specificity and ease of use, certain embodiments of the present invention are able to positively identify whether an individual is homozygous wild-type, homozygous mutant, or heterozygous (carrying one allele of each mutant and wild-type) in a single hybridization. In addition, the present invention is able to screen for greater than a single mutation, while still retaining the ability to determine whether an individual is heterozygous or homozygous for each allele and each mutation.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides a chemiluminescent image of six reverse dot blots. Each blot is hybridized with a different biotin-labeled target polynucleotide, amplified by PCR from a different patient nucleic acid sample. Following hybridization, each blot is stringently washed, and analyzed with the NEB Phototype Chemiluminescent Detection Kit. Specific hybridization events are thereby detected. Each membrane is derivatized with the same six allele-specific ohgonucleotides, laid out in two rows of three columns. Across the top row of each membrane, left to right, are allele-specific ohgonucleotides, specific for the wild-type alleles at the locations of the 185delAG and the 5382insC mutations of BRCAl, and the 6174delT mutation of BRCA2. Across the bottom row, left to right, are allele-specific ohgonucleotides, specific for the mutant alleles at the locations of the 185delAG and the 5382insC mutations of BRCAl, and the 6174delT mutation of BRCA2. The membrane on the top right is hybridized with a "normal" target polynucleotide, amplified from the patient sample containing only the wild- type alleles for BRCAl and BRCA2 genes at these locations. Membranes on the top left and middle left are hybridized with a target polynucleotide, amplified from a patient DNA sample containing, in addition to the three wild-type alleles, a mutant allele corresponding to a 5382insC mutation. Membranes on the bottom left, bottom right, and middle right are hybridized with a target polynucleotide, amplified from a patient DNA sample containing, in addition to the three wild-type alleles, a mutant allele corresponding to a 6174delT mutation.

Figure 2 provides an autoradiogram of a series of reverse dot blots. Figure 3 provides an autoradiograph of a series of reverse dot blots. An experiment titrating the concentration of allele-specific ohgonucleotides is shown. DNA from plasmid clones is used as the target polynucleotide in an RDB assay. An autoradiograph of plasmid clones isolated using RDB. The top two rows in each filter represent wild type signal while the bottom two rows represent a mutant plasmid.

Squares in which all rows light up indicate the presence of a mixed colony population. Each oligo probe is titrated to determine the optimal concentration from 1 ,2pmoles to O.Olpmoles.

Figure 4 provides an autoradiogram of a series of reverse dot blots. .An experiment comparing allele-specific ohgonucleotides and a determination of their optimal concentration is shown. In the first two columns are tests of 185 and 5382 probes where II, 12 and 13 are three different versions of oligo probe to detect the same mutation. The last column is a titration of two versions of the 6174 probe; the left side is a titration of the first oligo designed from 7.2 pmoles to 1.2 pmoles while the right side is an improved oligo titrated from 1.2 pmoles to 0.01 pmoles. A significant increase in sensitivity was obtained by redesigning the probe.

Figure 5 provides an autoradiogram of a series of reverse dot blots. A validation experiment is shown. Plasmids containing cloned BRCAl and BRCA2 DNA are used, demonstrating specificity and sensitivity of the allele-specific ohgonucleotides.

SUMMARY OF THE INVENTION

The invention provides a rapid, accurate, and simple assay for at least one of a group of mutations in the genes for BRCAl and BRC-A2, including the 185delAG and 5382insC of BRCAl and 6174delT of BRCA2. In addition, the assay also detects the wild-type allele of at least one of these mutations. In its most preferred formats, the assay contains a series of allele-specific ohgonucleotides that are specific for each allele, mutant and wild-type. The invention also provides a kit, suitable for genetic testing. Such a kit contains primers for amplifying regions of the BRCAl and the BRCA2 genes encompassing regions where the 185delAG, and 5382insC of BRCAl and 6174delT of BRCA2 mutations are found. The kit also contains allele-specific ohgonucleotides, specific for both mutant and wild-type alleles of at least one of these mutations. The kit may also contain sources of "control" target polynucleotides, as positive and negative controls. Such sources may be in the form of patient nucleic acid samples, cloned target polynucleotides, plasmids or bacterial strains carrying positive and negative control DNA.

The invention provides the allele-specific ohgonucleotides themselves, as well as plasmid constructs and bacterial strains carrying positive and negative control DNA for each of the alleles, mutant and wild-type, for regions of the human genome corresponding the locations of the 185delAG and 5382insC mutations of BRCAl and the 6174delT mutation of BRCA2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention concerns methods and reagents for detecting mutations in the BRCAl and BRCA2 genes of patients associated or correlated with familial breast and/or ovarian cancer.

It has been reported that in familial breast and ovarian cancer in Ashkenazi women, three mutations are of particular prevalence, the 185delAG and 5382insC mutations in BRCAl and the 6174delT mutation in BRCA2. Abeliovich et al, Am. J. Hum. Genet. 60:505-514 (1997). 20% of early onset breast cancer in this population is positive for the 185delAG mutation and 8% is positive for the 6174delT mutation. Approximately 2.6% of Ashkenazi Jewish women carry at least one of three inherited mutations in either BRCAl or BRCA2. There is a carrier frequency of 0.9%, of for the 185delAG mutation, 0.9% to 1.52% for the 6174delT mutation, and 0.13% for the 5382insC mutation in the general Ashkenazi Jewish population. Struewing et al, Nature Genet. JJ-198-200 (1995); Roa et al, Nature Genet. 14:185-187 (1996). It has been estimated that the contribution of the 185delAG mutation alone to breast cancer cases diagnosed before age 50 in Ashkenazi Jewish women to be approximately 19%. Roa et al, Nature Genet. 14:185-187 (1996).

Abeliovich et al. amplified the regions bearing the 185delAG, 5382insC, and the 6174delT mutations by multiplex PCR. The resultant products were then run on a denaturing polyacrylamide gel. Since each mutation is an insertion or a deletion, bands that migrate at an anomalous rate are suggestive of a mutation. However, this type of assay requires confirmation of specific amplification. Abeliovich addressed this problem by introducing a restriction site in primers specific of the 185delAG and the 6174delT mutations, allowing confirmation of a specific amplification product. Confirmation of a 5382insC mutation, however, required direct sequencing. There is a need in the art for improved assay for detection of these mutations. Present methods are time-consuming and frequently require verification by other means.

Others, such as Roa et al. have applied methods, such as allele specific oligonucleotide hybridization to detect BRCAl and BRCA2 mutations. The method of Roa requires that separate hybridizations be performed, using replicate filters of patient samples. Two different hybridizations must be performed for each mutation in question, one using a mutant-specific oligonucleotide and one using a wild-type oligonucleotide. Additional filters and additional hybridizations must be performed for each mutation to be studied.

A. Definitions

The term "BRCAl" as used herein refers to Breast Cancer gene 1. BRCAl is a tumor suppressor gene located on chromosome 17. Individuals carrying certain mutations in BRCAl are at increased risk for breast and ovarian cancer. A number of polymorphisms and mutations in BRCAl have been described. Many of these result in non-functional proteins or proteins with altered function. Nevertheless, it will be understood by those in the art that these and other genes and proteins are all BRCAl, whether genomic DNA, RNA, cDNA, etc.

The term "BRCA2" as used herein refers to Breast Cancer gene 2. BRCA2 is a tumor suppressor gene located on chromosome 13. Tavtigtan et al, Nature Genet. 12:333-337 (1996); Wooster et al, Nature 378:789-792 (1995). A number of polymorphisms and mutations in BRCA2 have been described. Tavtigtan et al, Nature Genet. 12:333-337 (1996). Individuals carrying certain mutations in BRCA2 are at increased risk for breast and ovarian cancer. Many of these result in nonfunctional proteins or proteins with altered function. Nevertheless, it will be understood by those in the art that these and other genes and proteins are all BRCA2, whether genomic DNA, RNA, cDNA, etc.

The term "185delAG" as used herein refers to a mutation in exon 2 of a BRCAl gene. The mutation corresponds to an "AG" deletion at or about a position corresponding to nucleotides 185 and 186 of a BRCAl gene. By way of reference, it will be understood by those in the art that the mutation corresponds to the 185delAG described, for example, by Simard et al, Nat. Genet. 8:392-398 (1994); Friedman et al, Am. J. Hum. Genet. 57:1284-1297 (1995); Takahashi et al, Cancer Res. 55:2998- 3002 (1995); Tonin et al, Am. J. Hum. Genet. 57:189 (1995); and Struewing et al, Nat. Genet. U :\ 98-200 (1995).

The term "5382insC" as used herein refers to a mutation in exon 20 of a

BRCAl gene. The mutation corresponds to the insertion of a "C" at or about a position corresponding to nucleotide 5382 of a BRCAl gene. By way of reference, it will be understood by those in the art that the mutation corresponds to the 5382insC mutation described, for example, by Benjamin et al, Nat. Genet. 14:185-187 (1996); and Oddoux et al, Nat. Genet. 14:188-190 (1996).

The term "6174delT" as used herein refers to a mutation exon 11Q of a

BRCA2 gene. The mutation is the deletion of a "T" at or about a position corresponding to nucleotide 6174 of a BRCA2 gene. By way of reference, it will be understood by those in the art that the mutation corresponds to the 6174delT mutation described, for example, by Neuhausen et al, Nat. Genet. JJJ26-128 (1996).

The term "biological sample" as used herein refers to any material containing nucleic acid, either DNA or RNA. Generally, such material will be in the form of a blood sample, tissue sample, cells, bacteria, or histology section, either fresh, fixed, frozen, or embedded in paraffin. The term "exon" as used herein is a term of art and refers to a segment or portion of a eukaryotic gene that is transcribed into an messenger RNA (mRNA).

The term "nucleotide" as used herein is intended to refer to ribonucleotides, deoxyribonucleotides, acyclic derivatives of nucleotides, and functional equivalents thereof, of any phosphorylation state. Functional equivalents of nucleotides are those that act as a substrates for a polymerase as, for example, in an amplification method. Functional equivalents of nucleotides are also those that may be formed into a polynucleotide that retains the ability to hybridize in a sequence specific manner to a target polynucleotide.

The term "oligonucleotide" as used herein is defined as a nucleic acid molecule comprised of less than 100 nucleotides. Preferably, ohgonucleotides are between 10 and 35 nucleotides in length. Most preferably, ohgonucleotides are 15 to 25 nucleotides in length. The exact length of a particular oligonucleotide, however, will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The source of the ohgonucleotides is not essential to the present invention. Ohgonucleotides may be synthesized chemically by any suitable means known in the art or derived from a biological sample, as for example, by restriction digestion.

The term "primer" as used herein refers to an oligonucleotide as defined herein, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, as for example, in a PCR reaction. Typically, such ohgonucleotides will be DNA molecules that are at least about 20 nucleotides in length and have a nucleotide sequence corresponding to a region of a BRCAl gene or a BRCA2 gene. Such molecules may be labeled, according to any technique known in the art, such as with radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags, etc.

The primers of the present invention are preferably single stranded, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The exact lengths of the primers will depend on many factors, including temperature and source of primer and use of the method. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain more or fewer nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template.

The primers herein are selected to be "substantially complementary" to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under environmental conditions wherein a polymerase or ligase is functional. Therefore, the 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 complementary to the strand. Alternatively, 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 strand to be amplified to hybridize therewith and thereby form a template for synthesis of the extension product of the other primer. However, for detection purposes, particularly using labeled sequence- specific probes, the primers typically have exact complementarity to obtain the best results. Alternatively, primers may be employed that will hybridize to their template only under conditions of high stringency.

"Target polynucleotide" as used herein refers to a nucleic acid sequence of interest. The target polynucleotide may be DNA or RNA and may be isolated from any suitable source, such as peripheral blood, tissue biopsies, paraffin-embedded histology sections, PCR reaction products, etc. Typically, the target polynucleotide will be amplified from a biological sample, as by PCR. Other suitable sources of nucleic acid will be readily apparent to those in the art. Generally, the target polynucleotide will contain at least a segment of a gene for BRCAl or BRCA2.

"Reverse dot blot" as used herein refers to an assay wherein a probe, such as an allele-specific oligonucleotide, is bound to a solid support, a target polynucleotide is hybridized to the allele-specific oligonucleotide, and a detectable signal is produced. As used herein, the terms "mutation" or "polymorphism" refer to the condition in which the identity of one or more nucleotides differ between two or more otherwise substantially similar target polynucleotides at a particular site in a nucleic acid sequence. "Mutations" or "polymorphisms" may be in the form of deletions, insertions, or base changes. Typically, the term "mutation" is used to denote a polymorphism that results in the gene coding for a non-functioning protein or a protein with a substantially altered or reduced function or that additionally contributes to a disease condition, such as cancer.

The term "allele-specific oligonucleotide" refers to an oligonucleotide that is able to hybridize to a region of a target polynucleotide spanning the sequence, mutation, or polymorphism being detected and is substantially unable to hybridize to a corresponding region of a target polynucleotide that either does not contain the sequence, mutation, or polymorphism being detected or contains an altered sequence, mutation, or polymorphism. As will be appreciated by those in the art, allele-specific is not meant to denote an absolute condition. Allele-specificity will depend upon a variety of environmental conditions, including salt and formamide concentrations, hybridization and washing conditions and stringency. Depending on the sequences being analyzed, one or more allele-specific ohgonucleotides may be employed for each target polynucleotide, as described further herein below.

"Allele-specific hybridization" as used herein refers to a hybridization involving at least one allele-specific oligonucleotide. Allele-specific hybridization also refers to an assay comprising such a hybridization step, including subsequent washing and/or detection steps. The specificity of such hybridizations are dependent upon environmental conditions of both the hybridization and subsequent washing steps, as well as the nucleotide sequence and composition of the allele-specific oligonucleotide.

The term "isolated" as used herein refers to the state of being substantially free of other material such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris or growth media with which the target polynucleotide, primer oligonucleotide, or allele-specific oligonucleotide may be associated. Typically, the term "isolated" is not intended to refer to a complete absence of these materials. Neither is the term "isolated" generally intended to refer to water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.

B. Amplification

Typically, a biological sample will the source of the nucleic acid. Nucleic acid utilized herein may be extracted from a biological sample, such as blood, tissue biopsy, histology section and the like. In certain embodiments of the present invention, the source of nucleic acid will be a plasmid or a bacterial strain as in where, for the purpose of validation controls, a region of a BRCAl or a BRCA2 gene has been cloned into a plasmid. The nucleic acid may be purified from any of the above- mentioned sources by a variety of techniques, such as are described in standard manuals such as Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), herein incorporated by reference; and Current Protocols in Molecular Biology (Eds. Ausubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc, Wiley-Interscience, NY, N.Y., 1992), herein incorporated by reference, or that are otherwise known in the art.

Generally, in performing the methods of the present invention, the target polynucleotide will be amplified from a nucleic acid source by any suitable means known in the art. Examples of preferred amplification means include the ligase chain reaction (LCR) Barany, F., Proc. Natl. Acad. Sci. (U.S.A.) 88J89-193 (1991), the oligonucleotide ligation assay (OLA) Landegren, U. et al, Science 241:1077-1080 (1988), and the polymerase chain reaction (PCR). PCR is the most highly preferred amplification means. Mullis, et al, U.S. Patent No. 4,965,188.

LCR uses two pairs of oligonucleotide probes to exponentially amplify a specific target. The sequences of each pair of ohgonucleotides is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependent ligase. As with PCR, the resulting products thus serve as a template in subsequent cycles and an exponential amplification of the desired sequence is obtained. In accordance with the present invention, LCR can be performed with ohgonucleotides having the proximal and distal sequences of the same strand of a polymorphic site. In one embodiment, either oligonucleotide will be designed to include the actual polymorphic site of the polymorphism. In such an embodiment, the reaction conditions are selected such that the ohgonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the polymorphic site present on the oligonucleotide.

The "Oligonucleotide Ligation Assay" ("OLA") shares certain similarities with

LCR and may also be adapted for use in polymorphic analysis. The OLA protocol uses two ohgonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. Unlike LCR, however, OLA results in

"linear" rather than exponential amplification of the target sequence.

Nickerson, D.A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D.A. et al, Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA. In addition to requiring multiple, and separate, processing steps, one problem associated with such combinations is that they inherit all of the problems associated with PCR and OLA.

Other known nucleic acid amplification procedures, such as transcription- based amplification systems (Malek, L.T. et al, U.S. Patent 5,130,238; Davey, C. et al, European Patent Application 329,822; Schuster et al, U.S. Patent 5,169,766; Miller, H.I. et al, PCT appln. WO 89/06700; Kwoh, D. et al, Proc. Natl. Acad. Sci. (U.S.A.) 86:1173 (1989); Gingeras, T.R. et al, PCT application WO 88/10315)), or isothermal amplification methods (Walker, G.T. et al, Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)) may also be used.

In a highly preferred embodiment of the present invention, the target polynucleotide is amplified by PCR from nucleic acid isolated from a biological sample. In one embodiment of the present invention pairs of primers are provided. Examples of suitable pairs of primers include:

BRCA1-2F: 5'-GAA GTT GTC ATT TTA TAA ACC TTT-3', (SEQ ID NOJ) and

BRCA1-2R: 5'-TCT CTT TTC TTC CCT AGT ATG T-3', (SEQ ID NO:2) or

BRCAl -20F: 5'-ATA TGA CGT GTC TGC TCC AC-3', (SEQ ID NO: 3) and

BRCA1-20R: 5'-GGG AAT CCA AAT TAC ACA GC-3', (SEQ ID NO: 4) or

BRCA2-QF: 5'-ACG AAA ATT ATG GCA GGT TGT-3, (SEQ ID NO: 5) and

BRCA2-QR: CTT GTC TTG CGT TTT GTA ATG-3', (SEQ ID NO: 6)

The designations BRCAl -2F and BRCAl -2R refer to sequences in the BRCAl gene, Exon 2, forward and reverse primers, respectively; BRCAl -20F and

BRCAl -20R refer to sequences in the BRCAl gene, Exon 20, forward and reverse primers, respectively; BRCaA -QF and BRCA2-QR refer to sequences in the BRCA2 gene, Exon 11Q, forward and reverse primers, respectively.

The precise sequence of the primers is not essential for the methods of the present invention. Alterations in the sequences of the primers are considered and do not depart from the nature of the invention. Primers of the present invention are those that allow the sequence-specific amplification of a region of a target polynucleotide encompassing a polymorphism or mutation addressed by the methods of the present invention. Preferred primers of the present invention are those that enable amphfication of regions comprising a portion of exon 2 of the BRCAl gene, exon 20 of the BRCAl gene, and exon 11Q of the BRCA2 gene. Particularly preferred primers of the present invention are those that allow for the sequence specific amplification of a region comprising the location of a 185delAG, a 5382insC, or a 6174delT mutation, whether or not such a mutation exists within the target polynucleotide. A most preferred embodiment of the present invention, comprises the use of a set of primers that allows the sequence-specific amplification of regions of the BRCAl and the BRCA2 genes comprising the location of the 185delAG, the 5382insC, and the 6174delT mutations, whether or not such mutations exist within the target polynucleotide. Such sequence-specific amplification may be performed as individual reactions for each region to be amplified. In a preferred embodiment of the present invention, however, greater than one such region is amplified in a single reaction, as by multiplex PCR. In a most preferred embodiment, all such regions will be amplified in a single reaction.

In a preferred embodiment of the present invention, the target polynucleotide is directly or indirectly labeled. The target polynucleotide may be labeled, whether or not the target polynucleotide is amplified. In addition, the target polynucleotide may be labeled either before amplification, during amplification, or subsequent to amplification. In a preferred embodiment of the methods of the present invention, the target polynucleotide is labeled during amplification through the use of labeled primers. Where labeled primers are used, either the forward primer or the reverse primer may be labeled. Alternatively, both forward and reverse primers may be labeled. Labeled nucleotides may also be incorporated during amphfication to produce a labeled target polynucleotide. Alternatively, the target polynucleotide may be reacted with or conjugated to a label. Such labels may be used either directly or indirectly to produce or amplify a detectable signal. Examples of suitable labels include enzyme labels, radiolabels, chromophores, fluorophores, chemiluminescent labels, components of amplified tags such antigen-labeled antibody, nucleotide sequence tags, biotin-avidin combinations etc. Examples of particularly preferred labels are biotin and digoxygenin. In certain embodiments of the present invention, the target polynucleotide may be attached to a solid support. In such embodiments, the allele-specific ohgonucleotides are preferably labeled. C. AHele-Specific Oligonucleotides

Allele-specific oligonucleotides may be biologically derived or chemically synthesized. Chemical synthesis is however preferred. Allele-specific oligonucleotides of the present invention are generally less than 50 nucleotides in length, preferably 10 to 35 nucleotides in length. Most preferably, allele-specific oligonucleotides will be 15 to 25 nucleotides in length. Particular lengths of allele- specific oligonucleotides will be guided by the desired hybridization and washing conditions.

1. Immobilization of AHele-Specific Oligonucleotides

Generally, allele-specific oligonucleotides will be attached to a solid support, though in certain embodiments of the present invention allele-specific oligonucleotides may be in solution. In such embodiments, the target polynucleotide is preferably bound to a solid support. In those embodiments where the allele-specific oligonucleotides or the target polynucleotides are attached to a solid support, attachment may be either covalent or non-covalent. Attachment may be mediated, for example, by antibody-antigen-type interactions, poly-L-Lys, streptavidin or avidin- biotin, salt-bridges, hydrophobic interactions, chemical linkages, UN cross-linking, baking, etc. In addition, allele-specific oligonucleotides may be synthesized directly on a solid support or attached to the solid support subsequent to synthesis. In a preferred embodiment, allele-specific oligonucleotides are chemically synthesized by phosphoramide chemistry and labeled on their 5' end with an amino group so as to facilitate covalent attachment to a solid support.

Suitable solid supports for the present invention include substrates constructed of silicon, glass, plastic (polystyrene, nylon, polypropylene, etc.), paper, etc. Solid supports may be formed, for example, into wells (as in 96-well dishes), plates, slides, sheets, membranes, fibers, chips, dishes, and beads. In certain embodiments of the present invention, the solid support is treated, coated, or derivatized so as to facilitate the immobilization of an allele-specific oligonucleotide or a target polynucleotide. Preferred treatments include coating, treating, or derivatizing with poly-L-Lys, streptavidin, antibodies, silane derivatives, low salt, or acid. A particularly preferred solid support is a positively-charged nylon membrane, derivatized with ethylendiamineacetylcarbomide (EDAC, Sigma Chemicals).

2. Design and Selection of AHele-Specific Oligonucleotides

In highly preferred embodiments of the present invention, allele-specific oligonucleotides specific for each allele are provided. Examples of suitable allele- specific oligonucleotides include:

185WT-1: 5'-ATCTTAGAGTGTCCCATCT-3', (SEQ ID NO: 7)

185WT-2: 5'-AAT CTT AGA GTG TCC CAT CTG-3', (SEQ ID NO: 8)

185WT-3: 5'-ATT CTT AGA GTG TCC CAT CTG T-3', (SEQ ID NO: 9)

185M-1: 5'-ATC TTA GTG TCC CAT CTG T-3', (SEQ ID NO: 10)

185M-2: 5'-AAT CTT AGT GTC CCATCT GTC-3', (SEQ ID NO: 11)

185M-3: 5'-AAT CTT AGT GTC CCATCT GTC T-3', (SEQ ID NO: 12)

5382WT-1: 5'-AGA GAATCC CAG GAC AG-3', (SEQ ID NO: 13)

5382WT-2: 5'-CAA GAG AAT CCC AGG ACA G-3', (SEQ ID NO: 14)

5382WT-3: 5'-AAG AGA ATC CCA GGA CAG A-3', (SEQ ID NO: 15)

5382M-1: 5'-AGA GAATCC CCA GGA C-3', (SEQ ID NO: 16)

5382M-2: 5'-AGA GAATCC CCA GGA CAG-3', (SEQ IDNO: 17)

5382M-3: 5'-AGA GAATCC CCA GGA CAG A-3', (SEQ ID NO: 18)

6174WT-1: 5'-ACA GCAAGT GGAAAATC-3', (SEQ ID NO: 19)

6174WT-2: 5'-ACA GCAAGT GGAAAATCT GT-3', (SEQ ID NO: 20)

6174M-1: 5'-CAC AGC AAGGGAAAAT-3', (SEQ ID NO: 21) and

6174M-2: 5'-CAC AGC AAG GGAAAA TC-3' (SEQIDNO.22) Those of ordinary skill in the art will readily comprehend that the oligonucleotides containing a sequence complementary the above-listed allele-specific oligonucleotides is also encompassed by this list.

The designations 185WT-1, 185WT-2, 185WT-3, 185M-1, 185M-2, and 185M-3 refer to allele-specific oligonucleotide sequences that hybridize to a region in exon 2 of a BRCAl gene corresponding to the location of a 185WT allele or a 185delAG mutation, respectively; the designations 5382WT-1, 5382WT-2, 5382WT- 3, 5382M-1, 5382M-2, and 5382M-3 refer to allele-specific oligonucleotide sequences that hybridize to a region in exon 20 of a BRCAl gene corresponding to the location of a 5382WT allele or a 5382insC mutation, respectively; the designations 6174WT-1, 6174WT-2, 6174M-1, and 6174M-2 refer to allele-specific oligonucleotide sequences that hybridize to a region in exon 11Q of a BRCA2 gene corresponding to the location of a 6174WT allele or a 6174delT mutation, respectively. The letters "WT" and "M" within the probe designation denote allele-specific oligonucleotides that specifically hybridize to the wild-type form of the allele and the mutant form of the allele, respectively.

D. AHele-Specific Hybridization

Generally, in performing the methods of the present invention, the target polynucleotide will be hybridized to at least one allele-specific oligonucleotide. Such hybridization may be by any suitable means known in the art. Examples of such methods are described in standard manuals such as Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor); and Current Protocols in Molecular Biology (Eds. Ausubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc, Wiley-Interscience, NY, N.Y., 1992) or that are otherwise known in the art. Examples of preferred hybridization methods include Southern, northern, and dot blot hybridizations, reverse dot blot hybridizations and hybridizations to allele-specific oligonucleotides that are immobilized in the form of a "DNA chip" and related formats. A particularly preferred hybridization method is a reverse dot blot. Sakai et al, Nucl Acids. Res. 86:6230-6234 (1989). The stringency of hybridization is highly dependent upon a variety of factors, including length of the allele-specific oligonucleotide, sequence composition, degree of complementarity (i.e. presence or absence of base mismatches), concentration of salts and other factors such as formamide, and temperature. These factors are important both during the hybridization itself and during subsequent washes performed to remove target polynucleotide that is not specifically hybridized. In practice, the conditions of the final, most stringent wash are most critical. In addition, the amount or percent of the target polynucleotide that is able to hybridize to the allele-specific oligonucleotide is also governed by such factors as the concentration of both the allele-specific oligonucleotide and the target polynucleotide, the presence and concentration of factors that act to "tie up" water molecules, so as to effectively concentrate the reagents (e.g., PEG, dextran, dextran sulfate, etc.), and the duration of hybridization and washing steps.

Consequently, the relative degree to which a target polynucleotide is able to specifically hybridize to two different allele-specific oligonucleotides is governed by a large number of factors. It is possible, therefore, to alter certain parameters, such as the sequence composition, length of oligonucleotide, or concentration of a particular allele-specific oligonucleotide so as to adjust the amount of the target polynucleotide that hybridizes and remains hybridized to a given allele-specific oligonucleotide. This then, affects the intensity of the resultant detectable signal.

In certain embodiments of the present invention, it is desirable therefore to adjust level of the resultant detectable signal, so as to enable each allele (mutant and wild-type) of a particular target polynucleotide to both hybridize specifically to its respective allele-specific oligonucleotide, and produce detectable signal of sufficient intensity so as to be readily detectable, yet be substantially unable to hybridize to other allele-specific oligonucleotides. In practice, there must be greater than a twofold difference between the level of the signal produced by the target hybridizing to its correct allele-specific oligonucleotide and the level of the signal produced by the target polynucleotide cross-hybridizing to the incorrect allele-specific oligonucleotide (e.g., a wild-type allele cross-hybridizing to an oligonucleotide specific for a mutant allele). In preferred embodiments of the present invention, there will be at least a five-fold difference in the level of the resultant signal. In highly preferred embodiments of the present invention, there will be an order of magnitude difference in the level of the resultant signal. A most highly preferred difference will be at least two orders of magnitude difference in the level of the resultant signal.

Preferably, where groups of allele-specific oligonucleotides are used in conjunction with one another, concentration and sequence composition of the allele- specific oligonucleotides are chosen so that the signal resulting from a hybridization with a target polynucleotide that is homozygous for wild-type alleles, homozygous for mutant alleles, and heterozygous for the alleles (i.e., containing one allele each of the wild-type and the mutant form) are readily distinguishable. Preferably, such differences will be readily distinguishable by eye. In a highly preferred embodiment, concentration and sequence composition of the allele-specific oligonucleotides are selected so that a target polynucleotide that is heterozygous for an allele will hybridize in roughly comparable amounts to allele-specific oligonucleotides that are specific for each allele, and will yield a detectable signal that is roughly comparable between the two forms of the allele. In preferred embodiments, there will be less that an order of magnitude difference in the signal produced under such circumstances. In highly preferred embodiments, there will be less than a 5-fold difference in the signal produced under such circumstances. In a more highly preferred embodiment, there will be less than a 2-fold difference in the signal produced under such circumstances.

Further, in those embodiments where greater than one mutation is assayed in a single hybridization, it is preferable for the level of the signal produced by each set of allele-specific oligonucleotides (mutant and wild-type) to be roughly comparable between allele-specific oligonucleotides specific for each mutation, when challenged with a target polynucleotide heterozygous for each mutation assayed.

E. Reverse Dot Blot (RDB)

The reverse dot blot or RDB assay is designed to detect known mutations, in this case, the 185delAG and 5382insC mutation in BRCAl as well as the 6174delT mutation in BRGA2. RDB is a mutation detection system that is sufficiently sensitive to detect mutations which vary from the normal DNA sequence by only a single nucleotide. Wall et al, Hum. Mut. 5:333-338 (1995); Sakai et al, Nucl. Acids. Res. 86:6230-6234 (1989). Amino-linked oligonucleotide probes, complementary to the mutations of interest, are covalently attached to a derivitized nylon membrane. Probes complementary to the wild type or non-mutated alleles are also attached to the membrane. Patient samples are amplified, using 5 '-biotinylated primers, via the polymerase chain reaction (PCR). The patient samples are denatured and hybridized to the membrane-bound allele-specific oligonucleotides. After a stringent wash, substantially only those sequences complementary to the attached probe are retained on the membrane. A solution of streptavidin is then added to the membrane. The streptavidin binds to the biotinylated portion of the amplified patient samples, at one of four binding sites. Biotinylated alkaline phosphatase is then added, which binds to one or more of the other three available sites on the streptavidin molecule. A chemical substrate, which produces light (chemiluminesence) in the presence of alkaline phosphatase is added to the membrane. The resulting chemiluminesence is then detected using standard autoradiography film. The nature of the present invention will be more readily understood by reference to the following illustrative examples:

EXAMPLE 1

Fixation of AHele-Specific Oligonucleotide Probes a Nylon Membrane

Oligonucleotides to be fixed to the nylon membrane are synthesized commercially using standard phosphoramide chemistry, containing a 5'-amino terminal group added post synthesis. Oligonucleotides are used directly. No downstream purification is necessary. Using the concentration provided by the manufacturer, each probe is diluted to a stock concentration of 100 μM. A portion of this stock is then diluted to 12 μM and aliquoted to individual tubes to serve as a working stock. These aliquots in turn are diluted to the final working concentrations.

Allele-specific oligonucleotides are fixed to positively charged nylon membranes (Biodyne C; Pall Inc.). To minimize the total volume of reagents necessary to cover each piece of filter membrane, preparation of the nylon membranes is performed in small polypropylene boxes with individual compartments. A 12.5 cm x 8.5 cm piece of membrane is cut, which is then subdivided (by pencil lines) into 48, 1 cm x 3 cm rectangles. The resultant grid is then cut into 6 strips of 8 rectangles each. Each membrane strip is then chemically derivitized as follows:

Membranes are first wet in 10 to 15 mL of deionized H₂O. The water is decanted and the membranes are then activated by incubating them in 10 mL of 0J M HC1 for 10 minutes at room temperature with gentle shaking. The HC1 is decanted and the membrane strips are washed twice in deionized H₂O. Each wash is five minutes at room temperature, with shaking. The membrane strips are then derivitized by incubating them in a solution of 7.5%> ethylendiamineacytylcarbomide (EDAC; Sigma Chemicals) for 15 minutes at room temperature with gentle shaking. The EDAC solution is then decanted. The strips are then washed three times at room temperature, 10 minutes each wash, with gentle shaking. The strips are then blotted between two pieces of Whatman 3MM paper. Using a template as a guide, 1 μL of the appropriate oligonucleotide solution is applied to each rectangle using a repeating pipetter. Each 1 cm x 3 cm rectangle is spotted with 2 rows and 3 columns. The oligonucleotides are allowed to react with the treated membrane for 15 minutes. After 15 minutes, the membrane is quenched by incubation in 0J N NaOH for 15 minutes at room temperature, with gentle shaking. The membrane strips are then washed three times with deionized H₂O, 10 minutes each wash. Following the last wash, the membranes are allowed to dry. Membranes are then dated and stored desicated at - 20°C.

EXAMPLE 2

Amplification of Target Polynucleotide from Peripheral Blood

White blood cells are collected from the patient and genomic DNA is extracted according to methods known in the art (Sambrook, et al, Molecular Cloning, A Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Laboratory Press, at 9J6- 9J9).

Exons 2 and 20 from BRCAl and exon 11Q from BRCA2 are amplified by PCR from patient total genomic DNA isolated from peripheral blood. The following 5 '-biotinylated forward and reverse primers are used: BRCA1-2F: 5'-GAA GTT GTC ATTTTATAA ACC TTT-3' (SEQ ID NO:1), and BRCA1-2R: 5'-TCT CTT TTC TTC CCT AGT ATGT-3' (SEQ ID NO: 2),

BRCA1-20F: 5'-ATATGA CGT GTC TGC TCC AC-3' (SEQ ID NO:3), and

BRCA1-20R: 5'-GGG AAT CCA AATTAC ACA GC-3' (SEQ ID NO:4)

BRCA2-QF: 5'-ACG AAA ATT ATG GCA GGT TGT-3 (SEQ ID NO:5) , and

BRCA2-QR: CTT GTC TTG CGT TTT GTA ATG-3' (SEQ ID NO: 6)

Each 50 μL PCR reaction contains the following components: 1 μL template (genomic DNA; 100 ng/μL), 5.0 μL 10 PCR buffer (Perkin-Elmer), 5.0 μL dNTP solution (2 mM each of dATP, dCTP, dGTP, and dTTP), 5.0 μL forward primer (10 mM), 5.0 μL reverse primer (10 mM), 0.5 μL Taq DNA polymerase (Perkin-Elmer). 25 mM MgCl₂ is added to each reaction according to requirements for each pair of PCR primers (3.2 mL for BRCAl -2F/BRCA1-2R, 2.4 mL for BRCAl -20F/BRCA1- 20R, etc.), and H₂O is added to 50 μL. All reagents for each exon, except the genomic DNA may be combined into a "master mix" and aliquoted into the reaction tubes as a pooled mixture.

For each exon amplified, the following control reactions are used:

(1) "Negative" DNA control (100 ng placental DNA; Oncor, Inc.)

(2) Three "no template" controls

PCR for all exons is performed using the following thermocycling conditions:

Temperature Time Number of Cvcles

1. 95°C 5 minutes 1 cycle

2. 95°C 30 seconds i

3. 55°C 30 seconds i

4. 72°C 1 minute (30 cycles of steps 2, 3, and 4)

5. 72°C 5 minutes 1 cycle

6. 4°C hold 1 cycle Following PCR amplification, the quality of the PCR products is examined prior to further analysis by electrophoresing an aliquot of each PCR reaction sample on an agarose gel. 5 μL of each PCR reaction is run on an agarose gel along side a DNA mass ladder (Low DNA Mass Ladder; Gibco BRL, Cat. No. 10068-013). Using the agarose gel as a reference, roughly equal amounts of each amplified exon are used in subsequent hybridization reactions.

EXAMPLE 3

Reverse Dot Blot Hybridization of Patient Samples

For each patient sample, all three amplified exons are combined into a clean

0.2 mL PCR tube, using a multichannel pipette. The tubes are then capped and the samples are then denatured in a PCR machine at 95°C for 5 minutes. Following denaturation, the samples are rapidly placed on ice.

A single, labeled, 1 cm x 3 cm filter strip, which contains the allele-specific oligonucleotides prepared by the procedure of example 2, is used for each patient sample. Each strip is prehybridized in an individual compartment, using 5 mL of 2x SSC + 0.1% SDS for 15 minutes at 45°C, with moderate shaking. Using the agarose gel of the amplified patient samples as a guide, 1 to 5 μL of each target polynucleotide is added to each compartment. The assay is relatively insensitive to the amount of input target polynucleotide. The target polynucleotide is then allowed to hybridize to the immobilized allele-specific oligonucleotides for 1 hour, at 45°C, with moderate shaking.

After 1 hour, the hybridization solution is aspirated and the membranes are washed with 10 mL of 2x SSC + 0.1% SDS for 5 minutes, at 45°C. The wash solution is then aspirated and the washing step is repeated. The membranes are then washed in a high stringency wash of 0.5x SSC + 0J%> SDS for 10 minutes. The high stringency wash solution is then aspirated and the final wash is repeated twice. EXAMPLE 4

Chemiluminscent Detection of Hybridized Target Polynucleotides

Detection of hybridized target polynucleotides is accomplished using a commercially available chemiluminescent detection kit (Phototype; NEB). All incubations and washes are performed at room temperature. Membranes are blocked for 15 minutes to 2 hours in the RDB blocking solution. Membranes are pooled into a single container at this step. Following the incubation, the blocking solution is decanted and 5 mL of the streptavidin solution is added to the filters and incubated with vigorous shaking for 5 minutes. The solution is then aspirated off and the filters are washed for 5 minutes in RDB Wash Solution I. The wash is repeated twice, aspirating as previously. 5 mL of alkaline phosphatase solution is then added to the filters and incubated with vigorous shaking for 5 minutes. This solution is then aspirated off and the filters are washed for 5 minutes in RDB Wash Solution II. The wash is then repeated twice, aspirating as previously. 5 mL of CDP-Star solution is added to the filters and incubated with vigorous shaking for 5 minutes. The filters are removed and blotted on Whatmann 3MM paper. The filters are then placed on a sheet of plastic film and covered with a second sheet of plastic film. Filters are exposed in the dark to Kodak Xomat-aAJR. film for 15 to 30 minutes. Following this exposure, the film is developed.

EXAMPLE 5

Titration of AHele-Specific Oligonucleotides Immobilized to a Nylon Membrane

A titration of allele-specific oligonucleotides of the present invention is performed to determine the optimal concentration to be immobilized to the derivatized nylon membrane (Figs. 3, 4).

Figure 3 shows an autoradiograph of a series of filters, using DNA from plasmid clones as the target polynucleotide in an RDB assay. The top two rows in each filter represent signal from hybridization to a plasmid containing a wild-type allele, while the bottom two rows represent signal from hybridization to a mutant plasmid. Squares in which all four rows light up indicate the presence of a mixed colony population. Each allele-specific oligonucleotide is titrated to determine the optimal concentration (left to right on each blot), from 1.2 pmoles to 0.01 pmoles.

Figure 4 shows a comparison of allele-specific oligonucleotides and a determination of their optimal concentration. In the first two columns are tests of allele-specific oligonucleotides specific for 185delAG and 5382insC and their respective wild-type alleles. Designations II, 12, and 13 indicate three different versions of allele-specific oligonucleotides to detect the same mutation. The last column is a titration of two versions of the 6174delT allele-specific oligonucleotide. The left side is a titration of the first oligo designed. Concentration from 7.2 pmoles to 1.2 pmoles are shown. The right side shows an improved allele-specific oligonucleotide titrated from 1.2 pmoles to 0.01 pmoles. A significant increase in sensitivity was obtained by redesigning the probes.

EXAMPLE 6

Validation plasmids

Six plasmids are constructed, each containing DNA from a single allele, either mutant or a wild-type, corresponding to the locations of the 185delAG or the 5382insC mutations of a BRCAl gene or the 6174delT mutation of a BRCA2 gene (Table 1).

TABLE 1

Validation Plasmids and Host Strains

The DNA from these plasmids is then used as a source of target polynucleotide in a series of validation experiments (Fig. 5). Plasmids are mixed in combinations, designed to simulate target polynucleotides amplified from patient samples.

Figure 5 is an autoradiogram of a series of reverse dot blots. Eight membranes are shown, each of which has been probed with a different set of plasmids from Table 1. Each membrane contains the same set of immobilized allele-specific oligonucleotides.

Target polynucleotide is amplified from each set of plasmids as in Example 2, hybridizations are performed as in Example 3, and chemiluminescent detection is performed as in Example 4. The membrane in the upper left, as well as the four membranes on the right have been hybridized with target polynucleotide derived from a set of plasmids designed to simulate various homozygous patient samples. All wild- type (upper left); all mutant (upper right); wild type/185delAG mutant (right side, second down); wild-type/5381 mutant (right side, third down); and wild-type/6174 mutant (lower right). The remaining three membranes are probed with a set of plasmids designed to simulate various heterozygous patient samples. Each wild-type plus 185delAG mutant (left side, second down); each wild-type plus 5382insC mutant (left side, third down); and each wild-type plus 6174delT mutant (lower left).

These plasmids and validation controls demonstrate the specificity and sensitivity of the allele-specific ohgonucleotides of the present invention.

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.

Claims

WHAT IS CLAIMED IS:

1. An allele-specific oligonucleotide for determining the identity of a nucleotide at a position in a BRCAl encoding target polynucleotide, wherein said allele- specific oligonucleotide specifically hybridizes to a region of said target polynucleotide if said region contains said nucleotide at said position, and does not specifically hybridize to said region if said target polynucleotide does not contain said nucleotide at said position, and thereby permits the determination of the identity of said nucleotide of said target polynucleotide at or about nucleotides corresponding to nucleotides 185 and 5382 of a BRCAl gene.

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

3. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO:7.

4. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO:8.

5. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO:9.

6. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO: 10.

7. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO: 11.

8. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO : 12.

9. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NOJ3.

10 The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO: 14.

11. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO: 15.

12. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO: 16.

13. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO: 17.

14. The allele-specific oligonucleotide of claim 1 having a nucleotide sequence comprising SEQ ID NO: 18.

15. An allele-specific oligonucleotide for determimng the identity of a nucleotide at a position in a BRCA2 encoding target polynucleotide, wherein said allele- specific oligonucleotide specifically hybridizes to a region of said target polynucleotide if said region contains said nucleotide at said position, and does not specifically hybridize to said region if said target polynucleotide does not contain said nucleotide at said position, and thereby permits the determination of the identity of said nucleotide of said target polynucleotide at nucleotides corresponding to nucleotide 6174 of a BRCA2 gene.

16. The allele-specific oligonucleotide of claim 15, wherein said allele-specific oligonucleotide is an isolated allele-specific oligonucleotide.

17. The allele-specific oligonucleotide of claim 15 having a nucleotide sequence comprising SEQ ID NO: 19.

18. The allele-specific oligonucleotide of claim 15 having a nucleotide sequence comprising SEQ ID NO:20.

19. The allele-specific oligonucleotide of claim 15 having a nucleotide sequence comprising SEQ ID NO:21.

20. The allele-specific oligonucleotide of claim 15 having a nucleotide sequence comprising SEQ ID NO:22.

21. A method for determining the identity of a nucleotide at a polymorphic site of a BRCAl gene or a BRCA2 gene, comprising:

a) incubating a 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 determination of the identity of a nucleotide in said target polynucleotide at a position selected from the group of nucleotide positions comprising positions corresponding to nucleotides 185 of a BRCAl gene, nucleotide 5382 of a BRCAl gene and nucleotide 6174 of a BRCA2 gene;

b) allowing said hybridization to occur; and

c) detecting said hybridization between said target polynucleotide and said allele-specific oligonucleotide.

22. The method of claim 21, wherein said target polynucleotide is amplified from a biological sample.

23. The method of claim 22, wherein said amplification is PCR.

24. The method of claim 23, wherein said target polynucleotide is labeled with a label selected from the group consisting of: radioisotope, protein, antibody, fluorophore, chromophore, enzyme, oligonucleotide, oligopeptide and hapten.

25. The method of claim 21 , wherein said target polynucleotide is labeled with a label selected from the group consisting of: radioisotope, protein, antibody, fluorophore, chromophore, enzyme, oligonucleotide, oligopeptide and hapten.

26. The method of claim 21, wherein said allele-specific oligonucleotide is attached to a solid support by a covalent attachment means.

7. The method of claim 26, wherein said solid support is a positively-charged nylon membrane solid support, said allele-specific oligonucleotide is an amino-linked oligonucleotide and said attachment means comprises:

a) activating said solid support;

b) incubating said solid support with ethylendiamineacetylcarbomide under conditions sufficient to allow said solid support to become derivatized with said ethylendiamineacetylcarbomide; and

c) reacting said allele-specific oligonucleotide with said derivatized solid support such that said amino group of said allele-specific oligonucleotide reacts with said ethylendiamineacetylcarbomide derivatized to said solid support, thereby forming a covalent linkage.

28. The method of claim 21, comprising allele-specific oligonucleotides specific for a 185delAG mutation and a 185WT allele.

29. The method of claim 21, comprising allele-specific oligonucleotides specific for a 5382insC mutation and a 5382WT allele.

30. The method of claim 21, comprising allele-specific oligonucleotides specific for a 6174delT mutation and a 6174WT allele.

31. The method of claim 21 , comprising allele-specific oligonucleotides specific for a 185delAG mutation, a 185WT allele, a 5382insC mutation, a 5382WT allele, a 6174delT mutation, and a 6174WT allele.

32. The method of claim 21, wherein said allele-specific oligonucleotide is attached to a solid support by a non-covalent attachment means.

33. The method of claim 27, comprising allele-specific oligonucleotides specific for a 185delAG mutation and a 185WT allele.

34. The method of claim 27, comprising allele-specific oligonucleotides specific for a 5382insC mutation and a 5382WT allele.

35. The method of claim 27, comprising allele-specific oligonucleotides specific for a 6174delT mutation and a 6174WT allele.

36. The method of claim 27, comprising allele-specific oligonucleotides specific for a 185delAG mutation, a 185WT allele, a 5382insC mutation, a 5382WT allele, a 6174delT mutation, and a 6174WT allele.