United States Patent [19J
`Lescallett et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006051379A
`[11] Patent Number:
`[45] Date of Patent:
`
`6,051,379
`*Apr. 18, 2000
`
`[54] CANCER SUSCEPTIBILITY MUTATIONS OF
`BRCA2
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`[75]
`
`Inventors: Jennifer Lee Lescallett, Great Falls,
`Va.; Tammy Lawrence, Laurel;
`Antonette Preisinger Allen, Severn,
`both of Md.; Sheri Jon Olson, Falls
`Church, Va.; Denise Bernadette
`Thurber, Silver Spring; Marga Belle
`White, Frederick, both of Md.
`
`4,683,195
`
`7/1987 Mullis eta!. ............................... 435/6
`
`OTHER PUBLICATIONS
`
`Panayiotiais P., et al. British Journal of Haematology
`97:844-847 (1997).
`
`[73] Assignee: Oncormed, Inc., Gaithersburg, Md.
`
`Tavitigian et al. Nature Genetics 12: 333-337 (1996).
`
`[ *] Notice:
`
`This patent issued on a continued pros(cid:173)
`ecution application filed under 37 CFR
`1.53( d), and is subject to the twenty year
`patent term provisions of 35 U.S.C.
`154(a)(2).
`
`Primary Examiner-Lisa B. Arthur
`Attorney, Agent, or Firm-Baker Botts
`
`[57]
`
`ABSTRACT
`
`[21] Appl. No.: 08/984,034
`
`[22] Filed:
`
`Dec. 2, 1997
`
`Related U.S. Application Data
`[60] Provisional application No. 60/059,595, Sep. 23, 1997.
`
`Int. Cl? .............................. C12Q l/68; C07H 21/04
`[51]
`[52] U.S. Cl. ......................... 435/6; 536/24.31; 536/24.33
`[58] Field of Search .............................. 536/24.33, 24.31,
`536/24.5; 435/6, 91.2, 320.1
`
`New mutations have been found in the BRCA2 gene. The
`mutations are located at nucleotide numbers 2192, 3772,
`5193, 5374, 6495 or 6909 of the published nucleotide
`sequence of BRCA2 gene. A process for identifying a
`sequence variation in a BRCA2 polynucleotide sequence is
`disclosed. The identification process includes allele specific
`sequence-based assays of known sequence variations. The
`methods can be used for efficient, and accurate detection of
`a mutation in a test BRCA2 gene sample.
`
`44 Claims, No Drawings
`
`GeneDX 1001, pg. 1
`
`

`

`1
`CANCER SUSCEPTIBILITY MUTATIONS OF
`BRCA2
`
`6,051,379
`
`This application is in part based on provisional patent
`application 60/059,595 filed Sep. 23, 1997, the contents are 5
`incorporated by reference.
`
`FIELD OF THE INVENTION
`
`This invention relates to the breast cancer succeptibility
`gene BRCA2. More specifically, this invention detects ger- 10
`mline mutations of the BRCA2 gene that are associated with
`a predisposition to breast, ovarian and associated cancers.
`Methods and reagents for detecting the presence of these
`mutations are included.
`
`BACKGROUND OF THE INVENTION
`
`2
`It is an object of the invention to provide a method for
`determining a predisposition or higher susceptibility to
`breast, ovarian and other cancers.
`It is another object of the invention to provide primers for
`detecting and amplifying a region of DNA which contains
`the BRAC2 mutations.
`It is another object of the invention to provide probes for
`detecting a region of DNA which contains the BRAC2
`mutations.
`It is a further object of the invention to provide a method
`of characterizing and classifying a tumor and determining a
`therapy dependant upon the type of mutation(s) present.
`It is also an object of the present invention to provide a
`mutant BRCA2 gene and expressed mutant protein for drug
`15 development, gene therapy and other uses to prevent or
`amelorate the effects of or resulting from the mutant BRCA2
`gene.
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`
`BRCA2, located on chromosome 13q12-q13, consists of
`over 70 kb of genomic DNA The coding sequence produces
`a protein of 3,418 amino acids. Although most of the exons
`are small, exons 10 and 11 represent approximately 60% of 20
`the entire coding region. BRCA2 is thought to be a tumor
`suppressor gene associated with breast and ovarian cancer.
`Thus mutantions which form an altered tumor suppressor or
`altered concentrations of tumor suppressor may be indica(cid:173)
`tive of a higher succeptibility to certain cancers.
`The nucleotide sequence for at least one BRCA2 gene is
`known and is reported in GENBANK accession Number
`U43746. The BRCA2 gene sequence is available on the
`Breast Cancer Information Core.
`Germline mutations of BRCA2 are predicted to account
`for approximately 35% of families with multiple case, early
`onset female breast cancer, and they are also associated with
`an increased risk of male breast cancer, ovarian cancer,
`prostrate cancer and pancreatic cancer.
`The location of one or more mutations of the BRCA2
`gene 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 such cases where one or
`only a few known mutations are responsible for the disease,
`such as testing family members, methods for detecting the
`mutations are targeted to the site within the gene at which
`they are known to occur.
`Many mutations and normal polymorphisms have already
`been reported in the BRCA2 gene. A world wide web site
`has been built to facilitate the detection and characterization
`of alterations in breast cancer susceptibility genes. Such
`mutations in BRCA2 can be accessed through the Breast
`Cancer Information Core at:
`HTTP://www.nchgr.nih.gov /dir/lab _transfer/bic.
`While mutations occur throughtout the BRCA2 gene,
`there is a need for a high sample number (throughput),
`sensitivity, accuracy and cost effectiveness. Identification of
`mutations of the BRCA2 gene would allow more wide(cid:173)
`spread diagnostic screening for hereditary breast and ovarian
`cancer than is currently possible and permit identification of
`functional areas deduced from the mutational spectrum
`observed.
`
`55
`
`25
`
`30
`
`For defining the present invention, the following nomen(cid:173)
`clature is used to describe the mutation due to an inconsis(cid:173)
`tency in the published literature. Beaudet et al, Human
`Mutations, 2: 245-248 (1993), Antonarakis et al, Human
`Mutations, 4: 166 (1994), Cotton, Human Mutations, 8:
`197-202 (1996), and Beutler et al, Human Mutations, 8:
`203-206 (1996). In defining the mutation, the number
`indicates the nucleotide number corresponding to the
`BRCA2 gene sequence where the mutation first occurs.
`Other BRCA2 sequences (haplotypes) which are polymor(cid:173)
`phisms or genetic variations of BRCA2 may used, in which
`a corresponding mutation at the corresponding nucleotide
`number are present. Different sequence variations in a
`normal BRCA1 gene have been discovered previously by
`the inventors (U.S. Pat. No. 5,654,155) and sequence varia-
`35 tions in a normal BRCA2 gene sequence are expected. Also
`note Shattuck-Eidens, et al, Journal of the American Medi(cid:173)
`cal Association, 278: p. 1242 (1997). Generally, the sense
`strand is referred to. For simplified identification purposes of
`this application, reference is to the BRCA2 sequence refer-
`40 enced above, however the invention is equally applicable to
`all of the normal BRCA2 sequences.
`Insertion mutations are indicated by "ins" and deletion
`mutations are indicated by "del". The letters after "ins" or
`"del" refer to the nucleotide(s) which were inserted or
`45 deleted. Insertions and deletions above two nucleotides are
`indicated by the number of nucleotides inserted or deleted.
`When the mutation results in one nucleotide being substi(cid:173)
`tuted for another, the nucleotide of the BRCA2 gene
`sequence is placed to the left of the number and the
`50 nucleotide found in the mutation is placed to the right of the
`number.
`The first mutation is referred to as C2192G. This mutation
`or genetic alteration causes a change in nucleotide number
`2192 from C to G resulting in codon 655 being changed
`from proline to arginine. Any amino acid change can have a
`dramatic change in biological activity. Some people believe
`that since proline can form a turn in the chain of amino acids
`in the protein, the removal of this turn, particularly when
`substituted with a charged amino acid may change the three
`dimentional configuration of the protein or at least may
`60 negatively affect on the biological activity of the resulting
`protein.
`The second mutation is referred to as 3772delTI. This
`mutation deletes TT at nucleotide number 3772 causing a
`frameshift mutation and forming an in-frame stop codon at
`codon 1182. It has been demonstrated that a truncated, and
`most likely non-functional, protein has been produced by
`this mutation.
`
`SUMMARY OF THE INVENTION
`The present invention is based on the discovery of six
`mutations in the BRCA2 gene sequence which is associated
`with susceptibility to and development of breast and ovarian
`cancer. Specifically, mutations located at nucleotide num- 65
`bers 2192, 3772, 5193, 5374, 6495 and 6909 have been
`discovered.
`
`GeneDX 1001, pg. 2
`
`

`

`6,051,379
`
`20
`
`25
`
`3
`The third mutation is referred to as C5193G. This muta(cid:173)
`tion substitutes G for C at nucleotide number 5193 causing
`a stop codon (TAG) to be formed at codon 1655. It has been
`demonstrated that a truncated, and most likely non(cid:173)
`functional, protein has been produced by this mutation.
`The fourth mutation is referred to as 5374del4. This
`mutation deletes TATG at nucleotide number 5374 causing
`a frameshift mutation and forming an in-frame stop at codon
`1723. It has been demonstrated that a truncated, and most
`likely non-functional, protein has been produced by this 10
`mutation.
`The fifth mutation is referred to as 6495delGC. This
`mutation deletes GC at nucleotide number 6495 causing a
`frameshift mutation and forming an in-frame stop codon at
`codon 2090. It has been demonstrated that a truncated, and 15
`most likely non-functional, protein has been produced by
`this mutation.
`The sixth mutation is referred to as 6909insG. This
`mutation inserts a G at nucleotide number 6909 causing a
`frameshift mutation and forming an in-frame stop codon at
`codon 2232. It has been demonstrated that a truncated, and
`most likely non-functional, protein has been produced by
`this mutation.
`The presence of truncated proteins was demonstrated by
`expression of overlapping portions of the mutant genes and
`measuring molecular weight by gel electrophoresis.
`Useful DNA molecules according to the present invention
`are those which will specifically hybridize to BRCA2
`sequences in the region of the C2192G, 3772delTT,
`C5193G, 5374del4, 6495delGC or 6909insG mutations.
`Typically these DNA molecules are 17 to 20 nucleotides in 30
`length (longer for large insertions) and have the nucleotide
`sequence corresponding to the region of the mutations at
`their respective nucleotide locations on the BRCA2 gene
`sequence. Such molecules can be labeled, according to any
`technique known in the art, such as with radiolabels, fino- 35
`rescent labels, enzymatic labels, sequence tags, biotin, other
`ligands, etc.
`According to another aspect of the invention, the DNA
`molecules, or oligonucleotides, contain one or more of the
`specific mutations. Generally it is preferred for each DNA 40
`probe to encompass only one mutation. Such molecules may
`be labeled and can be used as allele-specific oligonucleotide
`probes to detect the mutation of interest.
`Polynucleotide containing biological samples, such as
`blood, can be tested to determine whether the BRCA2 gene 45
`contains one of the specific mutations listed above. To
`amplify the BRCA2 gene, one may use polymerase chain
`reaction (PCR) using primers which hybridize to the ends of
`the exons or to the introns flanking the exons. In the situation
`of exon 11, the exon is so large that using plural pairs of
`primers to amplify overlapping regions is preferred. Such
`was actually used in the Examples below.
`Amplification may also be performed by a number of
`other techniques such as by cloning the gene and linking the
`BRCA2 gene or fragments thereof in the sample to a vector.
`"Shot gun" cloning is particularly preferred. For the pur- 55
`poses of this application, a vector may be any polynucleotide
`containing system which induces replication such as a
`plasmid, cosmid, virus, transposon, or portions thereof.
`In one embodiment of the invention a pair of isolated
`oligonucleotide primers are provided.
`BRCA2-11F 5'TGG TAC TTT AAT TTT GTC ACT T3'
`SEQ ID N0:1
`BRCA2-11R 5'TGC AGG CAT GAC AGA GAA T3' SEQ
`ID N0:2
`The designation BRCA2-11 refers to a sequence in or near 65
`exon 11 of the BRCA2 gene. F and R refer to forward and
`reverse.
`
`4
`The oligonucleotide primers are useful in directing ampli(cid:173)
`fication of a target polynucleotide prior to sequencing. These
`unique BRCA2 exon 11 oligonucleotide primers were used
`to scan the BRCA2 gene to find the mutations. From the
`5 sequence information, the probes were designed and pro(cid:173)
`duced to assay for the mutation based upon identification of
`the C2192G mutation.
`In another embodiment of the invention a pair of isolated
`allele specific oligonucleotide probes are provided.
`5'TGAAGAACC AAC TTT GT3' SEQ ID N0:3
`5'TGAAGAACG AAC TTT GT3' SEQ ID N0:4
`These allele specific oligonucleotides are useful in diag(cid:173)
`nosis of a subject at risk of having breast or ovarian cancer.
`The allele specific oligonucleotides hybridize with a target
`polynucleotide sequence containing the C2192G mutation.
`5'TGAAGAACC AAC TTT GT3', SEQ ID N0:3, hybrid(cid:173)
`izes preferentially to the wildtype sequence and is useful
`as a control sequence. 5'TGAAGAACG AAC TTT GT3',
`SEQ ID N0:4, is designed to hybridize preferentially to
`the mutant sequence.
`In a second embodiment of the invention a pair of isolated
`oligonucleotide primers are provided.
`BRCA2-11F 5'CTC AGA TGT TAT TTT CCAAGC3' SEQ
`ID N0:5
`BRCA2-11R 5'CTG TTA AAT AAC CAG AAG CAC3'
`SEQ ID N0:6
`The oligonucleotide primers are useful in directing ampli(cid:173)
`fication of a target polynucleotide prior to sequencing. These
`unique BRCA2 exon 11 oligonucleotide primers were used
`to scan the BRCA2 gene to find the mutations. From the
`sequence information, the probes were designed and pro(cid:173)
`duced to assay for the mutation based upon identification of
`the 3772delTT mutation.
`In another embodiment of the invention a pair of isolated
`allele specific oligonucleotides are provided.
`5'GCA AGC AAT TTG AAG GT3' SEQ ID N0:7
`5'GCA AGC AAT GAA GGT AC3' SEQ ID N0:8
`These allele specific oligonucleotides are useful in diag-
`nosis of a subject at risk of having breast or ovarian cancer.
`The allele specific oligonucleotides hybridize with a target
`polynucleotide sequence containing the 3772delTT muta(cid:173)
`tion. 5'GCA AGC AAT TTG AAG GT3', SEQ ID N0:7,
`hybridizes preferentially to the wildtype sequence and is
`useful as a control sequence. 5'GCAAGC AAT GAA GGT
`AC3', SEQ ID N0:8, is designed to hybridize preferentially
`to the mutant sequence.
`In a third embodiment of the invention a pair of isolated
`oligonucleotide primers are provided.
`BRCA2-11F 5'GCAAAG ACC CTAAAG TACAG3', SEQ
`ID N0:9
`BRCA2-11R 5'CAT CAAATA TTC CTT CTC TAA G3',
`SEQ ID N0:10
`The oligonucleotide primers are useful in directing ampli(cid:173)
`fication of a target polynucleotide prior to sequencing. These
`unique BRCA2 exon 11 oligonucleotide primers were used
`to scan the BRCA2 gene to find the mutations. From the
`sequence information, the probes were designed and pro(cid:173)
`duced to assay for the mutation based upon identification of
`the C5193G mutation.
`In another embodiment of the invention a pair of isolated
`allele specific oligonucleotides are provided.
`60 5'ACT TGT TAC ACAAAT CA3', SEQ 1D N0:11
`5'ACT TGT TAG ACAAAT CA3', SEQ ID N0:12
`These allele specific oligonucleotides are useful in diag-
`nosis of a subject at risk of having breast or ovarian cancer.
`The allele specific oligonucleotides hybridize with a target
`polynucleotide sequence containing the C5193G mutation.
`5'ACT TGT TAC ACAAAT CA3', SEQ ID N0:11, hybrid-
`izes preferentially to the wildtype sequence and is useful as
`
`50
`
`GeneDX 1001, pg. 3
`
`

`

`6,051,379
`
`5
`a control sequence. 5'ACT TGT TAG ACAAAT CA3', SEQ
`ID N0:12, is designed to hybridize preferentially to the
`mutant sequence.
`In a fourth embodiment of the invention a pair of isolated
`oligonucleotide primers are provided.
`BRCA2-11F 5'GAA AAT TCA GCC TTA GC3' SEQ ID
`N0:13
`BRCA2-11R 5'ATC AGA ATG GTA GGA AT3' SEQ ID
`N0:14
`The oligonucleotide primers are useful in directing ampli(cid:173)
`fication of a target polynucleotide prior to sequencing. These
`unique BRCA2 exon 11 oligonucleotide primers were used
`to scan the BRCA2 gene to find the mutations. From the
`sequence information, the probes were designed and pro(cid:173)
`duced to assay for the mutation based upon identification of
`the 5374del4 mutation.
`In another embodiment of the invention a pair of isolated
`allele specific oligonucleotides are provided.
`5'ATT ATT TGT ATG AAAAT3' SEQ ID N0:15
`5'ATT ATT TGAAAA TAA TT3' SEQ ID N0:16
`These allele specific oligonucleotides are useful in diag(cid:173)
`nosis of a subject at risk of having breast or ovarian cancer.
`The allele specific oligonucleotides hybridize with a target
`polynucleotide sequence containing the 5374del4 mutation.
`5'ATT ATT TGT ATG AAAAT3', SEQ ID N0:15, hybrid(cid:173)
`izes preferentially to the wildtype sequence and is useful as
`a control sequence. 5'ATT ATT TGAAAA TAA TT3', SEQ
`ID N0:16, is designed to hybridize preferentially to the
`mutant sequence.
`In a fifth embodiment of the invention a pair of isolated
`oligonucleotide primers are provided.
`BRCA2-11F 5'TAC AGC AAG TGG AAA GC3' SEQ ID
`N0:17
`BRCA2-11R 5'AAG TTT CAG TTT TAC CAA T3' SEQ ID
`N0:18
`The oligonucleotide primers are useful in directing ampli(cid:173)
`fication of a target polynucleotide prior to sequencing. These
`unique BRCA2 exon 11 oligonucleotide primers were used
`to scan the BRCA2 gene to find the mutations. From the
`sequence information, the probes were designed and pro(cid:173)
`duced to assay for the mutation based upon identification of
`the 6495delGC mutation.
`In another embodiment of the invention a pair of isolated
`allele specific oligonucleotides are provided.
`5'GAA CTG AGC ATA GTC TT3' SEQ ID N0:19
`5'GAA CTG AAT AGT CTT CA3' SEQ ID N0:20
`These allele specific oligonucleotides are useful in diag(cid:173)
`nosis of a subject at risk of having breast or ovarian cancer.
`The allele specific oligonucleotides hybridize with a target
`polynucleotide sequence containing the 6495delGC muta(cid:173)
`tion. 5'GAA CTG AGC ATA GTC TT3', SEQ ID N0:19,
`hybridizes preferentially to the wildtype sequence and is
`useful as a control sequence. 5'GAA CTG AAT AGT CTT
`CA3', SEQ ID N0:20, is designed to hybridize preferentially
`to the mutant sequence.
`In a sixth embodiment of the invention a pair of isolated
`oligonucleotide primers are provided.
`BRCA2-11F 5'ACT TIT TCT GAT GTT CCT GTG3' SEQ
`ID N0:21
`BRCA2-11R 5'TAAAAA TAG TGA TTG GCAACA3' SEQ
`ID N0:22
`The oligonucleotide primers are useful in directing ampli(cid:173)
`fication of a target polynucleotide prior to sequencing. These
`unique BRCA2 exon 11 oligonucleotide primers were used
`to scan the BRCA2 gene to find the mutations. From the
`sequence information, the probes were designed and pro(cid:173)
`duced to assay for the mutation based upon identification of
`the 6909insG mutation.
`In another embodiment of the invention a pair of isolated
`allele specific oligonucleotides are provided.
`
`5
`
`40
`
`15
`
`6
`5'CAG AAG CAG TAG AAA TT3' SEQ ID N0:23
`5'CAG AAG CAG GTA GAAAT3' SEQ ID N0:24
`These allele specific oligonucleotides are useful in diag(cid:173)
`nosis of a subject at risk of having breast or ovarian cancer.
`The allele specific oligonucleotides hybridize with a target
`polynucleotide sequence containing the 6909insG mutation.
`5'CAG AAG CAG TAG AAA TT3', SEQ ID N0:23, hybrid(cid:173)
`izes preferentially to the wildtype sequence and is useful as
`a control sequence. 5'CAG AAG CAG GTA GAA AT3',
`SEQ ID N0:24, is designed to hybridize preferentially to the
`10 mutant sequence.
`The primers of the invention embrace oligonucleotides of
`sufficient length and appropriate sequence to provide initia(cid:173)
`tion of polymerization on a significant number of nucleic
`acids in the polymorphic locus.
`Preferred sequences for the present invention are SEQ ID
`N0:1, SEQ ID N0:2, SEQ ID N0:5, SEQ ID N0:6, SEQ ID
`N0:9, SEQ ID N0:10, SEQ ID N0:13, SEQ ID N0:14,
`SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:21, and SEQ
`ID N0:22. Environmental conditions conducive to synthesis
`of extension products include the presence of nucleoside
`20 triphosphates, an agent for polymerization, such as DNA
`polymerase, and suitable conditions such as temperature,
`ionic strength 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
`25 extension products. The primer must be sufficiently long 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
`30 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
`35 complementary to hybridize with their respective strands
`under conditions which allow the agent for polymerization
`to perform. In other words, the primers should have suffi(cid:173)
`cient 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 polymor(cid:173)
`phic locus relative to the number of reaction steps involved.
`Typically, one primer is complementary to the negative (-)
`45 strand of the polymorphic locus and the other is comple(cid:173)
`mentary 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 +
`50 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)
`55 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
`prepared using any suitable method, such as conventional
`phosphotriester and phosphodiester methods or automated
`60 embodiments thereof. In one such automated embodiment,
`diethylphosphoramidites are used as starting materials and
`may be synthesized as described by Beaucage, et al., Tet(cid:173)
`rahedron Letters, 22:1859-1862, (1981). One method for
`synthesizing oligonucleotides on a modified solid support is
`65 described in U.S. Pat. No. 4,458,066.
`Any nucleic acid specimen, in purified or nonpurified
`form, can be utilized as the starting nucleic acid or acids,
`
`GeneDX 1001, pg. 4
`
`

`

`6,051,379
`
`20
`
`7
`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 10
`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, N.Y., 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 about 90°-100° C.
`from about 1 to 10 minutes, preferably from 1 to 4 minutes.
`This is sufficient to denature any double strands. After this
`heating period, the solution is allowed to cool at a rate which
`is 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. Ther(cid:173)
`mostable DNA polymerases, such as Taq polymerase may
`function at a higher 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 tempera(cid:173)
`tures sufficiently elevated to cause denaturation), such as
`Taq polymerase. The suitable enzyme will facilitate combi(cid:173)
`nation 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 syn(cid:173)
`thesis 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
`
`8
`step, the newly synthesized double-stranded molecule is
`subjected to denaturing conditions using any of the proce(cid:173)
`dures described above to provide single-stranded molecules.
`The steps of denaturing, annealing, and extension product
`5 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
`15 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 biolumines(cid:173)
`cent 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. In
`the preferred embodiment, the amplification products are
`25 determinable by separating the mixture on an agarose gel
`containing ethidium bromide which causes DNA to be
`fluorescent.
`Sequences amplified by the methods of the invention can
`be further evaluated, detected, cloned, sequenced, and the
`30 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., Rio/Technology, 3:1008-1012, 1985), allele-specific
`oligonucleotide (ASO) probe analysis (Conner, et. al., Proc.
`35 Natl. Acad. Sci. U.SA., 80:278, 1983), oligonucleotide liga(cid:173)
`tion 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
`40 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
`BRCA2 locus amplified by PCR using primers of the
`invention is similarly amplified by the alternative means.
`45 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 eDNA and
`degrades the RNA, followed by reverse transcriptase poly-
`50 merizing 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
`55 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(cid:173)
`stranded. Amplification is initiated by ligating a eDNA to the
`60 promoter olignucleotide and within a few hours, amplifica(cid:173)
`tion 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
`65 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
`
`GeneDX 1001, pg. 5
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`6,051,379
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`5
`
`9
`(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 complemen-
`tary and target-specific oligonucleotide probe pairs, thermo(cid:173)
`stable polymerase and ligase, and DNA nucleotides to
`geometrically amplify targeted sequences. A 2-base gap
`separates the oligonucleotide probe pairs, and the RCR fills