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`
`
`
`
`PATENT APPLICATION SERIAL NO.
`
`US. DEPARTMENT OF COMMERCE
`
`PATENT AND TRADEMARK OFFICE
`
`FEE RECORD SHEET
`
`
`x
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`GeneDX1004, pg. 5
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`GeneDX 1004, pg. 5
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`PATENT
`
`= E UTILITY PATENT
`'— APPLICATION
`' TRANSMITTAL
`
`.
`
`Attorney Docket No.
`
`A31420 - 2880/00002
`
`.
`
`First Named Inventor
`
`Jennifer L. Lescallet
`
`(Onlyfor new nonprovisional
`applications under 37 CFR I. 53(b))
`
`_
`Express Mail Label No.
`
`I
`EI476623229US
`
`Total Pages
`
`,
`
`98
`
`December 2, 1997
`
`
`
`
`BY EXPRESS MAIL
`
`Assistant Commissioner for Patents
`
`Box Patent Application .
`
`Washington, DC 20231
`
`Sir:
`
`Enclosed herewith for filing is a patent application of JENNIFER L. LESCALLET, ANTONETTE C..P
`ALLEN, TAMMY LAWRENCE, SHERI J. OLSON, DENISE B. THURBER, and MARGA B. WHITE
`entitled CANCER SUSCEPTIBILITY MUTATIONS OF BRCA2
`
`which includes:
`
`[X] Specification
`
`. _,_&3__ Total Pages
`
`[] Drawing(s)
`
`I _ Total Sheets
`
`[X] Combined Declaration and Power of'Attomey 4_ Total Pages
`
`.[X] Newly executed (original or copy)
`
`[] Copy from} a prior application
`(for continuation/divisional only)
`
`‘
`
`[] Divisional
`.
`[] Continuation
`of prior application No.
`/
`
`[] Continuation-In-Par’t (CIP)
`
`[X] An Assignment of the invention to
`
`[]
`
`is attached. A separate cover sheet in compliance with 37 CFR 3.28 and 3.31 is included.
`
`[X] will follow.
`
`[] Certified Copy of Priority Document(s) Country
`
`, No.
`
`,
`
`, filed
`
`[X] Small Entity Statement(s)
`'
`[]
`Small Entity Statement filed in prior application. Status still proper and desired.
`
`'124130.1
`12/97
`
`" '1'
`
`GeneDX1004, pg. 6
`
`GeneDX 1004, pg. 6
`
`
`
`
`
`BAKER & BOTTS, L.L.P.
`
`Appln. Trans.‘
`PATENT
`
`Attorney Docket No.
`
`' A31421
`
`'
`
`[]
`
`I Information Disclosure Statement (IDS) PTO-1449
`'[] Copies ofIDS Citations.
`
`,
`
`[X] Return Receipt Postcard
`
`[X] Other
`
`a diskette containing a computer readable copy of the Sequence ID Listing
`
`The filing fee has been calculated as shown below:
`
`Other Than A
`
`Small Entity
`Rate
`Fee
`
`X $22 =
`
`’
`
`x $82 =
`
`+$270=
`
`$790
`
`$
`
`$400
`
`$
`
`$
`
`(Col. 1)
`No.Filed
`
`(Col. 2)
`No. Extra
`
`Small Entity
`Rate
`Fee
`
`.
`
`OR
`
`FOR
`
`Basic Fee
`
`$395,
`
`$649
`
`$205
`
`Total Claims
`
`Ind. Claims
`
`79
`
`8
`
`-20=
`
`-3 =
`
`59
`
`5
`
`x $11:
`
`_x $41 ='
`
`
`
`
`Multiple Dependent Claim
`
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`
`$
`
`Total
`
`$1,249 *
`
`*
`
`If the difference in Col.
`
`1 is less than zero, enter "0" in Co]. 2.
`
`Fee Payment Being Made:
`
`[X ]
`
`Enclosed
`
`[X]
`
`Basic filing fee
`
`$1249
`
`‘Recording Assignment
`
`[$40.00; 37 CFR'1.21(h)]
`
`$
`
`Total fees enclosed
`
`$1,249.00
`
`1]
`
`[X1
`
`A check in the amountof $
`
`to coverefiling fee and assignment recordation fee is enclosed.
`
`The Commissioner is hereby authorized to charge payment of any additional filing fees required
`under 37 CFR 1.16, 1.17, and 1.21(h) associated with this communication'or credit any
`
`overpayment to Deposit Account No. 02-4377. Two copies of this sheet are enclosed.
`
`{/rfi'ixKER & B
`TS, L.L.P.
` i' r.
`
`PTO Registration No.
`
`Rochelle K. Seide
`
`: 32 300
`
`GeneDX1004, pg. 7
`
`Enclosures
`
`'
`
`1241301
`12/97
`
`GeneDX 1004, pg. 7
`
`
`
` m A
`
`BSTRACT OF THE DISQLOSURE
`
`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 BRCAZ gene sample.
`
`2:1
`fim".
`A";33’u..."x
`
`88
`
`GeneDX1004, pg. 8
`
`GeneDX 1004, pg. 8
`
`
`
`
`
`Attorney Docket No: PA—0107.
`
`CANCER SUSCEPTIBILITY MUTATIONS OF BRCA2
`
`This application is in part based on proVisional patent
`
`application 60/059,595 filed September 23, 1997,
`
`the contents-
`
`are incorporated by reference.
`
`FIELD'OF THE INVEETIOE
`
`
`
`
`
`This invention relates to the breast cancer
`
`succeptibility gene BRCA2. More specifically,
`
`this invention
`
`detects germline mutations of the BRCA2 gene that are
`
`associated with a predisposition to breast, ovarian and
`
`asociated cancers. Methods and reagents for detecting the
`
`presence of these mutations are included.
`
`BACKGROUND OF THE IEEENTIOE
`
`BRCA2,
`
`located on chromoéome 13q12-ql3, consists of over
`
`70kb 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 the entire
`
`coding-region.
`
`BRCA2 is thought to be a tumor suppressor gene
`
`asociated with breast and ovarian cancer.
`
`Thus mutantions
`
`which form an altered tumor suppressor or altered
`
`21
`
`tumor suppressor may be indicative of a
`concentrations Cf
`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
`
`26
`
`Information Core.
`
`GeneDX 1004, pg. 9
`
`GeneDX 1004, pg. 9
`
`
`
`
`
`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
`
`Hand 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 beenv
`
`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.p
`
`While mutations occur throughtout the BRCA2 gene,
`there
`is a need for a high sample number (throughput), sensitivity,
`
`26
`
`accuracy and cost effectiveneSs.
`
`Identification of mutations
`
`of the BRCA2 gene Would allow more widespread diagnostic
`
`screening for hereditary breast and ovarian cancer than is-
`
`currently possible and permit identification of functional
`
`'31
`
`areas deduced from the mutational spectrum observed.
`
`GeneDX 1004, pg. 10
`
`GeneDX 1004, pg. 10
`
`
`
`§UMMARY OF THE INZENTION
`
`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 numbers
`2192, 3772, 5193, 5374, 6495 and 6909 have been discovered.
`
`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.
`I
`
`‘
`
`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
`yr
`
`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
`
`development, gene therapy and other uses to prevent or
`
`amelorate the effects of or resulting from the mutant BRCA2
`
`gene.
`
`DEIAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
`
`For defining the preéent invention,
`
`the following
`
`nomenclature is used to describe the mutation due to an
`
`inconsistency 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
`
`GeneDX1004,pg.11
`
` 11
`
`21
`
`26
`
`A ‘
`
`K“’%fififil%§ini ON
`
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`
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`
`GeneDX 1004, pg. 11
`
`
`
`
`
`l
`
`(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 polymorphisms 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 BRCAl gene have been discovered‘previously by the
`
`inventors (U.S. Patent 5,654,155) and sequence variations in a
`normal BRCA2 gene sequence are expected. Also note Shattuck4
`
`Eidens) et al, J ur
`l of the
`gjgzip. 1242 (1997). Generally,
`
`'c n
`e i al Assoc'a
`the sense strand is referred
`
`to.
`
`For simplified identification purposes of this
`
`application, reference is to the BRCA2 sequence referenced
`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
`
`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 substituted
`for another,
`the nucleotide of the BRCA2 gene sequence is
`placed to the left of the number and the nucleotide found in
`
`the mutation is placed to the right of the number.
`
`26
`
`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
`
`31
`
`that since proline can form a turn in the chain of amino acids
`
`GeneDX 1004, pg. 12
`
`GeneDX 1004, pg. 12
`
`
`
`
`
`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
`
`negatively affect on the biological activity of the reSulting
`
`protein.
`
`‘
`
`The second mutation is referred to as 3772delTT. 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
`
`ll
`
`mutation.
`
`) l
`
`'33?H.331
`
`
`I1
`
`51'
`if
`
`flmflifithflfif
`if.1%1333???3:5$.73?=7
`
`The third mutation is referred to as C5193G. This
`
`mutation 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-functional,
`
`protein has been produced by this mutation.
`
`The fourth mutation is referred to as 5374del4.
`
`rThis
`
`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
`
`21
`
`likely non-functional, protein has been produced by this
`
`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
`
`26
`
`codon 2090.
`
`It has been demonstrated that a truncated, and
`
`most likely non—functional, protein has been produced by this
`
`mutationi
`
`The sixth mutation is referred to as 6909insG. This
`mutation inserts a G at nucleotide number 6909 causing a
`
`31
`
`frameshift mutation and forming an in—frame Stop codon at
`
`5
`
`GeneDX 1004, pg. 13
`
`GeneDX 1004, pg. 13
`
`
`
`
`
`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._
`I
`
`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 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, fluorescent 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
`
`
`
`a?!f”a?“
`
`
`‘mrzmI
`.2“...if}!:1
`
`
`5‘}
`
`21
`
`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
`
`26
`
`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
`
`GeneDX 1004, pg. 14
`
`GeneDX 1004, pg. 14
`
`
`
`
`
`
`
`iii
`
`imEZEEi
`
`“i?"mg,
`2:
`u«1 fix.«-
`
`yifififiim.
`
`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 purposes 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 S'TGG TAC TTT AAT TTT GTC ACT T3'
`
`SEQ ID NO:1
`
`BRCA2—11R S'TGC AGG CAT GAC AGA GAA T3'
`
`SEQ ID NO:2
`
`The designation BRCA2—ll refers to a sequence in or near
`
`exon 11 of the BRCA2 gene.
`
`F and R refer to forward and
`
`H OW
`
`reverse .
`
`The oligonucleotide primers are useful in directing
`
`amplification of a target polynucleotide prior to sequencing.
`
`21
`
`These unique BRCA2 exon.ll oligonucleotide primers were used
`to scan the BRCA2 gene to find the mutations.
`From the
`sequence information,
`the probes were designed and produced to
`assay for the mutation based upon identification of the CZl92G
`mutation.
`‘
`I
`
`In another embodiment of the invention a pair of isolated
`
`allele specific oligonucleotide probes are provided.
`
`26
`
`S‘TGA AGA ACC AAC TTT GT3'
`
`S'TGA AGA ACG AAC TTT GT35
`
`SEQ ID NO:3
`
`SEQ ID NO:4
`
`GeneDX 1004, pg. 15
`
`GeneDX 1004, pg. 15
`
`
`
`
`
`These allele specific oligonucleotides are useful in
`diagnosis 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'TGA AGA ACC AAC TTT GT3', SEQ ID NO:S, hybridizes
`
`preferentially to the wildtype sequence and is useful as a
`
`control sequence.
`
`5'TGA AGA ACG AAC TTT GT3', SEQ ID NO:4,
`
`is
`
`I
`
`designed to hybridize preferentially to the mutant sequence.
`In a second embodiment of the invention a pair of
`isolated oligonucleotide primers are provided.
`
`IBRCA2—llF 5'CTC AGA TGT TAT TTT CCA AGC3'
`
`SEQ ID NO:5
`
`BRCA2—11R 5'CTG TTA AAT AAC CAG AAG CAC3‘
`
`SEQ ID NO:6
`
`The oligonucleotide primers are useful in directing
`
`amplification of a target polynucleotide prior to sequencing.
`VThese unique BRCA2 exon lI oligonucleotide primers were, used
`
`to scan the BRCA2 gene to find the mutations.
`From the
`sequence information,
`the probes were designed and produced to
`
`assay for the mutation based upon identification of the 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 NO:7
`
`5'GCA AGC AAT GAA GGT AC3'
`
`SEQ ID NO:8
`
`These allele specific oligonucleotides are useful in
`
`diagnosis of a subject at risk of having breast or ovarian
`
`cancer.
`
`The allele specific oligonucleotides hybridize with a
`
`target polynucleotide sequence containing the 3772delTT
`
`8
`
`GeneDX 1004, pg. 16
`
`GeneDX 1004, pg. 16
`
`
`
`
`
`5'GCA AGC AAT TTG AAG GTS', SEQ ID NO:7, hybridizes
`mutation.
`preferentially to the wildtype sequence and is useful as a
`
`control sequence.
`
`5'GCA AGC AAT GAA GGT AC3', SEQ ID NO: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'GCA AAG ACC CTA AAG TAC AG3', SEQ ID NO:9
`
`BRCA2-11R
`
`5'CAT CAA ATA TTC CTT CTC TAA G3', SEQ ID NO:lO
`
`
`
`The oligonucleotide primers are useful in directing
`amplification 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 produced to
`
`assay for the mutation based upon identification of the C5193G
`
`mutation.
`
`In another embodiment of the invention a pair of isolated
`
`vallele specific oligonucleotides are provided.
`
`5'ACT TGT TAC ACA AAT CA3', SEQ ID NO:ll
`
`5'ACT TGT TAG ACA AAT CA3 '-,' SEQ ID NO:12
`
`These allele specific oligonucleotides are useful in
`diagnosis 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 ADA AAT CA3', SEQ ID NO:ll, hybridizes
`preferentially to the wildtype sequence and is useful as a
`control sequence.
`5'ACT TGT TAG ACA AAT CA3', SEQ ID NO:12,
`
`21
`
`26
`
`GeneDX 1004, pg. 17
`
`GeneDX 1004, pg. 17
`
`
`
`
`
`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 N0313
`
`BRCAZ—llR 5'ATC AGA ATG GTA GGA AT3'
`
`I
`
`SEQ ID NO:14
`
`The oligonucleotide primers are useful in directing
`
`Eamplification of a target polynucleotide prior to sequencing.
`
`These unique BRCA2 eXon ll oligonucleotide primers were used
`
`to-scan the BRCA2 gene to find the mutations.
`
`From the
`
`sequence information,
`
`the probes were designed and produced 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 AAA AI3'
`
`5'ATT ATT TGA AAA TAA TT3'
`
`SEQ ID NO:15
`
`SEQ ID NO:16
`
`These allele specific oligonucleotides are useful in
`
`diagnosis of a subject at risk of having breast or ovarian
`
`cancer.
`
`The allele specific oligonucleotides hybridize with a
`
`21
`
`target polynucleotide sequence containing the 5374del4
`
`mutation.
`
`5'ATT ATT TGT ATG AAA AT3', SEQ ID NO:15,
`
`hybridizes preferentially to the wildtype sequence and is
`
`useful as a control sequence.
`
`5'ATT ATT TGA AAA TAA TT3', SEQ
`
`ID NO:16,
`
`is designed to hybridize preferentially to the
`
`26
`
`mutant sequence.
`
`10
`
`GeneDX 1004, pg. 18
`
`GeneDX 1004, pg. 18
`
`
`
`
`
`In a fifth embodiment of the invention a pair of isolated
`
`oligonucleotide primers are proVided.
`
`BRCA2-11F S'TAC AGC AAG TGG AAA GC3'
`
`SEQ ID NO:17
`
`BRCA2-11R 5'AAG TTT CAG TTT TAC CAA T3' SEQ ID NO:18
`
`The oligonucleotide primers are useful in directing
`
`amplification 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 produced 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 NO:19
`
`5'GAA CTG AAT AGT CTT CA3P
`
`-
`
`SEQ ID NO:20
`
`These allele spedific oligonucleotides are useful in
`
`'diagnosis of a subject at risk of having breast or ovarian .
`
`cancer.
`
`The allele specific oligonucleotides hybridize with a'
`
`target polynucleotide sequence containing the 6495delGC
`mutation.
`5'GAA CTG AGC ATA GTC TT3', SEQ ID NO:19,
`
`
`
`
`21
`
`hybridiies preferentially to the wildtype sequence and is
`useful as a control sequence.
`5'GAA CTG AAT AGT CTT CA3', SEQ
`
`ID NO:20,
`
`is designed to hybridize preferentially to the
`
`mutant sequence.
`
`In a sixth embodiment of the invention a pair of isolated
`
`26
`
`oligonucleotide primers are provided.
`
`11
`
`GeneDX1004,pg.19
`
`GeneDX 1004, pg. 19
`
`
`
`
`
`BRCA2—11F 5'ACT TTT TCT GAT GTT CCT GTG3'
`
`’SEQ ID NO:21‘
`
`BRCA2—11R 5'TAA AAA TAG TGA TTG GCA ACA3'
`
`SEQ ID NO:22
`
`The oligonucleotide primers are useful in directing
`
`amplification 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 produced to
`
`assay for the mutation based upon identification of the
`6909insG mutation,
`I
`
`In another embodiment of the invention a pair of isolated
`
`allele specific oligonucleotides are provided.
`
`5'CAG AAG CAG TAG AAA TT3'
`
`SEQ ID NO:23
`
`5'CAG AAG CAG GTA GAA AT3'
`
`SEQ ID NO:24
`
`
`
`These allele specific oligonucleotides are useful in
`
`diagnosis of a_subject at risk of having breast or ovarian
`
`The allele specific oligonucleotides hybridize with a
`cancer.
`target polynucleotide sequence containing the 6909insG
`V
`
`mutation.q 5'CAG AAG CAG TAG AAA TT3', SEQ ID NO:23,
`
`hybridiZes preferentially to the wildtype sequence and is
`
`5'CAG AAG CAG GTA GAA AT3', SEQ
`useful as a control sequence.
`ID NO:24,
`is designed to hybridize preferentially to the
`
`21
`
`mutant sequence.
`The primers of the invention embrace oligonucleotides of
`sufficient length and appropriate sequence to-provide
`
`initiation of polymeriZation on a significant number of
`
`26'
`
`nucleic acids in the polymorphic locus.
`
`Preferred sequences for the present invention are SEQ ID
`
`NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID N026, SEQ ID NO:9, SEQ
`
`12
`
`GeneDX1004,pg.20
`
`GeneDX 1004, pg. 20
`
`
`
`
`
`l.
`
`ID NO:lO, SEQ ID NO:l3, SEQ ID NO:l4, SEQ ID NO:l7, SEQ ID
`
`NO:l8, SEQ ID NO:21, and SEQ ID NO:22. Environmental
`
`conditions cOnducive to synthesis of extension products
`
`include the presenCe of nucleoside triphoSphates, an agent for
`polymerization, such as DNA polymerase, and suitable
`
`I
`
`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
`
`The primer
`before being used to prepare extension products.
`must be sufficiently long to prime the synthesis of extension
`
`'——l H
`
`3593?if.’"lagHM$51:P3:25
`
`
`
`
`products in the presence of the inducing agent for
`
`polymerization.
`
`The exact length of primer will depend on_
`
`many factors,
`
`including temperature, buffer, and nucleotide
`
`composition.
`
`The oligonucleotide primer typically contains
`
`12-20 or more nucleotides, although it may contain fewer'
`
`nucleotides.
`
`Primers of the invention are designed to be
`“substantially” complementary to each strand of the genomic
`locus to be amplified. This means that the primers must be,
`
`sufficiently complementary to hybridize with their respective
`
`strands under conditions which allow the agent for-
`
`polymerization to perform.
`
`In other words,
`
`the primers should
`
`have suffiCient complementarity with the-5' and 3' sequences
`
`26
`
`flanking the mutation to hybridize therewith and permit
`amplification of the genomic locus.
`V
`Oligonucleotide primers of the invention are employed in
`
`the amplification process which is an enzymatic chain reaction-
`
`that produces exponential quantities of polymorphic locus
`relative to the number of reaction steps involved.’ Typically,.
`
`31
`
`one primer is complementary to the negative (—) strand of the
`
`13
`
`GeneDX1004,pg.21
`
`GeneDX 1004, pg. 21
`
`
`
`
`
`polymorphic locus and the other is complementary to the
`positive (+) strand. Annealing the primers to denatured
`
`nuc1eic acid followed by extension with an enzyme, such as the
`large fragment of DNA polymerase I
`(Klenow) and nucleotides,
`
`results in newly synthesized + and - strands containing the
`
`target polymorphic locus sequence. Because these newly
`
`synthesized Sequences are also templates, repeated cycles of
`
`denaturing, primer annealing, and extension results in
`
`exponential production of the region (i.e.,
`
`the target
`
`polymorphic locus sequence) defined by the-primers.
`
`The
`
`product of the chain reaction is a discreet nucleic acid
`
`duplex with termini corresponding to the ends of the specific
`
`primers employed.
`The oligOnucleotide primers of the invention may be
`
`prepared using any suitable method, such as conventional
`
`phosphotriester and phosphodieSter methods or automated
`embodiments thereof.
`In one such automated embodiment,
`
`diethylphosphoramidites are used as starting materials and may
`be synthesized as described by Beaucage, et al., Tetrahedron
`
`Letters, g;:1859—1862,
`(1981).
`One method for synthesizing
`oligonucleotides on a modified solid support is described in
`U.S. Patent No. 4,458,066.
`I
`
`Any nucleic acid specimen,
`
`in purified or nonpurified
`
`form, can be utilized as the starting nucleic acid or acids,
`
`providing it contains, or is suspected of containing,
`
`the
`
`
`
`26
`
`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
`
`31
`
`reverse transcribing the template to DNA would be utilized.
`
`14
`
`GeneDX1004,pg.22
`
`GeneDX 1004, pg. 22
`
`
`
`
`
`l
`
`In addition, a DNAsRNA hybrid which contains one strand of
`
`each may be utilized.
`
`A mixture of nucleic acids may also be
`
`employed, or the nucleic acids produced in a previous
`
`amplification reaction herein, using the same or different
`
`primers may be so utiliZed.
`
`The specific nucleic acid
`
`sequence to be amplified, i.e.,
`
`the polymorphic locus, may be
`
`a fraction of a larger molecule or can be present initially as
`
`a discrete molecule, so that the specific sequence constitutes
`
`the entire nucleic acid.
`
`It is not necessary that the
`
`sequence to be amplified be present initially in a pure form;
`
`it may be a minor fraction of a complex mixture, such as
`
`contained in whole human DNA.
`
`DNA utilized herein may be extracted from a body sample,
`such as blood,
`tissue material and the like by a variety of
`techniques such as that described by Maniatis, et. al.
`in
`
`' Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, NY,
`
`p 280—28;, 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
`
`
`
`
`
`26
`
`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
`
`31
`
`preferable for the primer hybridization.
`
`To the cooled
`
`15
`
`GeneDX 1004, pg. 23
`
`GeneDX 1004, pg. 23
`
`
`
`
`
`mixture is added an appropriate agent for effecting the primer
`extension reaction (called herein “agent for polymerization"l,
`
`and the reaction is allowed to occur under conditions known in
`
`the art.
`The agent for polymerization may also be added I
`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, Thermostable DNA polymerases,
`
`such as Taq polymerase may function at a higher temperature.
`The agent for polymerization may be any compound or
`I
`system which will function to accomplish
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