throbber
,,',60$1,37€
`
`E)(AMINER,4
`n?, l:{, ',1' o .
`*-r+**rez
`
`lfpr-rCffiof paf osq sqs
`HEDr +fizl+
`
`UTlLIW
`SERIAL
`NUMBER
`SERIAL NUMBER
`i::l t:j j':iit::td ., t.J ::.{di.
`
`P ..lfr:F.lhl.|. F EFi
`?,'i l',i-l- :::.rf,,if:'f -t-
`I Ur,,r I ;:,[:::lil].
`f ltti,=l|l]ii,:::;,i .i. r-.:
`
`FILING DATE I CTJSS
`I .'L i' ti.;
`';1'7 | ,4:::r.L,
`'r
`
`*: :i: l.-:iJltj'l- .i i.1ll.l I I'Jfi fti.:1f ft:t; :t: :{: :+: :+: :ti r+: :t: :t: :+; :r: :t: :+r ii: :t::+: tg; :*: :*: :{: :i:
`?BOUiEIONffL
`
`l.i' r1r Fr 'i:i.i
`A
`
`I8
`
`;.t: :.i: !::'t'-t [:i i::: I i:ii 1.,.1
`Uj:::.!:i I ;::: 1' [i':r
`
`r:.:if:rFil.. i i-:f:il- j. t::Jl\.iiji;:+: i+::+::+::i::t::t:,+::.t::+::{.::i.,
`
`1.... l.'-iH:iil:*i:r: t.:;irif'. | |"-'" l. t.J ::ii.r. Li::ii ri,:;r:::::
`
`:{.::+:;+::i:4, }.;::iF.lt:.\i...i....
`
`i::::hi.i ;."i...i :ii:i::,i.::+::ir
`
`DOCKETNO.
`
`rr..l .jt :i *i.:;:: i:t :;;::i::t:;:i,:.i
`
`ne ne-^
`(-Ak
`cPA,,
`
`irji.ti.:::i:::'Fi li.r Fil:..i-;-*
`
`oc
`
`t)ultro
`?
`
`l:i,j-i hl i:. fi:. l;: iiii t.. li:j t : F:' Fi l- T F-.,
`
`tl.il...r1'f.lt- l. r*rtrtli t..rr nftr.:iu
`
`{F
`
`PAI &TM-pIO436L
`
`Fom PIO.{36A
`(Rev.
`
`-\\ib
`s,s
`
`et'{\"fwut
`
`fthi
`0gtttt
`:. .. .
`.:ri,,
`..
`.::.
`.. .,,
`:..:,:.:
`-,- ;nij*-;, .,;..i* oqi :i ": i.l.lr *'-,:rr; *ti;i.
`
`GeneDX 1004, pg. 1
`
`

`

`TION
`
`| iltilt ililt ilil| ilil ililt tffi il||I iltil til iltl
`
`/t'a/ y-
`
`o
`
`ltl
`
`r(zzlfr
`
`15.
`
`16.
`
`lV.
`
`'18,
`
`19.
`,,
`20,
`
`27;
`
`?2.
`
`23,.
`
`i4.,,
`
`30;
`
`31.
`
`32.
`
`i+:*-*
`
`:".
`
`'
`
`a':
`
`'.'
`
`GeneDX 1004, pg. 2
`
`

`

` ORIGINAL CLASSIFICATION
`I
`_SUBCLASS
`
`llllllllfifififififififillllllll
`cmsIIIIIIIIImaaIIIIIIII
`
`
`
`34.3! , 34.99-—
`f Le‘SCaHaIUV m2.
`
`
`
`
`APPLICANT'S NAME (PLEASE PRINT)
`
`
`Issue CLASSIFICATION SLIP
`
`US DEPARTMENT OF COMMERCE
`PATENT AND TRADEMARK OFFICE
`
`.
`
`II
`
`,
`
`
`
`
`
`
`
`s IIIIIII-
`
`_
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`£39452: :1}
`f;
`g E“ 24.24%
`M n.»—'
`é‘finfi'fir
`III‘jqflIIE
`Iz‘IIIIIIII
`mull-III
`A-:::=:==--
`IIII-IIIIIIIIIII
`fiflllllII—I-
`IIIIIII
`m-7 IIIIIII v
`mIIIIIII
`7 a IIIIIIII=-
`I”:- -=
`[annulu—
`E'II‘II-"IEE-‘E-
`IIIIIIIIII===.
`II I
`'
`‘
`II
`III
`II
`r-IIIlIl-==-
`IIIIIIIIIIII
`Eil- lIlII-----
`IMIIMIII = II
`aaI IIIII----
`IIIIIIIIIIIIII
`Emballllll- ---
`Ia -16IIIIIIII
`many-III.-
`IIII.IIIIIIIII
`InIIIIIIIII-I
`Is IIIIIIII
`MEIIlm----
`IIIIIIIIIIIII
`a - Ill-IE.-
`IIIIIIIIIIfi
`.EIIIIIIII
`-
`Il21IIIIIIII
`ImIIIIIIII
`M-I'IIII=.---
`EHEEEEEEE
`ll'l-Il
`I—
`--
`-I24 I III
`Ell-EH.-
`iigan‘I-I'IE—n-E
`.IgIgeug-gggg...
`ELI-IIIIIII-
`m- ------
`IE‘iIIlI-——-
`I--------
`EIIIIIIII-
`, z; I
`---
`IEélIIII-II-I
`..80 -====---
`EEE‘IIEEEEII- “ """""""2353:?
`' IIIIIIIII-
`
`1
`1
`f i'T'I'I'EougII numbeIIIINCanceIed ----------
`IIIIIIIIIIII- I
`
`.
`.
`-
`as IIIIIII-
`IE-IIIIIIIIII- I
`Iggggggg
`--------
`
`
`IE-IIIIIIIIII-
`932124;;d I-
`---------------------------------
`IEIIIIIIIIIII o
`.
`I a --------
`IEE-II-I-I-I
`-II:-a-------- .
`I'm-l”..-
`- 3; IIIIIII-
`IIIIIIIIIII-
`Ifi--------
`Mi-I-----
`IEIIIIIII-
`mflMIIIII-II—
`I
`‘ “
`91 IIIIIII-
`IIIIIIIIIII-
`II
`'
`92 IIIIIII-
`IIIIIIIIIIIIII-
`I5 -------
`IIIIIIIIIIIII-
`I.
`' I _
`94 IIIIII-
`" m." ---
`--------
`13“ -"'---=E
`.m-I- III=
`993”” "I...
`----I III-
`.I IIIIIIII-
`EBB-III...-
`“’494IIII-I-I—
`-_
`1-9-1---=4-----
`=-=§¥H§E==
`51:1 SOIIIIIIII-
`
`
`
`. We:
`

`
`
`
`z 3
`
`
`
`
`
`1
`
`
`
`
`
`GeneDX 1004, pg. 3
`
`

`

`
`
`
`
`GeneDX 1004, pg. 4
`
`

`

`
`
`PATENT APPLICATION SERIAL NO.
`
`US. DEPARTMENT OF COMMERCE
`
`PATENT AND TRADEMARK OFFICE
`
`FEE RECORD SHEET
`
`
`x
`3f
`354%: a
`
`03 $01202
`03 0402203»
`
`IWHHG 00000009 00984034
`assooos
`205.00 OP
`605.00 0P
`
`as ‘qu m: 0210311090 «Mme ooosmao,
`M's-E0337? mm. "1090:00984034
`o
`scar: m
`. 0:04.00 m
`
`PTO-1556
`
`(5/87)
`
`GeneDX1004, pg. 5
`
`GeneDX 1004, pg. 5
`
`

`

`
`
`.
`BAKER 5. BOTTS, L.L.F?
`:8 '
`—— m
`
`.\
`
`‘
`
`\
`
`.11....1,"Lb/CU/6|,
`
`g g
`E
`.1.— .C
`E— E”
`
`BAKER & BOTTE’ '
`30 ROCKEFELLERPLAJ
`NEWYORK,NEWYORKIOIla-Ozza
`v
`
`p.
`
`Zia-7055000
`FACSIMILE2I2> 705.5020
`
`/'
`
`, [fix g“"7 f7
`'Appln. Trans.
`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
`
`+ $135 =
`
`$
`
`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
`
`3..
`
`Zfififi‘
`Vin“
`‘5‘.1!’
`m.m
`
`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

This document is available on Docket Alarm but you must sign up to view it.


Or .

Accessing this document will incur an additional charge of $.

After purchase, you can access this document again without charge.

Accept $ Charge
throbber

Still Working On It

This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.

Give it another minute or two to complete, and then try the refresh button.

throbber

A few More Minutes ... Still Working

It can take up to 5 minutes for us to download a document if the court servers are running slowly.

Thank you for your continued patience.

This document could not be displayed.

We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.

Your account does not support viewing this document.

You need a Paid Account to view this document. Click here to change your account type.

Your account does not support viewing this document.

Set your membership status to view this document.

With a Docket Alarm membership, you'll get a whole lot more, including:

  • Up-to-date information for this case.
  • Email alerts whenever there is an update.
  • Full text search for other cases.
  • Get email alerts whenever a new case matches your search.

Become a Member

One Moment Please

The filing “” is large (MB) and is being downloaded.

Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.

Your document is on its way!

If you do not receive the document in five minutes, contact support at support@docketalarm.com.

Sealed Document

We are unable to display this document, it may be under a court ordered seal.

If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.


Access Government Site

We are redirecting you
to a mobile optimized page.





Document Unreadable or Corrupt

Refresh this Document
Go to the Docket

We are unable to display this document.

Refresh this Document
Go to the Docket