MYR 1034
`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
`
`Page 1 of 15
`
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`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
`
`Page 1 of 15
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`
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`photoluminescent dye at one ofthe 5’ or 3’ ends and a quenching agentat the opposite 5’
`
`or 3’Xend, wherein the loop comprises 16 base pairs and has a Tm of 50-51°C, and
`
`wherein\he stem comprises 4 base pairs having a sequence 5’-CACG-3’.
`
`
`
`
`66.
`
`The molecular beacon probe of claim 65, wherein the probe detects
`
`a wild-type nucleic\acid better than a mutant nucleic acid.
`
`67.
`
`(New)
`
`
`olecular beacon probe of claim 65, wherein the probe detects
`
`a mutant nucleic acid better than
`a wild-type nucleic acid.
`
`
`68.
`
`(New)
`
`A moleculaybeagon probe comprising:
`
`an oligonucleotide comprising a stem and a loop structure and having a
`
`photoluminescent dye at one ofithe 5’ or
`
`3>énds and a quenching agentat the opposite 5’
`
`
`
`or 3’ end, wherein the loop comprises 19-20 base pairs and has a Tm of 54-56°C, and
`
`wherein the stem comprises 4 base pairs having a séquence 5’-CACG-3’.
`
`
`A pair ofmolecular beacon probes
`
`69.
`
`(New)
`
`comprising:
`
`a first oligonucleotide comprisinga first stem anda firdt
`loop structure and having
`a photoluminescent dye at one ofthe 5’ or 3’ ends and a quenching agentat the opposite
`
`
`
`5’ or 3° end, wherein the first loop comprises 16 base pairs and has a ‘{m of 50-51°C, and
`
`wherein the first stem comprises 4 base pairs having a sequence 5’-CACG-3’; and
`
`a second oligonucleotide comprising a second stem and a second loop structure
`
`
`ge
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`Page 2 of 15
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`Page 2 of 15
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`loop comprises 19-20 base pairs and has a
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`NN
`5°-CACG-3’,
`‘tem comprises
`
`
`
`
`e of the 5’ or 3’ ends and a quenching agentat
`
`¢ pairs having a sequence
`
`IN THE SPECIFICATION
`
`Please replace the paragraph beginning on page 4, line 5, with the following
`
`paragraph,
`Fic. vA, 1B, ic! Schematic ofexperimental design. (A) The basic two steps involved:
`PCR on diluted DNA samples is followed by addition of fluorescent probes which
`
`discriminate between WT and mutantalleles and subsequentfluorometry. (B) Principle
`
`
`
`of molecular beacon analysis. In the stem-loop configuration, fluorescence from a dyeat
`
`the 5’ end of the oligonucleotide probe is quenched by a Dabcyl groupat the 3’ end.
`
`Upon hybridization to a template, the dye is separated from the quencher,resulting in
`
`a” increased fluorescence. ModifiedfromMarras etal. . (C) Oligonucleotide design.
`
`Primers F1 and R1 are used to amplify the genomic region of interest. Primer INTis
`
`used to produce single stranded DNA from the original PCR products during a
`
`subsequent asymmetric PCR step (see Materials and Methods). MB-REDis a Molecular
`
`Beacon which detects any appropriate PCR product, whether it is WT or mutantat the
`
`queried codons. MB-GREENis a Molecular Beacon which preferentially detects the WT
`
`Page3 of 15
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`PCRproduct.
`TT
`a]
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`
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`
`
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`Page 3 of 15
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`
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`}_Please-replace the paragraphbeginning onpage 14,line 29 with the following paragraph.
`
`Oligonucleotides and DNA sequencing. Primer F1:
`
`iis
`
`5’-CATGTTCTAATATAGTCACATITITCA-3’ (SEQ ID NO: 1); Primer R1:
`
`5’-TCTGAATTAGCTGTATCGTCAAGG-3’ (SEQ ID NO: 2); Primer INT:
`
`5’-TAGCTGTATCGTCAAGGCAC-3’ (SEQ ID NO: 3); MB-RED:
`
`5’-Cy3-CACGGGCCTGCTGAAAATGACTGCGTG-Dabcyl-3’ (SEQ ID NO: 4);
`
`MB-GREEN:5’-Fluorescein-CACGGGAGCTGGTGGCGTAGCGTG-Dabcyl-3’ (SEQ
`
`ID NO: 5). Molecular Beacons (33,34) were synthesized by Midland Scientific and other
`
`oligonucleotides were synthesized by Gene Link (Thornwood, NY). All were dissolved
`
`at 50 uM in TE (10 mM Tris, pH 8.0/ 1 mM EDTA)and keptfrozen and in the dark until
`
`3
`ik
`
`use. PCR products were purified using QlAquick PCRpurification kits (Qiagen). In the
`relevant experiments described in the text, 20% ofthe product from single wells was used
`
`for gel electrophoresis and 40% was used for each sequencing reaction. The primer used
`
`for sequencing was 5’-CATTATTTTTATTATAAGGCCTGC-3’ (SEQ ID NO: 6).
`
`Sequencing was performed using fluorescently-labeled ABI Big Dye terminators and an
`
`ABI 377 automated sequencer.
`
`
`SEQUENCELISTING
`
`Please enter the enclosed paper copy ofthe Sequence Listing after the claims. A
`
`computer readable copy ofthe Sequence Listing is also enclosed herewith to comply with
`
`37 § CFR 1.821(e). The content of the paper and computer readable copyofthe
`4
`
`oe
`
`
`
`ob
`
`,
`, (,i
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`Page 4 of 15
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`
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`Sequence Listing, submitted in accordance with 37 CFR § 1.821 (c) and (e), respectively,
`
`are identical. The submitted Sequence Listing,filed in accordance with 37 CFR § 1.821
`
`(g) herein does not include new matter.
`
`REMARKS
`
`The Invention
`
`The invention is directed to a method for determining the ratio of a selected
`
`genetic sequence in a population of genetic sequences. Nucleic acid template molecules
`
`in a biological sample are diluted to form a set comprisinga plurality of assay samples.
`
`The diluted nucleic acid template molecules are amplified to form a population of
`
`amplified molecules in the assay samples of the set. The amplified molecules are
`
`analyzed to determine a first number of assay samples which contain the selected genetic
`
`sequence and a second number of assay samples which contain a reference genetic
`
`sequence. Thefirst number and the second number are compared to ascertain a ratio that
`
`reflects the composition ofthe biological sample (claim 1).
`
`The invention is also drawn to a method for determining the ratio of a selected
`
`genetic sequence in a population of genetic sequences, Template molecules within a set
`
`which comprises a plurality of assay samples are amplified to form a population of
`
`amplified molecules in each of the assay samples of the set. The amplified molecules in
`
`the assay samplesofthe set are analyzed to determine a first number of assay samples
`
`which contain the selected genetic sequence and a second umber of assay samples which
`
`contain a reference genetic sequence. At least one-fiftieth of the assay samples in the set
`5
`
`
`
`« Page5of 15_
`ey
`
`Page 5 of 15
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`
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`comprise a number (N) of molecules such that 1/N is larger than the ratio of selected
`
`genetic sequences to total genetic sequences required to determine the presence ofthe
`
`selected genetic sequence. The first number is compared to the second number to
`
`ascertain a ratio which reflects the composition of the biological sample (claim 38).
`
`The invention is also drawn to molecular beacon probes. The molecular beacon
`
`probe comprises an oligonucleotide with a stem-loopstructure having a photoluminescent
`
`dye at one of the 5’ or 3’ ends and a quenching agentat the opposite 5’ or 3’ end, The
`
`loop consists of 16 base pairs and has a T,, of 50-51°C, The stem consists of 4 base pairs
`
`and has a sequence 5’-CACG-3’ (claim 33), The loop ofthe molecular beacon probe
`
`may alternatively consist of 19-20 base pairs and have a Tm of 54-56°C (claim 36),
`
`The invention also is drawn to a pair of molecular beacon probes comprising a
`
`first and second probe. This first probe comprises an oligonucleotide with a stem-loop
`
`structure having a photoluminescent dye at one of the 5’ and 3’ ends and a quenching
`
`agent at the opposite 5’ or 3’ end. The loop consists of 16 base pairs and has a Tn, of 50-
`
`51°C, The stem consists of 4 base pairs and has a sequence 5’°-CACG-3°. The second
`
`probe comprises an oligonucleotide that has a stem-loop structure having a
`
`photoluminescent dye at one of the 5’ and 3’ ends and a quenching agentat the opposite
`
`5’ or 3’ end. The loop consists of 19-20 base pairs and has a Ty of 54-56°C, The stem
`
`consists of4 base pairs and has a sequence 5’CACG-3’ (claim 37).
`
`Information Disclosure Statement
`
`The Office Action asserts that the listing ofreferences in the specification is not a
`
`6
`
`Page 6 of 15
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`
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`Page 6 of 15
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`
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`proper information disclosure statement (IDS). Thelisting of references in the
`
`specification is not intended as the IDS forthe application. Applicants have made two
`
`submissions on Form PTO-1449, in compliance with 37 CFR 1.98(b) on December 15,
`
`2000 and March 7, 2001. A copy of each IDS submitted is attached along with the
`
`postcard receipts, at Tabs A and B. Clearly the PTO received at least one sheet of PTO-
`
`1449, as this has been returned to applicant, albeit entirely crossed out. No explanation is
`
`provided for the failure to consider the references. A new set of references is included in
`
`case these were lost in PTO handling. Liet al. is not included with this response, but will
`
`be sent in a separate mailing. Applicants recognizethat the twolists ofreferences are
`
`almost identical but for the Brown patent which was only listed on the March 7, 2001
`
`submission, Applicants request an initialed copy of the PTO-1449indicating
`
`consideration of each reference.
`
`Objections to the Specification
`
`The Office Action has objected to the specification for reciting “Figure 1” in the
`
`Brief Description of the Drawings, while no Figure 1 exists in the drawings. The
`
`specification has been amended to properly recite Figure 1A, 1B, 1C in the Brief
`
`Description of the Drawings.
`The specification was further objected to for improper disclosure ofnucleotide
`
`sequences. The sequences referenced in the Office Action (at page 14,lines 30 and 31,
`
`as well as sequences not referenced in the Office Action at page 15,lines 1, 2, 4, and 13
`
`ofthe specification) were entered into a SequenceListing as they appear in the
`
`Page7 of15 ©
`Hy
`
`Page 7 of 15
`
`
`
`application, and thus contain no new matter. A paper and computer readable form ofthe
`
`SequenceListing are submitted with this amendment.
`
`The Rejection of Claims 1-64 under 35 U.S.C. § 112
`
`Claims 1-64 have been rejected under 35 U.S.C. § 112, second paragraph, as
`
`being indefinite for failing to particularly point out and distinctly claim the subject matter
`
`which the applicant regardsas his invention.
`
`A.
`
`The rejected claims are allegedly incomplete for omitting essential steps. The
`
`Office Action identifies the omitted steps as “serially diluting to form aset ofassay ~
`
`samples and testing by PCR.” (Page4, lines 3-4.) Applicants respectfully traverse.
`
`Claim 1 recites both a diluting step and an amplifying step at lines 3 and 5,
`
`respectively. Claim 38 recites an amplifying step at line 3. Thus the only step that could
`
`possibly be missing is diluting in claim 38. However, this step is neither essential nor
`
`required. Claim 38 requires a certain concentration of template which may, but need not,
`
`be achieved bydilution. If samples are initially sufficiently dilute, no dilution is
`
`required. Thus dilution is not a necessary step.
`
`The Office Action points to the specification at page 13, lines 17-19, to
`
`demonstrate that claims 1-32 and 38-64 omit the essential steps ofserially diluting and
`
`testing via PCR. Thecitation is to example 1. The examples, however, are provided “for
`
`purposesofillustration only, annd are not intended to limit the scope of the invention.”
`
`(Page 13, lines 6-7.) Nothing in the exampleindicates that dilution isessential, and as
`
`discussed above, it is not.
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`Page 8 of 15
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`
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`Page 8 of 15
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`
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`B.
`
`The Office Action asserts that the claims omit linear amplification by PCR, which
`
`is allegedly a critical step of the invention. The PTO supports this assertion byciting the
`specification at page 14, line 18 where an example of sample analysis is disclosed in
`
`which linear amplification is used to enhance the signal provided by molecular beacon
`
`probes. Applicants respectfully traverse.
`
`Claim 1 recites:
`
`analyzing the amplified molecules in the assay samples of
`the set to determine a first number of assay samples which
`contain the selected genetic sequence and a second number
`of assay samples which contain a reference genetic
`sequence,
`
`Claim 38 recites:
`
`analyzing the amplified molecules in the assay samples of
`the set to determine a first number of assay samples which
`contain the selected genetic sequence and a second ymber
`of assay samples which contain a reference genetic
`sequence, wherein at least one-fiftieth of the assay samples
`in the set comprise a number (N) of molecules such that
`1/N is larger than the ratio of selected genetic sequences to
`total genetic sequences required to determine the presence
`of the selected genetic sequence.
`
`In each claim, the amplified molecules in the assay samples ofthe set are analyzed, but a
`
`particular analysis methodis not required.
`
`Linear amplification can be performed as part ofthe step of analyzing butit need
`
`not be. The specification teaches that: “Although the working examples demonstrate the
`
`use ofmolecular beacon probes as the meansofanalysis of the amplified dilution
`
`samples, other techniques can be used as well.” (Emphasis added, page 12, lines 29-31.)
`
`Since linear amplification was taughtto enhancethe signal ofmolecular beacon probes,
`
`9
`
`Page9 of 15
`
`
`
`
`Page 9 of 15
`
`
`
`which are notessential, clearly linear amplification is not essential either, Therefore, the
`
`claimed method does not require linear PCR.
`
`Nothingin the specification indicates that linear amplification by PCR is
`
`essential. Rather, linear amplification by PCR is disclosed as an enhancementto the
`
`analysis step when molecular beacon (MB) probes are used. The specification states,
`
`“fluorescent signals obtained could be considerably enhanced if several cycles of
`
`asymmetric, linear amplification were performed in the presence of the MB probes.”
`
`(Page 19, lines 9-11.} Thus linear amplification is not essential to the method ofthe
`
`invention.
`
`C.
`
`The Office Action asserts that the use of the term “consists” is confusing because
`
`“[i]t cannot be determined whether the claim intends open or closed language for the
`
`limitation ofthe sequence. Proper Markush language is required.” (Page 4, lines 14-15.)
`
`Applicant’s respectfully traverse.
`
`Each of claims 33, 36, and 37 recite “the stem consists of 4 base pairs having a
`
`sequence 5’-CACG-3.” ‘Whenthe phrase ‘consists of? appears in a clause of the body of
`
`a claim, rather than immediately following the preamble,it limits only the elementset
`
`forth in that clause.” Manesmann Demag Corp. v. Engineered Metal Products Co., 793
`
`F.2d 1279, 230 USPQ 45 (Fed. Cir. 1986). Therefore, the term “consists” is closed. The
`
`stem contains the four recited base pairs 5’°-CACG-3’ and no others. No Markush group
`
`is present in claims 33, 36, and 37.
`
`D.
`
`The Office Action asserts that “fcjlaim 2 is confusing because it is unclear as to
`
`whether each sample ofthe fraction of one outten (sic) are to contain N molecules.”
`
`10
`
`Page 10 of 15
`
`J
`
`4
`
`Page 10 of 15
`
`
`
`(Page 4, lines 16-17.) Applicant’s respectfully traverse.
`
`Theclaim recites, “at least one-tenth of the assay samples in the set comprise a
`
`number (N) of molecules.” (Claim 2, lines 2-3.) The claim positively recites that at least
`
`one outof ten of the assay samples in the set have a number (N) molecules. The claim is
`
`not confusing or unclear. The language of the claim affirmatively answers the question
`
`ofthe Office Action. Each of the 1/10 fraction of samples comprise a number (N)
`
`molecules. N is defined so that 1/N is larger than the ratio of selected genetic sequences
`
`
`to total genetic sequences. Thusall of the 1/10 samples need not have the same number
`
`ofmolecules, but a number thatfits the definition. Nonetheless, if samples are formed by
`
`dilution, as in claim 2, the samples should have roughly identical numbers of molecules.
`
`Withdrawal of the 35 U.S.C. §112 rejection of claims 1-64 is respectfully
`
`requested as all claims are clear and definite.
`
`Rejection of claims | and 3-32 under 35 U.S.C. §103(a)
`
`Lapidus (U.S, 5,928,870) and Ruano (P.N.A.S., vol. 87, pp. 6296-6300, August
`
`1990) in combination are cited as teaching the invention of claims 1, 3, 4-11, 14-16, and
`
`19-32, Tyagi (U.S. 5,925,517) is further combined to allegedly teach the invention of
`
`claims 12, 13, 17, and 18, These rejections are respectfully traversed.
`
`It is axiomatic that all elements of a claim must be taught or suggested by the
`
`priorart for a primafacie case of obviousness to be proper. MPEP §2143. The present
`
`rejection fails to fulfill this “all elements”rule and thusfails to present aprimafacie case.
`
`Claim 1 requires four steps: diluting, amplifying, analyzing, and comparing.
`
`1
`
`Page 11 of 15
`
`Page 11 of 15
`
`
`
`Neither Lapidus nor Ruano teach the step of analyzingor the step of comparing as
`
`specified in claim 1. Claim 1, steps 3 and 4,recite:
`
`analyzing the amplified molecules in the assay
`samples of the set to determine a first number of assay
`samples which contain the selected genetic sequence and a
`second number of assay samples which contain a reference
`genetic sequence;
`comparing the first number to the second number to
`ascertain a ratio which reflects the composition of the
`biological sample.
`
`Emphasis added. The Office Action fails to point to any portion in either Lapidus or
`
`Ruano which teach these two steps. Lapidus does not teach determining a number of
`
`assay samples containing genetic sequences. Lapidus instead teaches determining
`
`concentration, The Office Action refers to this teaching of Lapidus as “enumerating
`
`number moleculesof a target,” citing col. 2, lines 58-66. This, however,is different from
`
`determining the number of assay samples containing a genetic sequence. Since the
`
`numbers of assay samples are not determined according to Lapidus,neither are the
`
`numbers compared, as required in step 4.
`
`This difference leads to an advantageofthe present invention over Lapidus.
`
`Digital amplification, as claimed, converts “the intrinsically exponential nature of PCR to
`
`a linear one.” Specification at page 8, lines 17-18. Thus the present invention eliminates
`
`the quantitative bias which exponential amplification introducesinto a nucleic acid
`
`sample. Since neither Lapidus nor Ruano teach these elements ofthe claims, the prima
`
`facie case mustfail.
`
`12
`
`Page 12 of 15
`
`
`
`Page 12 of 15
`
`
`
`Tyagi teaches molecular beacon probes. Tyagi is cited in combination with
`
`Lapidus and Ruanoto allegedly render claims 12, 13, 17 and 18 obvious. (Claim 12 does
`
`not employ a molecular beaconprobeatall, so its inclusion in this rejection is improper.)
`
`Like the primary references, Tyagi does not teach the element of “determining a
`
`first number of assay samples” nor of comparingthefirst and second numbers. Thus
`
`Tyagi does not remedy the defect of the primary references. Again, the primafacie case
`
`fails to teach all elements of the claimed invention and must therefore be withdrawn as
`
`improper.
`
`A speedy allowanceofall pending claims is respectfully requested.
`
`Respectfully submitted,
`
`Date:
`
`July 12, 2001
`
`By:
`
`Sarah A. Kagan
`Registration No. 32,141
`
`BaNnneR & WITCOFF, LTD.
`1001 G STREET, NW
`WASHINGTON, DC 20001
`202-508-9100
`
`13
`
`Page 130f15 >
`
`. ek _
`
`
`
`Page 13 of 15
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`
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`MARKED UP VERSION TO SHOW CHANGES MADE
`
`Replacement paragraph beginning on page4,line 5.
`
`
`Fic. 1A, 1B,1C. Schematic of experimental design. (A) The basic two steps
`
`involved: PCR on diluted DNA samples is followed by addition of fluorescent probes
`
`which discriminate between WT and mutantalleles and subsequentfluorometry. (B)
`
`Principle of molecular beacon analysis. In the stem-loop configuration, fluorescence
`
`from a dye at the 5’ end ofthe oligonucleotide probe is quenched by a Dabcyl group at
`
`the 3’ end. Upon hybridization to a template, the dye is separated from the quencher,
`
`resulting in increased fluorescence. Modified from Marras et al. . (C) Oligonucleotide
`
`design. Primers F1 and R1 are used to amplify the genomic region of interest. Primer
`
`INTis used to produce single stranded DNA from the original PCR products during a
`
`subsequent asymmetric PCR step (see Materials and Methods). MB-REDis a Molecular
`
`Beacon which detects any appropriate PCR product, whether it is WT or mutant at the
`
`queried codons. MB-GREEN is a Molecular Beacon which preferentially detects the WT
`
`PCRproduct.
`
`Replacement paragraph beginning on page 14,line 29.
`
`Oligonucleotides and DNA sequencing. Primer F1:
`
`5’-CATGTTCTAATATAGTCACATTTTCA-3’ (SEQ ID NOz1); Primer R1:
`
`5’-TCTGAATTAGCTGTATCGTCAAGG-3’ (SEO ID NO: 2); Primer INT:
`
`5°-TAGCTGTATCGTCAAGGCAC-3’ (SEQID NO;3); MB-RED:
`
`14
`
`Page 14 of 15
`
`Page 14 of 15
`
`
`
`5’-Cy3-CACGGGCCTGCTGAAAATGACTGCGTG-Dabcyl-3’ (SEO ID NO: 4);
`
`MB-GREEN:5’-Fluorescein-CACGGGAGCTGGTGGCGTAGCGTG-Dabcyl-3’ (SEO
`
`ID NO: 5). Molecular Beacons (33,34) were synthesized by Midland Scientific and other
`
`oligonucleotides were synthesized by Gene Link (Thomwood, NY). All were dissolved
`
`at 50 uM in TE (10 mM Tris, pH 8.0/ 1 mM EDTA)and keptfrozen and in the dark until
`
`use. PCR products were purified using QIAquick PCR purification kits (Qiagen). In the
`
`relevant experiments describedin the text, 20% of the product from single wells was used
`
`for gei electrophoresis and 40% was used for each sequencing reaction. The primer used
`
`for sequencing was 5°-CATTATTTTTATTATAAGGCCTGC-3? (SEO ID NO: 6).
`
`Sequencing was performed using fluorescently-labeled ABI Big Dye terminators and an
`
`ABI 377 automated sequencer.
`
`15
`
`Page 15 of 15
`
`Page 15 of 15
`
`
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