PATENT
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
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`In re Application of
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`Bert VOGELSTEIN et al.
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`Serial No. 13/071,105
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`Filed: March 24, 2011
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`For: DIGITAL AMPLIFICATION
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`Examiner: WOOL WINE, Samuel C.
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`Group Art Unit: 163 7
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`Confirmation No. 3361
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`Atty. Dkt. No. 001107.00866
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`RESPONSE TO OFFICE ACTION
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`Commissioner of Patents
`P.O. Box 1450
`Alexandria, VA 22313-14 50
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`Sir:
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`In response to the office action mailed June 27, 2013, applicants request entry of the
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`amendment and reconsideration of the patentability of the claims in view of the remarks.
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`A request for consideration under the AFCPP 2.0 accompanies this paper. No petition for
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`extension of time accompanies this submission. The Commissioner is authorized to charge any
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`fees which may be required or credit any overpayment to our Deposit Account 19-0733.
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`1
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`MYR 1037
`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
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`Page 1 of 11
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`IN THE CLAIMS
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`Please replace the following claim set for that currently of record.
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`1. -48. (Cancelled)
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`49. (Currently amended)
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`A method for detecting quantity of a genetic sequence in a mixed
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`population of human genomic nucleic acid sequences comprising at least a first and a second
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`human genomic sequence, wherein the first sequence is a wild-type sequence of an allele and a
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`second sequence is a mutant sequence of the allele, comprising:
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`distributing or diluting a mixed population of cell-free, human genomic nucleic acid
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`template molecules from a sample in which the fraction of mutant alleles is less than 20 %, into a
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`set comprising at least Wil fifteen assay samples such that said at least Wil fifteen assay samples
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`each comprises less than ten template molecules;
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`amplifying the template molecules in the assay samples, wherein an assay sample with a
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`single template molecule forms homogeneous amplification products in the assay sample;
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`analyzing by determining nucleic acid sequence of amplification products in the assay
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`samples of the set with homogeneous amplification products to determine a first number of assay
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`samples in the set which contain the first sequence and a second number of assay samples in the
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`set which contain the second sequence;
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`comparing the first number to the second number to ascertain a ratio which reflects the
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`composition of the mixed population;
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`identifying a mutation in the mixed population if a statistically significant fraction of
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`assay samples comprises the second sequence.
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`50. (Previously Presented)
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`The method of claim 49 wherein the assay samples of the set have
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`on average 0.5 molecules of template.
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`51. (Previously Presented)
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`The method of claim 49 wherein between 0.1 and 0.9 of the assay
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`samples yield an amplification product.
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`2
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`52. (Previously Presented)
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`The method of claim 49 wherein the mixed population of nucleic
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`acid sequences is distributed or diluted to a single template molecule level in the assay samples.
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`53. (Currently amended)
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`The method of claim 49 wherein the mixed population of nucleic
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`acid sequences is from a ti88lt€: @f body sample.
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`54. (Currently amended)
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`The method of claim ~ 53 wherein the mixed population of
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`nucleic acids sequences is from a body sample selected from the group consisting of stool, blood,
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`and lymph nodes.
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`55. (Cancelled)
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`56. (Previously Presented)
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`The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least twenty assay samples comprise less
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`than ten template molecules.
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`57. (Previously Presented)
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`The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least twenty-five assay samples comprise
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`less than ten template molecules.
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`58. (Previously Presented)
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`The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least thirty assay samples comprise less than
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`ten template molecules.
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`59. (Previously Presented)
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`The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least forty assay samples comprise less than
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`ten template molecules.
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`60. (Previously Presented)
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`The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least fifty assay samples comprise less than
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`ten template molecules.
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`3
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`Page 3 of 11
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`61. (Previously Presented) The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least seventy-five assay samples comprise
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`less than ten template molecules.
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`62. (Previously Presented) The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least one hundred assay samples comprise
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`less than ten template molecules.
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`63. (Previously Presented) The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least five hundred assay samples comprise
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`less than ten template molecules.
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`64. (Previously Presented) The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least one thousand assay samples comprise
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`less than ten template molecules.
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`65. (Currently amended)
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`The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least one thousand assay samples are
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`distributed or diluted to a single template molecule level.
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`66. (Previously Presented) The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that at least one thousand assay samples has on
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`average 0.5 molecules of template.
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`67. (Previously Presented) The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that between 0.1 and 0.9 of at least one thousand
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`assay samples yield an amplification product.
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`68. (Previously Presented) The method of claim 49 wherein the mixed population of nucleic
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`acids sequences is distributed or diluted such that one half of at least one thousand assay samples
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`have one template molecule.
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`4
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`69. (New) The method of claim 49 wherein the mutation is a somatic mutation.
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`70. (New) The method of claim 49 wherein the mutation is a cancer gene mutation.
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`71. (New) The method of claim 49 wherein the template molecules are from a population of
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`cells which are not purely tumor cells.
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`72. (New) The method of claim 49 wherein between 1% and 10 % of the alleles in said human
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`genomic nucleic acid template molecules are the mutant sequence of the allele.
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`73. (New) The method of claim 49 wherein the mixed population of nucleic acid sequences is
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`from a tissue.
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`74. (New)
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`A method for detecting quantity of a genetic sequence in a mixed population of
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`human genomic nucleic acid sequences comprising at least a first and a second human
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`genomic sequence, wherein the first sequence is a wild-type sequence of an allele and a
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`second sequence is a mutant sequence of the allele, comprising:
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`distributing or diluting a mixed population of cell-free, human genomic nucleic acid
`
`template molecules into a set comprising at least fifteen assay samples such that said at least
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`fifteen assay samples comprises an average of0.5 molecules of template.;
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`amplifying the template molecules in the assay samples, wherein an assay sample with a
`
`single template molecule forms homogeneous amplification products in the assay sample;
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`analyzing by determining nucleic acid sequence of amplification products in the assay
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`samples of the set with homogeneous amplification products to determine a first number of assay
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`samples in the set which contain the first sequence and a second number of assay samples in the
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`set which contain the second sequence;
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`comparing the first number to the second number to ascertain a ratio which reflects the
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`composition of the mixed population;
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`identifying a mutation in the mixed population if a statistically significant fraction of
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`assay samples comprises the second sequence.
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`5
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`Page 5 of 11
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`IN THE SPECIFICATION
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`Please substitute at page 3, last paragraph, and first paragraph on page 4, with the
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`following:
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`According to another embodiment of the invention, a molecular beacon probe is
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`provided. It comprises an oligonucleotide with a stem-loop structure having a photoluminescent
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`dye at one of the 5' or 3' ends and a quenching agent at the opposite 5' or 3' end. The loop
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`consists of 16 base pairs which has a T m of 50-51 § ~C. The stem consists of 4 base pairs having
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`a sequence 5'-CACG-3'.
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`A second type of molecular beacon probe is provided in another embodiment. It
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`comprises an oligonucleotide with a stem-loop structure having a photoluminescent dye at one of
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`the 5' or 3' ends and a quenching agent at the opposite 5' or 3' end. The loop consists of 19-20
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`base pairs and has aT m of 54-56§ ~C. The stem consists of 4 base pairs having a sequence 5'(cid:173)
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`CACG-3'.
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`Please substitute the paragraph spanning pages 7 and 8, with the following:
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`Digital amplification can be used to detect mutations present at relatively low levels in
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`the samples to be analyzed. The limit of detection is defined by the number of wells that can be
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`analyzed and the intrinsic mutation rate of the polymerase used for amplification. 384 well PCR
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`plates are commercially available and 1536 well plates are on the horizon, theoretically allowing
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`sensitivities for mutation detection at the .about.O.l% level. It is also possible that Digital
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`Amplification can be performed in microarray format, potentially increasing the sensitivity by
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`another order of magnitude. This sensitivity may ultimately be limited by polymerase errors. The
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`effective error rate in PCR as performed under our conditions was 1.1 %, i.e., four out of 351
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`PCR products derived from WT DNA sequence appeared to contain a mutation by RED/GREEN
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`ratio criteria. However, any individual mutation (such as a G toT transversion at the second
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`position of codon 12 of c-Ki-Ras), are is expected to occur in <1 in 50 of these polymerase-
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`6
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`generated mutants (there are at least 50 base substitutions within or surrounding codons 12 and
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`13 that should yield high RED/GREEN ratios). Determining the sequence of the putative mutants
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`in the positive wells, by direct sequencing as performed here or by any of the other techniques,
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`provides unequivocal validation of a prospective mutation: a significant fraction of the mutations
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`found in individual wells should be identical if the mutation occurred in vivo. Significance can
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`be established through rigorous statistical analysis, as positive signals should be distributed
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`according to Poisson probabilities. Moreover, the error rate in particular Digital Amplification
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`experiments can be precisely determined through performance of Digital Amplification on DNA
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`templates from normal cells.
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`7
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`Page 7 of 11
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`Remarks
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`The amendments to claim 49 are fully supported and do not add new matter. The
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`recitation of fifteen assay samples was formerly recited in claim 55. The recitation of fraction of
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`mutant alleles is supported in the specification at page 2, lines 5-7.
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`Claim 53 is amended to separate alternative recitations. Claim 54 is amended to clarify
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`its intended meaning.
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`Claim 55 are cancelled in view of the amendment to independent claim 49 which
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`incorporates its recitation.
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`Claim 65 is amended for internal consistency of its recitations.
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`New claims 69-73 are fully supported in the application as originally filed. Claim 69 is
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`supported at page 1, line 12, and page 2, line 15. Claim 70 is supported at page 9, in the table at
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`row 2, column 2. Claim 71 is supported at page 10, last full sentence. Claim 72 is supported at
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`page 21, last paragraph. Claim 73 is supported by claim 54 prior to its amendment.
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`Claim 74 is supported by claim 49, prior to its amendment and claim 50.
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`The rejection of claims 49 and 51-53 under § 1 03(a)
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`Claims 49 and 51-53 stand rejected as unpatentable over Ruano (PNAS 87:6296-6300,
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`1990). Ruano is cited as having met the limitations of the first three steps of (a) distributing or
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`diluting, (b) amplifying, and (c) analyzing. The last two steps of (d) comparing and (e)
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`identifying were asserted as not taught, but as just being obvious. The Patent and Trademark
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`Office's construction of the claim terms "wild- type" and "mutant" as encompassing two
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`different SNPs was a necessary part of its rejection. While applicant does not concur in the
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`construction of these terms, it has added further terms to independent claim 49 to clarify the
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`context and scope of the claimed method.
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`Claim 49 as amended recites that the fraction of mutant alleles in the sample is less than
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`20%. This is a limitation that distinguishes over Ruano's mere SNPs, one on each of two
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`homologous chromosomes, present in a 1:1 ratio; the mutant alleles of the present invention are
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`present in a minority of nucleic acids in a sample. Claim 49 as amended also recites a minimum
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`of 15 assay samples that are amplified and analyzed and compared. Ruano did not teach more
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`than 10 samples, and so did not teach the number of samples now recited in claim 49. See office
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`action at page 6, lines 12-13.
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`8
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`Ruano's determination of a haplotype does not teach or suggest a mutant allele which is
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`present at less than 20% in the sample. SNPs, as taught by Ruano, are typically present at a
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`fraction of ~50%.
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`Finally, there would be no reason to assay fifteen assay samples for Ruano's method. If
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`two alleles are present in a 1: 1 ratio in a sample, then ten assay samples are more than sufficient
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`to achieve excellent detection, as Ruano demonstrated.
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`The obtaining of a haplotype, as Ruano taught, is conceptually distinct and does not
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`suggest detection of rare alleles in a mixed population present at <20%.
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`The method of independent claim 49 as amended is not obvious over Ruano. For at least
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`these reasons dependent claims 51, 52, and 53 are also not obvious.
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`Please withdraw this rejection.
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`Rejection of claims 50 and 55-68 under § 1 03(a)
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`Claims 50 and 55-68 stand rejected as obvious over Ruano (PNAS 87:6296-6300, 1990)
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`in view of Stephens (Am. J. Hum. Gen 46:1149-1155, 1990). Claim 55 is cancelled.
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`Claim 50 recites (as do claims 66 and 74) that the assay samples have on average 0.5
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`molecules of the template. Claims 56-68 recite at least 20 and up to at least 1000 assay samples.
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`The Patent and Trademark Office acknowledges that Ruano does not teach either of these
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`parameters. However, Stephens is cited to remedy the deficiency of Ruano.
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`Stephens is cited as teaching in Table 1 the probability of success at having one or more
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`vials having one but not both haplotypes in it. Stephens teaches that for 10 vials and 1 haploid
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`equivalent in a vial, as used by Ruano, one has a 0.9985 chance of success. In fact, ofthe
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`choices shown in Table 1, it appears that Ruano used the optimal conditions. Contrary to the
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`assertion of the Patent and Trademark Office, Table 1 would not have motivated one of ordinary
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`skill in the art to use even more assay vials. A 0.9985 chance of success would be considered to
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`be quite high and increasing the number of vials would likely yield a very small increase.
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`Stephens, contrary to the Patent and Trademark Office's assertion, would lead one of skill in the
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`art to do exactly what Ruano had done- I 0 samples and single molecule dilution. No
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`modification of the method of Ruano would have been suggested.
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`Similarly, contrary to the assertion of the Patent and Trademark Office, Stephens would
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`not motivate one of skill in the art to modify Ruano's teaching of single molecule dilution to use
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`0.5 molecules, as recited in claim 50. Inspection of Stephens' Table 1 would lead one of skill in
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`the art to realize that modifying Ruano's method in that way would lead to a diminished success
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`rate.
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`One of ordinary skill in the art would not have found Stephens' teaching to be
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`motivational for any empirical optimization. Stephens explicitly taught the theoretical
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`underpinnings for the parameters that Ruano chose-they were the optimum for the method that
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`Ruano was performing. Once optimized, those of skill in the art are not likely to seek variations.
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`Moreover, for at least the same reasons as for independent claim 49, dependent claims 50
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`and 56-68 are not obvious over the combination of Ruano and Stephens. Stephens does not
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`address any of the deficiencies of Ruano with regard to claim 49 as amended.
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`Please withdraw this rejection.
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`Rejection of claims 55-68 under § 103 (a)
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`Claims 55-68 stand rejected over Ruano (PNAS 87:6296-6300, 1990), as applied above,
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`in view ofKruglyak (Nature Genetics 22:139-144, 1999). Claim 55 is cancelled.
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`As acknowledged by the Patent and Trademark Office, Ruano did not teach more than 10
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`assay samples. Use of more than 10 assay samples is recited in each of claims 56-68.
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`Kruglyak is cited as suggesting haplotype analysis at many different SNPs. This, the
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`Patent and Trademark Office asserts, would have made it obvious to use the method of Ruano
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`for haplotype analysis at many different sites throughout the genome. The rejection concludes
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`that this would have necessitated the use of up to 1000 or even more "single molecule" samples.
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`Page 8, lines 3-4.
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`What the Patent and Trademark Office proposes is that a sample would be distributed
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`into 1000 or more assays, which might have, arguendo, fulfilled the limitations of the step of
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`"distributing or diluting." It appears that the hypothesized experiment would determine different
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`SNPs in different subsets of the 1000 assay samples. As discussed above, optimized results for
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`the Ruano type haplotype assay for one SNP (allele) can be determined with just 10 assay
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`samples. Thus it appears that many subsets of 10 assay samples would be used to determine
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`different SNPs. The hypothesized experiment would not fulfill the recitations of the step of
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`"analyzing by determining nucleic acid sequence." That step requires that the number of assay
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`samples with the first sequence (wild type allele) and the number of assay samples with the
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`10
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`Page 10 of 11
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`second sequence (mutant allele) be determined in the assay samples of the set. The set is defined
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`with a minimum of at least 20, 25, 30, 40, 50, 75, 100, 500, or 1000 assay samples in claims 56-
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`68. The hypothesized experiment would not, therefore, fulfill all elements of the claims.
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`Moreover, for at least the same reasons as for independent claim 49, dependent claims
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`56-68 are not obvious over the combination of Ruano and Kruglyak. Kruglyak does not address
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`any of the deficiencies of Ruano with regard to independent claim 49 as amended.
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`Please withdraw this rejection.
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`The rejection of claim 54 under§ 103(a)
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`Dependent claim 54 stands rejected as unpatentable over Ruano (PNAS 87:6296-6300,
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`1990) in view ofKulozik. (Am J. Hum Gen 39:239-244, 1986). Kulozik is cited as teaching
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`blood as a source of genomic DNA for haplotyping. Kulozik does not remedy the deficiencies of
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`Ruano with regard to independent claim 49. Kulozik teaching nothing about assaying for alleles
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`present at less than 20% using at least 15 assay samples.
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`For at least the same reasons as claim 49, claim 54 is patentable over Ruano in view of
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`Kulozik. Please withdraw this rejection.
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`Date: 25 Seytember, 2013
`
`By:
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`Banner & Witcoff, Ltd.
`Customer No. 11332
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`Respectfully submitted,
`
`/Sarali A. Xagan/
`Sarah A. Kagan
`Registration No. 32,141
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`Page 11 of 11
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