Ex Parte Ling et al

Patent Trial and Appeal Board

Oct 20, 2017

11538189 (P.T.A.B. Oct. 20, 2017)

United States Patent and Trademark Office
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O.Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
11/538,189 10/03/2006 Xinsheng Sean Ling NAB-003 1590
51414 7590 10/24/2017
GOODWIN PROCTER LLP
PATENT ADMINISTRATOR
100 Northern Avenue
BOSTON, MA 02210
EXAMINER
KAPUSHOC, STEPHEN THOMAS
ART UNIT PAPER NUMBER
1634
NOTIFICATION DATE DELIVERY MODE
10/24/2017 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
PATENTBOS @GOODWINPROCTER.COM
PSOUSA-ATWOOD@GOODWINPROCTER.COM
GLENN.WILLIAMS@GOODWINPROCTER.COM
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte XINSHENG SEAN LING and BARRETT BREADY1
Appeal 2017-001668
Application 11/538,189
Technology Center 1600
Before DONALD E. ADAMS, JOHN G. NEW, and
JOHN E. SCHNEIDER, Administrative Patent Judges.
NEW, Administrative Patent Judge.
DECISION ON APPEAL
SUMMARY
Appellants file this appeal under 35 U.S.C. § 134(a) from the
Examiner’s Final Rejection of claims 1, 3—14, 16—23 and 25—28 as
unpatentable under 35 U.S.C. § 103(a) as being obvious over the
combination of Deamer (US 6,617,113 B2 , September 9, 2003) (“Deamer”),
Branton et al. (US 2003/0104428 Al, June 5, 2003) (“Branton”), K.R.
Khrapko et al., An Oligonucleotide Hybridization Approach to DNA
appellants state that the real party-in-interest is Nabsys 2.0 LLC. App. Br.
3.
Appeal 2017-001668
Application 11/538,189
Sequencing, 256(1,2) FEBS Letts. 118-122 (1989) (“Khrapko”), and S.
Hannenhalli et al., Positional Sequencing by Hybridization, 12(1) CABIOS
19-24 (1996) (“Hannenhalli”).
We have jurisdiction under 35 U.S.C. § 6(b).
We AFFIRM.
NATURE OF THE CLAIMED INVENTION
Appellants’ invention is directed to a method of employing a
nanopore structure in a manner that allows the detection of the positions
(relative and/or absolute) of nucleic acid probes that are hybridized onto a
single-stranded nucleic acid molecule. Abstract.
REPRESENTATIVE CLAIM
Claim 1 is representative of the claims on appeal and recites:
1. A method for determining the sequence of a biomolecule
strand of interest, comprising the steps of:
a. providing a sequencing apparatus having a first fluid
chamber, a second fluid chamber, a membrane positioned
between said first and second chambers and a nanopore
extending through said membrane such that said first and second
chambers are in fluid communication via said nanopore;
b. providing a single-stranded biomolecule comprising at
least one of DNA or RNA and having an unidentified sequence;
c. providing a first plurality of probes, the first plurality of
probes comprising matching probes having a same known
sequence;
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d. hybridizing said first plurality of probes with said
single-stranded biomolecule such that said first plurality of
probes attach to portions of said single-stranded biomolecule
to produce a partially hybridized biomolecule;
e. introducing said partially hybridized biomolecule into
said first chamber;
f. translocating said partially hybridized biomolecule from
said first chamber through said nanopore and into said second
chamber;
g. monitoring changes in an electrical property across said
nanopore as said partially hybridized biomolecule is translocated
therethrough, said changes in the electrical property
corresponding to locations along said partially hybridized
biomolecule containing one of said first plurality of probes;
h. recording said changes in the electrical property as a
function of time to determine relative positions of the hybridized
regions of the biomolecule
i. repeating steps c. to h. using at least a second plurality
of probes having a known sequence different than said known
sequence of said first plurality of probes, wherein the known
sequences of the at least second plurality of probes at least
partially overlap the known sequences of the at least first
plurality of probes; and
j. determining an at least partial sequence of said single-
stranded biomolecule using said changes in the electrical
property,
wherein the at least partial sequence of the single-stranded
biomolecule comprises a number of consecutive bases larger
than that of any one of the probes.
App. Br. 14-15
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Application 11/538,189
ISSUES AND ANALYSES
We agree with, and adopt, the Examiner’s findings of fact and
conclusions that the appealed claims are obvious over the cited prior art.
We address the arguments raised by Appellants below.
Issue 1
Appellants argue the Examiner erred because the combined cited prior
art references neither teach nor suggest the claimed sequencing method
where the sequence of a target segment longer than the length of any of the
individual probes is obtained by determining the relative position of bound
hybridized probes, and where the probes have overlapping sequences. App.
Br. 12.
Analysis
The Examiner finds that Deamer teaches the analysis of double
stranded nucleic acids via translocation of the nucleic acid through a
nanopore. Final Act. 3. The Examiner finds, however, that Deamer does
not specifically teach the analysis of a hybridized biomolecule that is a
partially hybridized biomolecule relevant to the limitation, or that changes in
electrical potential correspond to locations along a partially hybridized
biomolecule containing a probe, as required by claim 1. Id. at 5. The
Examiner finds Branton teaches methods for characterizing a single stranded
biomolecule using hybridized probes, where the location of hybridization is
determined upon passing the biomolecule through a nanopore. Id.
The Examiner finds that, although the combination of Deamer and
Branton does not expressly teach methods in which overlapping probes are
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Application 11/538,189
used to determine a sequence of the target that is longer than any of the
probes, the use of probe hybridization using multiple overlapping probes
was well known in the prior art. Final Act. 6. Specifically, the Examiner
finds Khrapko teaches sequencing by hybridization as a method for DNA
sequencing, by which hybridization of a DNA fragment to be sequenced
with multiple oligonucleotide probes that are shorter in length than the DNA
fragment. Id. The Examiner finds Hannenhalli teaches that ambiguities in
sequence reconstruction can be reduced by providing a relative position of
each hybridized probe. Id. at 9.
The Examiner concludes that it would have been prima facie obvious
to a person of ordinary skill in the art to have combined the multiple probe
hybridizations and sequence compilation methods of Khrapko with the
sequence analysis methods taught by Deamer and Branton. Final Act. 8.
The Examiner finds that such an artisan would have been motivated to
combine the sequencing method of Khrapko with the analysis methods
taught by Deamer and Branton because sequencing by hybridization was a
method known in the prior art, and the issue of identifying the relative
position of probe hybridization was similarly known in the art. Id. at 9.
Appellants argue that Deamer neither teaches nor suggests: (1) the
analysis of a partially hybridized biomolecule; (2) that changes in electrical
potential correspond to locations along a partially hybridized biomolecule
containing a probe; (3) capacitance measurement; (4) mapping of probe
locations, (5) the use of a plurality of probes with different sequences; and
(5) using changes in an electrical property to determine at least a partial
sequence of a single-stranded biomolecule where that partial sequence is
longer than the length of any individual probe as required by each of the
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Application 11/538,189
pending claims; or (6) use of relative positional information to determine a
sequence. App. Br. 6—7.
Appellants argue that Branton fails to cure the alleged deficiencies of
Deamer. App. Br. 7. Specifically, Appellants contend Branton does not
teach or suggest a method for determining the sequence of a biomolecule as
recited in claim 1. Id. at 8. Specifically, Appellants argue, Branton neither
teaches nor suggests how to determine the sequence of a biomolecule. Id.
By way of example, Appellants point to Branton’s teaching Oligonucleotide
Hybridization Encoded Analysis (“OHEA”) that can use mixtures of either
genome specific, allele specific, or other sets of universal oligonucleotides.
Id. (citing (Branton, 179). According to Appellants, Branton relates to
obtaining sufficient information such that a comparison can be made to
known data obtained from “predetermined agents” in order to
unambiguously determine that genetic material from that agent is present in
the sample. Id. As such, argue Appellants, Branton does not teach or
suggest a method for determining the genetic sequence of an unknown
target, but, rather, teaches using genetic data to identify a target by
comparing that data to previously determined data. Id. Appellants therefore
assert that a person of ordinary skill would have understood that a method
for determining the length of a restriction fragment, such as an RFLP, does
not equate to a teaching of the determination of the sequence of that
fragment, much less the sequence of the biomolecule, as recited in the
claims. Id.
Furthermore, Appellants argue, whereas the claims on Appeal seek to
determine positions of probes relative to one another, Branton teaches
determining the interactions of individual nucleotides of the sample with the
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Application 11/538,189
detector. App. Br. 9 (citing, e.g., Branton 114) (“Because the individual
nucleotides of the nucleic acid molecule interact with the detector in
sequential order, information regarding the location and composition of a
plurality of modified sites along a single molecule can be obtained”).
Consequently, Appellants argue, Branton neither teaches nor suggests
Appellants’ claimed method, which does not determine the sequence by
analyzing individual nucleotides by passing them through a detector in
sequential order. Id.
The Examiner responds that Deamer was not relied upon by the
Examiner as teaching the limitations recited by Appellants supra. Rather,
the Examiner cites Deamer to establish that the detection of nucleic acids
using nanopores, and in particular the detection of double-stranded versus
single-stranded nucleic acids, was known in the art at the time the invention
was made. Ans. 3 (citing Deamer Fig. 2B).
With respect to Appellants’ argument that a person of ordinary skill
would not have understood from the teachings of Branton that changes in
electrical properties within a nanopore can correspond to the location of a
hybridized probe, the Examiner finds that Branton expressly teaches that
analysis of electrical changes within a nanopore provides information with
respect to the location of hybridized probes along the target nucleic acid.
Ans. 3 (citing, e,g., Branton 114). The Examiner finds Branton teaches a
method of analysis by which the relative locations of probes hybridized to a
target are identified from measurements of electrical changes within a
nanopore. Id. (citing Branton Figs. 3A, 3B).
Finally, the Examiner finds that, contrary to Appellants’ arguments,
Branton is not cited by the Examiner for the purpose of teaching any specific
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Application 11/538,189
sequencing by hybridization embodiments, but only for its teaching that the
nanopore-based method can be effectively used to identify the relative
locations of probes hybridized to a target. Ans. 4.
We agree with the Examiner. As an initial matter, “one cannot show
non-obviousness by attacking references individually where ... the
rejections are based on combinations of references.” In re Keller, 642 F.2d
413, 426 (C.C.P.A. 1981). Furthermore, and by the same logic, Appellants
cannot claim a given reference fails to teach or suggest a specific limitation
of the claim when the Examiner did not, in fact, rely upon the reference as
teaching that limitation.
Deamer teaches:
[Njucleic acids present in a fluid sample are translocated through
a nanopore, e.g. by application of an electric field to the fluid
sample. The current amplitude through the nanopore is
monitored during the translocation process and changes in the
amplitude are related to the passage of single- or double-
stranded molecules through the nanopore. The subject methods
find use in a variety of applications in which the detection of the
presence of double-stranded nucleic acids in a sample is desired,
e.g. in hybridization assays, such as Northern blot assays,
Southern blot assays, array based hybridization assays, etc.
Deamer Abstr. (emphases added). Deamer further teaches that, during
translocation of a nucleic acid through the nanopore:
In many embodiments, the measured data values, e.g.
current amplitudes, are then manipulated to produce a current
blockade profile or similar output capable of being compared
against reference outputs such that the nature of the nucleic acid,
i.e. the single or double strandedness of the nucleic acid passing
through the pore can be determined.
Id. at col. 5,11. 20—25. Finally, Deamer teaches:
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Application 11/538,189
The subject methods find use in a variety of different
applications where on wishes to determine the presence or
absence of double stranded nucleic acids in a sample. For
example, the subject methods find use in detecting hybridization
events in assays where complementary nucleic acids are
hybridized to each other and are detected. Examples of such
hybridization assays include assays where one or more probes
are combined with target nucleic acid and the occurrence of
hybridization events in solution is detected. In such assays,
unlabeled probe is contacted with the target nucleic acid sample.
Next, the fluid sample is assayed according to the subject
methods, and the presence of double-stranded nucleic acids in
the sample is determined. The presence of double-stranded
nucleic acids indicates that hybridization between the probe and
target has occurred.
Id. at col. 6,11. 8—19. Deamer thus teaches hybridizing probes to a single-
stranded nucleic acid and translocating the nucleic acid through a nanopore
to detect whether hybridization (i.e., binding of the probe to a
complementary sequence on the nucleic acid) has occurred, resulting in a
double-stranded sequence that is measurable in a current conducted across
the nanopore.
Branton teaches:
In one aspect of the invention, a method is provided for
characterizing a nucleic acid molecule. In this aspect of the
invention, a property of at least one defined local area of the
nucleic acid molecule is modified.... The modified nucleic acid
molecule traverses a defined and preferably molecular
dimensioned (e.g. very small) volume on the substrate so that
nucleotides of the modified nucleic acid molecule interact with
the detector in sequential order, whereby data correlating with
the cross-sectional area, local charge, or local chemistry of the
nucleic acid molecule are obtained.... Because the individual
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Application 11/538,189
nucleotides of the nucleic acid molecule interact with the
detector in sequential order, information regarding the location
and composition of a plurality of modified sites along a single
molecule can be obtained.
Branton 114. Branton further teaches:
In one or more embodiments, the identifier is a
hybridizable probe. The probe binds to or modifies the nucleic
acid molecule to form a nucleic acid:probe complex at locations
where the specific nucleic acid sequence is present. The presence
and location of binding in the nucleic acid:probe complex is
detected by contacting the nucleic acid:probe complex with a
substrate, the substrate including a detector capable of
identifying a characteristic of a nucleic acid molecule, or a
characteristic of the probe, or a characteristic of the nucleic acid:
probe complex, and causing the nucleic acid:probe complex to
traverse a defined volume of the substrate, preferably molecular
dimensioned (e.g. very small) volume, so that nucleotides of the
nucleic acid interact with the detector in sequential order,
whereby data correlating with the presence and/or location of
binding or modification are obtained. The relationship between
multiple loci on one nucleic acid molecule, or among different
nucleic acid molecules in a mixture is established by identifying
and distinguishing among the different nucleic acid:probe
complexes in the mixture. Information that can be obtained from
a nucleic acid molecule or population of nucleic acid molecules
population of nucleic acid molecules includes the number of a
selected nucleic acid:probe or identifier complexes, and the
relative location or locations of probe or identifier binding on a
nucleic acid molecule.
Id. at 120. We consequently agree with the Examiner’s findings that
Deamer and Branton together teach the use of nanopore translocation
technology, and in particular the detection of double-stranded versus single-
stranded nucleic acids, and that changes in electrical properties within a
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Application 11/538,189
nanopore can correspond to the location of a hybridized probe,
corresponding to limitations a—h of Appellants’ claim 1. See Ans. 2—A.
Issue 2
Appellants argue further that the Examiner erred because an artisan of
ordinary skill would not have combined the teachings of Khrapko and
Hannenhalli with those of Deamer. App. Br. 9.
Analysis
Appellants contend Khrapko teaches a sequencing method based on
analyzing dissociation curves of the labeled DNA hybridized with
oligonucleotides immobilized on a glass plate. App. Br. 9-10. In contrast,
Appellants argue, the claimed method requires monitoring changes in an
electrical property across a nanopore as the partially hybridized biomolecule
is translocated through the nanopore, where the changes in the electrical
property correspond to locations along the partially hybridized biomolecule
containing the probes. Id. at 10. Appellants therefore argue that Khrapko
neither teaches nor suggests determining the sequence of a single-stranded
biomolecule using changes in an electrical property, as recited in claim 1.
Id.
Similarly, argue Appellants, Hannenhalli teaches methods of attaching
a large set of single-stranded oligonucleotides to a substrate to form a
sequencing chip. App. Br. 10 (citing Hannenhalli 19). Appellants assert
Hannenhalli also teaches algorithms for positional sequencing by
hybridization (PSBH) to improve the resolving power of sequencing by
hybridization. Id. (citing Hannenhalli Abstr.). However, Appellants
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Application 11/538,189
contend, both Khrapko and Hannenhalli relate to sequencing methods where
targets are hybridized to probes immobilized on a surface, whereas the
claims require translocation through a pore. Id. Appellants therefore
contend that a person of ordinary skill in the art would not combine the
teachings of Khrapko and Hannenhalli which require immobilized probes
with methods that employ translocation of molecules through nanopores,
because to do so would fundamentally change the principle of operation of
the references. Id.
We are not persuaded by Appellants’ arguments. The Examiner cites
Khrapko as teaching sequencing by hybridization as a method for DNA
sequencing comprising hybridization of a DNA fragment to be sequenced
with multiple oligonucleotide probes that are shorter in length than the DNA
fragment, as recited in claims b—d of claim 1. Final Act. 7. Similarly, the
Examiner finds Hannenhalli teaches determining relative positions of
hybridized probes as recognized difficulty in sequencing by hybridization: a
problem addressed by the teachings of Deamer and Branton with respect to
nanopore translocation. Ans. 5.
“The test for obviousness is not whether the features of a secondary
reference may be bodily incorporated into the structure of the primary
reference.... Rather, the test is what the combined teachings of the
references would have suggested to those of ordinary skill in the art.” In re
Keller, 642 F.2d at 425. The Examiner cites to Khrapko and Hannenhalli as
teaching methods of sequencing by hybridization, and notes that a difficulty
of this technique is ascertaining the location of the hybridized sequence.
See, e.g., Hannenhalli 19. Branton expressly teaches determining the
position of hybridized sections of otherwise single-stranded nucleic acids via
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nanopore translocation and consequent alteration of the electrical signal at
the nanopore. See Branton 120. Because it is the concept of sequencing by
hybridization that the Examiner relies upon Khrapko and Hannenhalli as
teaching, and not the remainder of the limitations of claim 1 with respect to
translocation and detection through the nanopore, for which the Examiner
relies upon the teachings Deamer and Branton, we are not persuaded by
Appellants’ argument that a person of ordinary skill in the art would have
understand that the teachings of the references could not be combined.
Issue 3
Appellants argue the Examiner erred because unexpected results
overcome the Examiner’s prima facie case of obviousness. App. Br. 12.
Analysis
Appellants point to the Declaration of John S. Oliver, Ph.D. (the
“Oliver Declaration,” Appellants’Exhibit A). App. Br. 12. According to
Appellants, Dr. Oliver states that, at the time of the invention of Appellants’
claimed method, the use of nanopores to provide positional information and
thus circumvent the limitations of sequencing by hybridization had not been
envisioned. Id. (citing Oliver Deck Tflf6—7). Dr. Oliver further opines that,
at the time of the invention, sequencing by hybridization would have been
constrained in the length of sequence that could be determined because of
sequence repeats. Id. By way of example, Dr. Oliver states that repeated
sequences would limit the reconstructable sequence length resulting from
SBH with a library of 8-mers (i.e., 65,536 different oligonucleotides)
constructed from cognate bases to less than 256 bases. Id. (see Oliver Deck
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1 6). Dr. Oliver states that the addition of positional information would have
allowed one to sequence targets of effectively infinite length from the same
library of 8-mers and that without the knowledge that nanopores could
provide sufficiently accurate positional information in combination with the
reconstruction of sequence based on overlapping probes, as with classical
array-based sequencing by hybridization, that technique would have failed to
sequence through the repeat structure. Id. (citing Oliver Deck 1 8).
Appellants therefore contend that obtaining positional information by
the use of nanopores in combination with sequencing by hybridization
would not have been obvious in view of the cited prior art. App. Br. 12.
According to Appellants, Hannenhalli and the statements of the Oliver
Declaration evince a long-felt need existed for the methodology of the
claimed invention. Id.
The Examiner responds that cited prior art, and particularly Branton,
teach using nanopore translocation to provide positional information
pertaining to hybridized probes. Ans. 6. The Examiner finds that, although
Branton does not expressly teach sequencing by hybridization, Hannenhalli
teaches that determining the relative positions of hybridized probes as a
recognized difficulty in sequencing by hybridization, the application of
nanopore detection of probe hybridization to the process would have been an
improvement to sequencing by hybridization methods that one of ordinary
skill in the art would have motivated to use. Id.
With respect to paragraph 8 of the Oliver Declaration, which,
Appellants argue, opines that the addition of positional information allows
one to sequence targets of effectively infinite length using an 8-mer library,
the Examiner finds that Appellants’ argument is not commensurate in scope
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with the required limitations of the claims, which do not require any
minimum target length, or any particular probe library. Ans. 6.
We are not persuaded by Appellants’ arguments. “[W]hen
unexpected results are used as evidence of nonobviousness, the results must
be shown to be unexpected compared with the closest prior art.” In re
Baxter TravenolLabs., 952 F.2d 388, 392 (Fed. Cir. 1991). Appellants
adduce no additional art to which to compare their allegedly unexpected
results. Consequently, we rely upon the prior art cited by the Examiner.
Branton teaches that nanoprobe translocation can be used to identify the
location(s) of a probe hybridized to a single-stranded nucleic acid, which, we
agree with the Examiner, teaches limitations b-h of claim 1. Appellants
adduce no experimental evidence or data to support their contention that
their results are unexpected when compared to the teachings of the prior art.
Instead, Appellants rely upon the Oliver Declaration. Dr. Oliver is
Vice President of Research and Development at NABsys, Inc., received his
Ph.D. in 1988 in chemistry from Northwestern University, and has been
working in the field of sequencing by hybridization since 2000, authoring a
number of papers in this field. Oliver Decl. Tflf 1—3. We therefore accept
that Dr. Oliver is an expert, if an interested one, in this field of endeavor and
is qualified to provide a probative opinion.
Dr. Oliver opines that:
At the time of invention, [sequencing by hybridization]
would have been constrained in the length of sequence that could
be determined because of sequence repeats. This issue has been
evident since the mid-1990[ ]s. As one example of the inherent
deficiencies of [sequencing by hybridization], repeated
sequences would limit the reconstructable sequence length
resulting from SBH with a library of 8-mers (i.e., 65,536
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different oligonucleotides) constructed from cognate bases to
less than 256 bases.
[ ] I am not aware of anyone, at the time of the invention
claimed in the '189 application, using nanopores to provide
positional information during [sequencing by hybridization] in
order to circumvent the limitations of [sequencing by
hybridization],
[ ] The addition of positional information allows one to
sequence targets of any length from the same library of 8-mers.
Oliver Decl. ^fl[ 6—8.
We accord Dr. Oliver’s Declaration due evidentiary weight, but we do
not find it sufficiently probative of nonobviousness to overcome the
Examiner’s prima facie conclusion. As we have explained supra, Branton
teaches detection of the location of hybridized probes to a single stranded
nucleic acid via nanopore translocation. See Branton 120. Khrapko and
Hannenhalli teach hybridizing by sequence, by hybridizing probes of known
sequences to single stranded nucleic acids. See, e.g., Khrapko 119. Dr.
Oliver points to no evidence to suggest that any results provided by
Appellants’ Specification would be unexpected in view of the teachings of
the combined cited prior art. Moreover, we agree with the Examiner’s
finding that the limitations of the technique described in the Oliver
Declaration are not commensurate with the scope of the claims, which do
not require any minimum target length, or any particular probe library. See
Ans. 6.
Finally, Dr. Oliver attests: “I am not aware of anyone, at the time of
the invention claimed in the ’ 189 application, using nanopores to provide
positional information during SBH in order to circumvent the limitations of
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SBH.” Oliver Dec. 17. However, that is not determinative, or even
probative, of obviousness. Rather, the proper test for obviousness: “is what
the combined teachings of the references would have suggested to those of
ordinary skill in the art.” Keller, 642 F.2d at 425. In the appeal before us,
and for the reasons we have explained supra, we agree with the Examiner
that the combined teachings of the cited prior art would have obviously
suggested Appellants’ claimed invention.
We consequently affirm the Examiner’s rejection of the claims.
DECISION
The Examiner’s rejection of claims 1, 3—14, 16—23, and 25—28 as
unpatentable under 35 U.S.C. § 103(a) is affirmed.
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a)(1). See 37 C.F.R.
§ 1.136(a)(l)(iv).
AFFIRMED
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