Ex Parte Chee et al

Board of Patent Appeals and Interferences

Sep 17, 2009

09513362 (B.P.A.I. Sep. 17, 2009)

UNITED STATES PATENT AND TRADEMARK OFFICE
____________________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
____________________

Ex parte MARK S. CHEE
and
JOHN R. STUELPNAGEL,
Appellants
____________________

Appeal 2009-000287
Application 09/513,3621
Technology Center 1600
____________________

Decided: September 17, 2009
____________________

Before CAROL A. SPIEGEL, ROMULO H. DELMENDO, and
RICHARD M. LEBOVITZ, Administrative Patent Judges.

SPIEGEL, Administrative Patent Judge.

DECISION ON APPEAL

1 Application 09/513,362 ("the 362 Application"), Nucleic Acid Sequencing Using
Microsphere Arrays, was filed 25 February 2000 and claims benefit under 35
U.S.C. § 119 of the 22 October 1999 filing date of provisional applications
60/160,917 and 60/161,148, of the 18 October 1999 filing date of provisional
application 60/160,027, of the 20 May 1999 filing date of provisional applications
60/135,123, 60/135,053, and 60/135,051, and the 20 April 1999 filing date of
provisional application 60/130,089. The real party in interest is ILLUMINA, INC.
(Appeal Brief filed 14 December 2007 ("App. Br.") at 1).
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I. Statement of the Case
Appellants appeal under 35 U.S.C. § 134 from an Examiner's final rejection
of all pending claims, claims 1-38 and 40-50 (App. Br. 3). An oral hearing was
held May 20, 2009. We have jurisdiction under 35 U.S.C. §§ 6(b) and 134. We
AFFIRM.
The subject matter on appeal is directed to microsphere or bead array based
pyrosequencing methods and kits therefor. Claims 10 and 18 are illustrative and
read (App. Br. Claims App'x. 28-30).
10. A method of sequencing a plurality of target
nucleic acids each comprising a first domain and an
adjacent second domain, said second domain comprising a
plurality of target positions, said method comprising:
a) providing [a] first hybridization complex
comprising a first target sequence and a first sequencing
primer that will hybridize to the first domain of said first
target sequence,
b) providing a second hybridization complex
comprising a second target sequence and a second
sequencing primer that will hybridize to the second
domain of said second target sequence,
wherein said first and second sequencing primers
are covalently attached to microspheres distributed at
discrete sites on a surface of a substrate, said discrete sites
having an attached enzyme used to generate a signal from
pyrophosphate;
c) determining the identity of a plurality of
bases at said target positions, wherein said determining
comprises simultaneously extending said first and second
sequencing primers by the addition of a first nucleotide to
a first detection position using a first enzyme to form first
and second extended primers, respectively; and
d) detecting the release of pyrophosphate (PPi)
with said enzyme attached at said discrete sites within a
common reaction chamber of said simultaneous
extensions to determine the type of said first nucleotide
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added onto said first and second sequencing primers,
respectively.

18. A kit for nucleic acid sequencing comprising:
a) a composition comprising:
i) a substrate with a surface comprising
discrete sites;
ii) a population of microspheres
distributed on said sites; wherein said
microspheres comprise different
capture probes, wherein said array is
configured for simultaneous contact of said
different capture probes with a common
reaction chamber; and
iii) an enzyme attached at said discrete
sites, wherein said enzyme is used to
generate a signal from pyrophosphate;
b) a first extension enzyme; and
c) dNTPs.
The Examiner has finally rejected the pending claims as unpatentable under
35 U.S.C. § 103(a) as follows:
(i) claims 1-16, 22-27, 31-38, 40-42, 44-47, 49, and 50 over Rothberg2 and
Walt3 (FR4 5-12; Ans.5 4-11);
(ii) claim 17 over Rothberg, Walt, and Nyren6 (FR 12; Ans. 11-12);

2 U.S. Patent 6,274,320 B1, Method of Sequencing a Nucleic Acid, issued 14
August 2001, based on application 09/398,833, filed 16 September 1999, to
Rothberg et al. ("Rothberg").
3 U.S. Patent 6,327,410 B1, Target Analyte Sensors Utilizing Microspheres, issued
4 December 2001, based on application 09/151,877, filed 11 September 1998, to
Walt et al. ("Walt").
4 Final Office Action mailed 30 May 2007 ("FR").
5 Examiner's Answer mailed 17 March 2008 ("Ans.").
6 International patent application WO 98/13523, Method of Sequencing DNA,
published 2 April 1998 by Nyren et al. ("Nyren").
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(iii) claims 18, 19, 28-30, 43, and 48 over Rothberg, Walt, Nyren, and
Stratagene7 (FR 12-16; Ans. 12-16); and,
(iv) claims 20 and 21 over Rothberg, Walt, Nyren, Stratagene, and Ross8
(FR 16-17; Ans. 16-17). The Examiner also relies on Michael9 and Ronaghi10 as
evidence of unpatentability (Ans. 2-3, 20, and 22).
According to the Examiner, Rothberg teaches attaching reactants of a
pyrosequencing method to the surface of a fiber optic bundle, but does not teach
using microspheres to attach the reactants (Ans. 4-9). According to the Examiner,
Walt teaches microsphere-based analytical chemistry systems comprising optically
identifiable populations of microspheres carrying bioactive agents which are
randomly distributed on a fiber optic bundle or array and using the array for
sequencing (Ans. 9-10). The Examiner concluded that it would have been obvious
to combine the microspheres of Walt with the fiber optic bundle used in Rothberg's
sequencing method to allow the generation of large fiber optic arrays comprising
randomly distributed microspheres that can be encoded or decoded in order to
provide a fast and inexpensive method compared to either the in situ synthesis or
spotting techniques of the prior art (Ans. 10-11).
Appellants acknowledge that Walt describes the use of beads on a fiber optic
surface but argue that, absent any articulated reason for using the beads in an array
for the pyrosequencing method of Rothberg, the Examiner has failed to establish a

7 STRATAGENE 1988 CATALOG, "Gene Characterization Kits," 39
("Stratagene").
8 International patent application WO 91/06678, DNA Sequencing, published 16
May 1991 by Ross et al. ("Ross").
9 Michael et al., "Randomly Ordered Addressable High-Density Optical Sensor
Arrays," 70 Analytical Chemistry 1242-48 (1998) ("Michael").
10 Ronaghi et al., "Real-Time DNA Sequencing Using Detection of Pyrophosphate
Release," 242 Analytical Chemistry 84-89 (1996).
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prima facie case of obviousness (App. Br. 14-15; Reply Br.11 1-3). Appellants
further argue that Rothberg teaches away from bead usage, e.g., bead loss during
washing limited the ability to sequence longer stretches of nucleic acid (App. Br.
15-17; Reply Br. 3-6). Appellants rely on the prosecution history of a
continuation-in-part application of Rothberg, i.e., application 09/814,338 ("the
Rothberg CIP application") to support their position that Rothberg not only
believed that bead loss limited previous pyrosequencing methods, but also had to
physically alter its substrate to allow for the use of beads (App. Br. 17-21; Reply
Br. 6-7). In particular, Appellants rely on the following exhibits made of record in
its March 8, 2007 response: the Rothberg CIP application,12 Rothberg CIP Notice
of Allowability,13 Rothberg CIP Amendment and Response,14 Margulies
Declaration I,15 and Margulies Declaration II.16
Thus, at issue is whether Appellants have shown that the Examiner failed to
articulate a reason for combining the optically identifiable microspheres of Walt
with the pyrophosphate sequencing method of Rothberg and/or that Rothberg
teaches away from such a combination.

11 Reply Brief to Examiner's Answer filed 19 May 2008 ("Reply Br.").
12 U.S. application 09/814,338, Method of Sequencing a Nucleic Acid, filed 21
March 2001, now U.S. Patent 7,244,559 B2, issued 17 July 2007, to Rothberg et al.
("the Rothberg CIP application"), which Appellants filed as Exhibit A.
13 Notice of Allowability of application 09/814,338 mailed 7 February 2007
("Rothberg CIP Notice of Allowability"), which Appellants filed as Exhibit B.
14 Amendment and Response to the Non-Final Office Action Dated November 6,
2003 filed in the Rothberg CIP application on 23 April 2004 ("Rothberg CIP
Amendment and Response"), which Appellants filed as Exhibit C.
15 Declaration of Marcel Margulies, Ph.D., Under 37 C.F.R. § 1.132, dated 20 April
2004 ("Margulies Decl. I"), which Appellants filed as Exhibit D.
16 Declaration of Marcel Margulies Under 37 C.F.R. § 1.132, dated 22 December
2006 ("Margulies Decl. II"), which Appellants filed as Exhibit E.
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Appellants have presented a single set of patentability arguments.
Therefore, we decide this appeal on the basis of claim 10. 37 C.F.R.
§ 41.37(c)(1)(vii).
II. Findings of Fact ("FF")
The following findings of fact are supported by a preponderance of the
evidence of record.
A. The 362 application ("Spec.")
[1] According to the "Background of the Invention" section of the 362
application (Spec. 1:21-3:2), a pyrophosphate (PPi) is released during a
DNA polymerase reaction as a nucleotriphosphate (dNTP) is added a
growing nucleic acid chain (Spec. 2:14-16).
[2] Several known DNA sequencing systems, including Nyren, are based on
detecting base (A, G, C, T) incorporation by measuring released PPi (Spec.
2:14-20).
[3] In one known method, for example,
the four deoxynucleotides (dATP, dGTP, dCTP and
dTTP; collectively dNTPs) are added stepwise to a
partial duplex comprising a sequencing primer
hybridized to a single stranded DNA template and
incubated with DNA polymerase, ATP sulfurylase,
luciferase, and optionally a nucleotide-degrading enzyme
such as apyrase. A dNTP is only incorporated into the
growing DNA strand if it is complementary to the base in
the template strand. The synthesis of DNA is
accompanied by the release of PPi equal in molarity to
the incorporated dNTP. The PPi is converted to ATP and
the light generated by the luciferase is directly
proportional to the amount of ATP. In some cases the
unincorporated dNTPs and the produced ATP are
degraded between each cycle by the nucleotide degrading
enzyme. [Spec. 2:21-29.]
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[4] The invention of the 362 application is characterized as directed to methods
using the detection of PPi generated during nucleic acid synthesis
(pyrosequencing), particularly to methods using microsphere arrays (Spec.
1:17-19).
[5] The 362 application discloses several ways to attach a DNA template (target
nucleic acid) to the surface of an array, such as an array formed on the
surface of a biosensor (Spec. 1:18-19; 2, (as amended) ¶ 2). Figure 1B, for
example, depicts a target nucleic acid 25 comprising a first portion (domain
for which sequence information is not desired), which is hybridized to a
nucleotide sequence primer 20 (which also serves as a capture probe), and an
adjacent second portion containing nucleotide target positions 31, 32, 33,
and 34 for which sequencing is desired. The sequence primer 20 is tethered
to a microsphere 10 by means of a linker 15. The microsphere 20 is in a
well (site) in the surface of a substrate 5 of an array. [Spec. amended 2, ¶ 2;
9:4-6.] Figure 1B is reproduced below.

{Figure 1B of the 362 application depicts one configuration for
attaching a target sequence to a discrete site (well) on an array
surface.}
[6] In one embodiment, the substrate, comprising microspheres with attached
hybridization complexes of target nucleic acids and primers, is dipped or
contacted with a reaction chamber containing a single type of dNTP, an
extension enzyme, such as DNA polymerase, and the reagents and enzymes
necessary to detect PPi (Spec. 10:19-21; 10:37-11:1; 12:13-16).
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[7] If the dNTP is complementary to the base of the target position of the target
nucleic acid, the dNTP is added (extending the sequence primer as a
growing nucleic acid chain), thereby releasing PPi and generating detectable
light. If the dNTP is not complementary to the base at the target position, no
PPi is released and no light is generated. The process is repeated to obtain
the sequence of the target position(s) on the target nucleic acid. (Spec.
12:16-23.)
[8] Preferably, the reaction chamber is washed between dNTP additions to
remove unreacted dNTP and decrease background signal (Spec. 12:25-30).
[9] In a preferred embodiment, the enzymes used to detect PPi (the secondary or
signal generating enzymes) are attached to sites on the array, e.g., the
microspheres, preferably using flexible linkers (Spec. 14:34-15:2).
[10] Attaching the signal generating enzymes to the sites on the array is said to
increase the concentration of the enzymes in the vicinity of the PPi detection
reaction, thereby allowing use of less enzyme and faster reaction rates for
detection (Spec. 15:2-4).
[11] In a preferred embodiment, the substrate is an optical fiber bundle or array
(Spec. 24:18).
[12] According to the 362 application, at least one surface of the substrate is
modified to contain discrete, individual sites for later association of
microspheres such that the microspheres do not move during the course of
the assay. The sites may be physically altered sites, e.g., wells or
depressions, or chemically altered sites, e.g., spots of adhesive and sites with
functional groups, such as amino, carboxy, oxo, and thiol groups, for
covalently attaching microspheres. (Spec. 24:27-32; 25:28-26:8; 26:31-33.)
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[13] When microsphere arrays are used, an encoding/decoding system must be
used to correlate a functionality on the bead with its location since the beads
are generally distributed randomly onto the substrate (Spec. 30:4-8).
[14] For example, a coding/decoding system may use a decoding binding ligand
(DBL), generally directly labeled with an optical signature such as a
fluorescent dye, that binds to either the capture probe or to identifier binding
ligands (IBLs) attached to the beads (Spec. 30:22-30).
[15] The coding/decoding system allows nucleic acid synthesis to occur on the
beads after random distribution on an array, thereby providing a faster and
less expensive process as compared to either the in situ synthesis or spotting
techniques of the prior art. After sequencing, the random array is decoded to
determine the functionality of each bead (Spec. 30:8-22).
B. Rothberg
[16] Rothberg discloses a method of determining the sequences of target nucleic
acid sequences (templates) attached to a solid substrate such as optical fiber
bundle surfaces, wherein one or more of the surfaces have an array of
anchored primers (capture probes) (Rothberg 2:62-3:6; 3:41-45; 5:6-16;
6:62-66).
[17] In some embodiments, the anchor primers are linked directly to the termini
of the optical fibers which have been etched to form cavities therein which
are, preferably, one-half to three times the diameter of the optic fiber
(Rothberg 20:15-24).
[18] Rothberg also discloses a sequencing apparatus comprising, in one
embodiment, (i) a perfusion chamber including a sealed compartment
designed to allow laminar flow of solution, e.g., reagents and washes, over a
substrate, such as a wafer formed from the termini of the optical fibers, (b)
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the substrate wafer, and (c) a CCD imaging system (Rothberg 19:26-20:13;
Figures 2 and 3).
[19] In use, an anchor primer is annealed to at least one nucleic acid template to
form a hybridization complex and then a sequencing primer is annealed to
the template and extended with a polymerase and a predetermined dNTP. If
the predetermined dNTP is incorporated into the primer, PPi is generated
and determined, e.g., by enzymatic conversion to detectable light using
sulfurylase and luciferase. Additional predetermined dNTPs are added
sequentially and the presence or absence of PPi generation associated with
each synthesis reaction is determined. (Rothberg 3:18-45; 14:13-41, 50-52,
64-66; 16:7-8, 13-16; 17:1-12, 24-36.) Sulfurylase can be utilized to
generate ATP with the liberated PPi; the thus produced ATP is converted to
light using enzymatic reactions, such as luciferase. (Id. at 14:64-66; 16:7-
20.)
[20] To increase the effective concentration of sulfurylase and to mitigate the
generation of background light, the enzymes used to detect PPi maybe
immobilized in the region of the nucleic acid template (Rothberg 27:58-62;
28:60-67).
[21] In addition to the above two tactics for optimizing the pyrosequencing
analysis, Rothberg also suggests detecting only early light, increasing the
concentration of sulfurylase in the reaction mixture, increasing the time it
takes for the PPi to diffuse out of the reaction area by adding a viscosity
increasing agent to the pyrosequencing reagents, and physically preventing
the PPi from diffusing laterally, e.g., by increasing the depth of the etched
cavities (Rothberg 27:46-52; 28:13-18, 24-41).
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[22] Rothberg also provided the following discussion of the historical
development of pyrophosphate sequencing:
Solid-phase pyrophosphate sequencing was
initially developed by combining a solid-phase
technology and a sequencing-by-synthesis technique
utilizing bioluminescence (see e.g., Ronaghi, et al., 1996,
Real-time DNA sequencing using detection of
pyrophosphate release. Anal. Biochem. 242: 84-89). In
the solid-phase methodology, an immobilized, primed
DNA strand is incubated with DNA polymerase, ATP
sulfurylase, and luciferase. By stepwise nucleotide
addition with intermediate washing, the event of
sequential polymerization can be followed. A
remarkable increase in signal-to-noise ratio was obtained
by the use of α-thio dATP in the system. This dATP
analog is demonstrated to be efficiently incorporated by
DNA polymerase while being silent for luciferase,
allowing the sequencing reaction to be performed in real-
time. In these early studies, sequencing of a PCR
product using streptavidin-coated magnetic beads as a
solid support was presented. However, it was found that
the loss of beads during washing, which was performed
between each nucleotide and enzyme addition, was the
limiting factor to sequence longer stretches. (Rothberg
21:14-34, emphasis added.)
C. Appellants' Evidentiary Exhibits from the prosecution of the Rothberg
CIP Application
[23] The Rothberg CIP application provided the same discussion of the historical
development of pyrophosphate sequencing quoted in fact 22 (Rothberg CIP
application 39:12-24).
[24] According to the Rothberg CIP Amendment and Response and the testimony
of Marcel Margulies, the Rothberg CIP application "expressly recognizes the
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problem of bead/sample loss during the sequencing reaction" (Rothberg CIP
Amendment and Response, p. 20, ll. 14-16; Margulies Decl. I ¶17).
[25] Marcel Margulies, Ph.D., testified on behalf of the exclusive licensee of the
Rothberg CIP application that the superior performance of the Rothberg CIP
application invention can be attributed to 1) compact wafers, 2) attachable
optical fibers, 3) flow chamber and fluid means, and 4) specific fiber and
well sizes (Margulies Decl. I ¶¶ 1, 2, 4).
[26] According to Dr. Margulies, continuous plunging of an optic fiber into
nucleotide solutions would tend to dislodge any beads from the wells,
whereas use of a flow chamber allows rapid and efficient delivery of
sequencing reagents and washes (Margulies Decl. I ¶ 12-14).
[27] Dr. Margulies testified that using wells with a depth of one-half to three
times the diameter of the optic fibers allows for more effective sequence
analysis (Margulies Decl. I ¶¶ 17-18).
[28] According to Dr. Margulies,
[w]ell depth is selected on the basis of a number of
competing requirements in a nucleic acid sequencing
application: (1) wells need to be deep enough for DNA-
carrying beads to remain in the wells in the presence of
convective transport past the wells; (2) the wells must be
sufficiently deep to provide adequate isolation against
diffusion of by-products from a well in which
incorporation is taking place to a well where no
incorporation is occurring; (3) they must be shallow
enough to allow rapid diffusion of nucleotides into the
wells and rapid washing out of remaining nucleotides at
the end of each flow cycle to enable high sequencing
throughput and reduced reagent use; and (4) they must
not be so deep that it would be easy for more than one
bead to fit in a well (Margulies Decl. II ¶8).
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[29] The Examiner's reasons for allowing the Rothberg CIP application are
reproduced in toto below:
The diameter range, the depth, and well-depth of
the claimed cavitated fiber optic wafer, said wafer being
formed from a fused bundle of a plurality of individual
optical fibers, are deemed non-obvious over the general
teaching provided for by Chee et al. (of record), based on
Margulies Declaration, as each of the above-mentioned
parameters are critical to the laminar flow of the reaction
reagents, allowing for rapid diffusion and washing out of
the nucleotides, enabling a high cycle sequencing (see
page 3 bottom paragraph to page 4, top paragraph;
Margulies Declaration).
Hence, the parameters are no longer deemed
arbitrary, not involving routine optimization, as the
parameters are specifically adapted to the way in which
the disclosed invention operates. [Rothberg CIP Notice
of Allowability p. 2, ¶¶ 1-2.]
D. Walt
[30] Walt describes an analytical system and method based on
1) the development of a bead-based analytic chemistry
system in which beads, also termed microspheres,
carrying different chemical functionalities may be mixed
together while the ability is retained to identify the
functionality of each bead using an optically
interrogatable encoding scheme (an "optical signature");
and 2) the use of a substrate comprising a patterned
surface containing individual sites that can bind or
associate individual beads. This allows the synthesis of
the bioactive agents (i.e. compounds such as nucleic
acids and antibodies) to be separated from their
placement on an array, i.e. the bioactive agents may be
synthesized on the beads, and then the beads are
randomly distributed on a patterned surface. Since the
beads are first coded with an optical signature, this means
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that the array can later be "decoded", i.e. after the array is
made, a correlation of the location of an individual site
on the array with the bead or bioactive agent at that
particular site can be made. This means that the beads
may be randomly distributed on the array, a fast and
inexpensive process as compared to either the in situ
synthesis or spotting techniques of the prior art. Once the
array is loaded with the beads, the array can be decoded,
or can be used, with full or partial decoding occurring
after testing . . . . (Walt 4:35-58.)
[31] Preferably, the substrate is a surface of a fiber optic bundle having one
terminal end of the individual core fibers have been modified with small
wells or cavities to provide discrete, individual sites for later association
with the microspheres (Walt 5:57-63; 7:61-66).
[32] Preferably, the optical signature is a mixture of fluorescent dyes (Walt
13:25-26).
[33] The sites may be physically altered sites, e.g., wells or depressions that can
retain the beads, or chemically altered sites, e.g., spots of adhesive and sites
with functional groups, e.g., amino, carboxy, oxo, and thiol groups (Walt
5:61-6:2; 6:48-7:10).
[34] "[A] key component . . . is the use of a substrate/bead pairing that allows the
association or attachment of the beads at discrete sites on the surface of the
substrate, such that the beads do not move during the course of the assay"
(Walt 7:50-54).
[35] For example, the beads are first distributed into the wells and subsequently
exposed to an aqueous buffer which causes the beads to swell, thereby
physically entrapping them in the wells (Walt 18:3-12). Alternatively,
photoactivable attachment linkers, adhesives, or masks could be used to fix
the beads inside the wells (id. 18:41-47).
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[36] In one embodiment, the probes to detect a nucleic acid are in an array and
used for sequencing by hybridization (Walt 21:25-28; 24:51-52).
E. Ronaghi
[37] Ronaghi is the reference cited by Rothberg in discussing early studies in
pyrophosphate sequencing (see FF 22).
[38] According to Ronaghi, "[t]he decrease in signal due to loss and aggregation
of beads during the washing procedure . . . has been compensated for"
(Ronaghi 87, col. 2, ¶1).
[39] Ronaghi suggests that immobilizing the DNA template in a capillary could
avoid the template loss observed for the paramagnetic beads (Ronaghi 88,
col. 2, ¶2).
A discussion of Nyren, Ross, and Stratagene is not necessary to our decision.
III. Discussion
In order to establish a prima facie case of obviousness, the Examiner must
show that each and every limitation of the claim is described or suggested by the
prior art or would have been obvious based on the knowledge of those of ordinary
skill in the art. In re Fine, 837 F.2d 1071, 1074 (Fed. Cir. 1988). Moreover,
"[n]onobviousness cannot be established by attacking references individually
where the rejection is based upon the teachings of a combination of references." In
re Merck & Co., Inc., 800 F.2d 1091, 1097 (Fed. Cir. 1986). It is also proper to
"take account of the inferences and creative steps that a person of ordinary skill in
the art would employ." KSR Int'l Co. v. Teleflex, Inc., 550 U.S. 398, 418 (2007).
See also id. at 421 ("A person of ordinary skill is also a person of ordinary
creativity, not an automaton."). Furthermore, it is well settled that optimization of
a result effective variable is within ordinary skill. In re Peterson, 315 F.3d 1325,
1330 (Fed. Cir. 2003).
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Here, the Examiner found that Rothberg describes every step of the
pyrosequencing method of claim 10 but using microspheres (Ans. 4-9). The
Examiner found that Walt teaches randomly distributing populations of
microspheres comprising a bioactive agent and an identifying optical signature on
the surface of a fiber optic array which can be used for sequencing analysis (Ans.
9-10). The Examiner concluded that it would have been obvious to combine the
microspheres of Walt with the fiber optic bundle used in Rothberg's sequencing
method to allow the generation of large fiber optic arrays comprising randomly
distributed microspheres that can be encoded or decoded in order to provide a fast
and inexpensive method compared to either the in situ synthesis or spotting
techniques of the prior art (Ans. 10-11). Thus, contrary to Appellants' argument
(App. Br. 14-15; Reply Br. 1-3), the Examiner expressly articulated a reason for
combining the teachings of Rothberg and Walt. Specifically, according to the
Examiner, "[t]he advantage of using beads of Walt . . . is that the system provides
an enormous flexibility and ease of use, as the fiber optic surface is simply filled
with a different set of beads if an assay needs to be changed, saving time and
money in building a sensor" (Ans. 19).
By way of emphasis, we note that Rothberg and Walt both relate to methods
of determining nucleic acid sequences (FF 16, 19, 36). Rothberg and Walt both
attach analytical reactants to the surface of fiber optic arrays (FF 16, 31) and both
conduct analytical reactions in wells or cavities formed in the termini of the optical
fibers (FF 17, 31). Walt teaches that since each population of its microspheres is
individually encoded with its own optical signature, various populations of
microspheres can be randomly distributed on the surface of the fiber optic array
and decoded after loading, thereby providing a fast and inexpensive process as
compared to either the in situ synthesis or spotting techniques of the prior art (FF
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30). It is these benefits offered by the microspheres of Walt that the Examiner
articulates as a reason for combining the teachings of Rothberg and Walt (Ans. 18-
19).
Furthermore, while there must be some articulated reasoning with some
rational underpinning to support the legal conclusion of obviousness, "the analysis
need not seek out precise teachings directed to the specific subject matter of the
challenged claim, for a court can take account of the inferences and creative steps
that a person of ordinary skill in the art would employ." KSR, 550 U.S. at 418.
When a work is available in one field of endeavor, design
incentives and other market forces can prompt variations
of it, either in the same field or a different one. If a
person of ordinary skill can implement a predictable
variation, § 103 likely bars its patentability. For the same
reason, if a technique has been used to improve one
device, a person of ordinary skill in the art would
recognize that it would improve similar devices in the
same way, using the technique is obvious unless its
actual application is beyond his or her skill. Id. at 417.
Thus, although Appellants complain that the Examiner did not articulate a reason
to combine pyrophosphate sequencing with beads (App. Br. 15; Reply Br. 2), a
precise teaching is unnecessary because it was well-established that pyrophosphate
sequencing was conventional (FF 2, 3, 19) and microspheres (beads) had been
suggested by Walt (FF 36) as suitable for carrying out sequencing. Therefore, in
this case, combining the microsphere-based analytical system of Walt with the
pyrosequencing method of Rothberg is no more than the predictable use of prior art
elements according to their established functions.
Appellants also argue that Rothberg teaches away from bead usage because
in early studies loss of beads during washing limited the ability to sequence longer
stretches of nucleic acid (App. Br. 15-17; Reply Br. 3-6) (FF 22). However,
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[a] reference may be said to teach away when a person of ordinary
skill, upon reading the reference, would be discouraged from
following the path set out in the reference, or would be led in a
direction divergent from the path that was taken by the applicant. The
degree of teaching away will of course depend on the particular facts;
in general, a reference will teach away if it suggests that the line of
development flowing from the reference’s disclosure is unlikely to be
productive of the result sought by the applicant.

In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994). Here, the Examiner contends that
(i) Ronaghi is discussing homogeneous assays where the microspheres are
suspended in solution, (ii) the claims are not limited to sequencing nucleic acids of
any particular length, (iii) Ronaghi suggests immobilizing the DNA template to
prevent it from being washed away, and (iv) Walt teaches a number of ways to
immobilize microspheres in the wells (Ans. 20-22).
While we do not find factual support for the Examiner's first contention in
the record before us, we do agree with the Examiner's remaining contentions. By
way of emphasis we note that the rejection is based on the combined teachings of
Rothberg and Walt, not just Rothberg. We also note that Walt not only expressly
uses substrate/bead pairings such that the beads do not move during the course of
the assay (FF 34), but also provides a number of ways by which the beads can be
covalently or non-covalently immobilized within the wells of the substrate (FF 35).
Appellants' rebuttal evidence drawn from the prosecution history of the Rothberg
CIP application does not convince us otherwise. At issue is what the combined
teachings of Rothberg and Walt would have suggested to one of ordinary skill in
the art, not to the inventors of the Rothberg CIP application. Allowance of the
Rothberg CIP application was based on a different record than the one before us.
For example, Dr. Margulies offered no opinion regarding the disclosure of Walt.
In addition, both Rothberg and the Rothberg CIP application provided ample,
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similar teachings on how to optimize assay parameters (compare FF 20-21 with FF
27-28). Finally, Appellants cite no authority holding that the Board is bound by
the patentability determinations of an Examiner, particularly on a different record
in a different case.
In conclusion, Appellants have not shown that the Examiner failed to
articulate a reason for combining the optically identifiable microspheres of Walt
with the pyrophosphate sequencing method of Rothberg or that Rothberg teaches
away from such a combination. Consequently, we sustain the rejection of claims
1-38 and 40-50 under § 103.
IV. Order
Upon consideration of the record, and for the reasons given, it is
ORDERED that the decision of the Examiner rejecting claims 1-16, 22-27,
31-38, 40-42, 44-47, 49, and 50 over Rothberg and Walt is AFFIRMED;
FURTHER ORDERED that the decision of the Examiner rejecting claim 17
over Rothberg, Walt, and Nyren is AFFIRMED;
FURTHER ORDERED that the decision of the Examiner rejecting claims
18, 19, 28-30, 43, and 48 over Rothberg, Walt, Nyren, and Stratagene is
AFFIRMED;
FURTHER ORDERED that the decision of the Examiner rejecting claims
20 and 21 over Rothberg, Walt, Nyren, Stratagene, and Ross is AFFIRMED; and,
FURTHER ORDERED that no time period for taking any subsequent action
in connection with this appeal may be extended under 37 C.F.R. § 1.136(a).
AFFIRMED

Appeal 2009-000287
Application 09/513,362

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