Ex Parte Haefner et al

Patent Trial and Appeal Board

Feb 28, 2017

13466656 (P.T.A.B. Feb. 28, 2017)

United States Patent and Trademark Office
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
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APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
13/466,656 05/08/2012 Stefan Haefner 213218-0001-US-287396 1573
123223 7590 03/02/2017
Drinker Biddle & Reath LLP (WM)
222 Delaware Avenue, Ste. 1410
Wilmington, DE 19801-1621
EXAMINER
PRIEST, AARON A
ART UNIT PAPER NUMBER
1637
NOTIFICATION DATE DELIVERY MODE
03/02/2017 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):
IPDocketWM @ dbr.com
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PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte STEFAN HAEFNER, ULRICH SCHWANEBERG,
JAN MARIENHAGEN, ALEXANDER DENNIG, and
AMOL V. SHIVANGE1
Appeal 2016-002968
Application 13/466,656
Technology Center 1600
Before JEFFREY N. FREDMAN, ROBERT A. POLLOCK, and
DEVON ZASTROW NEWMAN, Administrative Patent Judges.
POLLOCK, Administrative Patent Judge.
DECISION ON APPEAL
Appellants appeal under 35 U.S.C. § 134(a) from the final rejection of
claims 1—14, 16, and 17. We have jurisdiction under 35 U.S.C. § 6(b).
We affirm and enter a new ground of rejection with respect to claim
14.
STATEMENT OF THE CASE
Appellants’ invention relates to “methods involving the joining of
nucleic acids and uses of such joined nucleic acids, for example for creating
1 Appellants identify the real party-in-interest as RWTH Aachen University.
App. Br. 2.
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Application 13/466,656
transformed microorganisms.” Spec. 1, 13—15. In general, such methods
comprise:
a) providing a set of at least three nucleic acid fragments to
be joined, wherein the nucleic acid fragments comprise
pairwise compatible ends, and wherein at least two of the
nucleic acid fragments are double stranded at least in the region
of the respective compatible end, and wherein each counter­
strand of the respective compatible end comprises nucleotides
configured for nonenzymatic removal,
b) non-enzymatically removing of such nucleotides
configured for non-enzymatic removal [and],
c) mixing of the nucleic acid fragments under hybridizing
conditions.
Id. at 2:19—3:3. The above recited “nucleotides configured for non­
enzymatic removal” may be introduced into the nucleic acids to be joined as
primers in a PCR reaction. Id. at 17:8—15. The primers may comprise “a
modified phosphodiester bond . . . wherein phosphorothioates are most
preferred.” Id. at 9:6—12. After incorporation into the nucleic acid
fragments by PCR, the “nucleotides comprising a phosphorothioate group
are preferably removed non-enzymatically by exposure to elementary iodine
and a molar surplus of an alcohol,” thereby producing terminal, single-
stranded regions available for inter-strand hybridization and subsequent
transformation into competent cells. Id. at 2:4—12, 21:3—5, 22:24—23:20,
33:26—34: 34:27—35:23. The resulting nucleic acid fragments may be
mixed in a single mixture kept at 4 °C to ensure that no
significant spontaneous separation of hybridized fragments will
occur. After mixing of all fragments, the mixture is heated to
break up hybridizations of the fragments, preferably by raising
the temperature of the mixture to 70-80 °C for 3 minutes. If
lower temperatures are employed, more time is required for
breaking up the hybridization of fragments, if higher
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temperatures are employed, spontaneous degradation of
fragments can occur. After heating, the mixture temperature is
lowered to the hybridization temperature as indicated above,
and the mixture is maintained at that temperature for a
preselected time, preferably for 5 min at 20 °C. Surprisingly,
with such mixing, heating and hybridizing protocol no
preferential hybridization of fragments occurs. This is very
advantageous, as the method of the present invention thus
allows to prepare a fair mixture of joined fragments.
Id. at 18:25-19:8.
The Specification describes the invention with reference to the
phosphorothioate-based ligase-independent gene cloning (PLICing) method
of Blanusa et al.2 See e.g., 33:25—34:10, 35:1—23, 39:4—9, 42:1—3, 44:9—12.
According to the Specification, although Blanusa “describe [s] a
phosphorothioate-based ligase independent gene cloning method .... [that]
relies on the nonenzymatic removal of phosphorothioate modified
nucleotides to generate compatible sticky ends of a vector and an insert........
[Blanusa] does not disclose or suggest that three or more fragments could be
joined and transformed with high efficiency to create a cloned library of
different nucleic acids.” Id. at 21:24—22:1.
Claims 1—13 and 16—17 stand rejected under 35 U.S.C. § 103(a) as
unpatentable over Blanusa and Church,3 as evidenced by Elledge,4 Li,5
2 Blanusa et al., “Phosphorothioate-based ligase-independent gene cloning
(PLICing): An enzyme-free and sequence-independent cloning method,” 406
Anal. Biochem. 141^46 (2010).
3 Church et al., US 2006/0281113 Al, published Dec. 14, 2006.
4 Elledge, US 2007 /0292954 Al, published Dec. 20, 2007.
5 Li and Elledge, Harnessing homologous recombination in vitro to generate
recombinant DNA via SLIC, 4(3) Nature Methods 251—56 (2007).
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Quan,6 Nour-Eldin,7 and Clontech.8
Claim 14 stands rejected under 35 U.S.C. § 102(b) as anticipated by
Church.9
ANALYSIS
Claims 1—13 and 16—17
The Examiner rejects claims 1—13 and 16—17 under 35 U.S.C.
§ 103(a) as unpatentable over Blanusa and Church, as evidenced by Elledge,
Li, Quan, Nour-Eldin, and Clontech. Appellants do not argue the claims
separately. Claim 1 is representative:
1. A nucleic acid joining method, comprising the steps of:
a) providing a set of at least three nucleic acid fragments to
be joined, wherein the nucleic acid fragments comprise
pairwise compatible ends, and wherein at least two of the
nucleic acid fragments are double stranded at least in the
region of the respective compatible end, and wherein each
counter-strand of the respective compatible end comprises
nucleotides configured for non-enzymatic removal,
b) non-enzymatically removing the nucleotides configured
for non-enzymatic removal, and
c) mixing the nucleic acid fragments under hybridizing
conditions so that the nucleic acid fragments are joined by
6 Quan and Tian, Circular Polymerase Extension Cloning of Complex Gene
Libraries and Pathways, 4(7) e6441 PLOS One 1—6 (2009).
7 Nour-Eldin et al., USER Cloning and USER Fusion: The Ideal Cloning
Techniques for Small and Big Laboratories, 643 Plant Secondary
Metabolism Engineering; Methods in Molecular Biology 185-200
(2010).
8 In-Fusion ® Multiple Fragment Cloning manual, Clonetch Labs. Inc.,
(2013).
9 The Examiner has withdrawn the anticipation rejection of claims 1—13 and
16—17 over Church. See Ans. 12.
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hybridization, wherein the method does not comprise a step
of ligation;
wherein the process comprises mixing the fragments
obtained in step b) in a single mixture at 4 °C, heating the
mixture by raising the temperature of the mixture to 70—80°C
for 3 minutes, and lowering the mixture temperature to the
hybridizing conditions.
We have reviewed Appellants’ contentions that the Examiner erred in
rejecting claims 1—13 and 16—17 as unpatentable over the cited art. (App.
Br. 5—15; Reply 4—8.) With respect to this rejection, we disagree with
Appellants’ contentions and adopt the findings and analysis set forth in the
Examiner’s Answer and Final Rejection dated December 2, 2014 (“Fin.
Rej.”). For emphasis, we highlight and address the following:
Blanusa discloses a “phosphorothioate-based ligase-independent gene
cloning (PFICing) method, which involves a chemical cleavage reaction of
phosphorothioate bonds in an iodine/ethanol solution. Blanusa, Abstract.
“PFICing starts with amplification of the target gene and the vector by PCR
using primers with complementary phosphorothioated nucleotides at the 5 ’
end (Fig. 2B). The PCR products are cleaved in an iodine/ethanol solution,
producing single-stranded overhangs. Subsequently, these ends hybridize at
room temperature (RT), and the resulting DNA constructs can be directly
transformed into competent host cells.” Id. at 142; see also Blanusa Figure 2
(illustrating steps).
With respect to the iodine/ethanol cleavage step, “[sjamples [of the
PCR reaction] were cleaved (70 °C, 5 min), mixed in a 1:1 ratio, and
incubated (RT, 5 min). Then 2 pi of this reaction was transformed using 40
pi of chemically competent E.coli XFIO-Gold cells.” Id. at 144; see id. at
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143 (“Samples were incubated (70 °C, 5 min, Eppendorf MasterCycler) and
subsequently kept on ice until further use.”).
As noted by the Examiner, “[t]he only differences between Blanusa
and the instant claims are that Blanusa does not explicitly teach: (1) the first
temperature of 4 °C; (2) 5 minutes at 70 °C; and (3) three inserts. However,
all of these techniques were routine and/or routinely optimized.” (Ans. 13).
With respect to the time and temperature limitations of claim 1, the
Examiner reasonably finds that:
As to the 4 °C mixing step, Applicants have merely mixed
the reagents on ice/4 °C to avoid premature reactions
(presumably performed in Blanusa but not explicitly stated
because this is routinely performed as seen at pg. 143, col. 2
explaining that reactions were kept on ice until further use),
then optimized the 5 minutes at 70°C of Blanusa to 3
minutes. Absent secondary evidence, this is prima facie
obvious.
Id. at 14—15; see also id. at 16—17 (routine to use low temperature to avoid
unwanted reactions; “4 °C is a result-effective variable routinely applied in
the art during mixing stages such as the mixing stages in Blanusa.”).
With respect to the multiplicity of inserts, the Examiner relies on
Church, Li, Quan, Nour-Eldin, Sleight, and Clontech as disclosing multi­
insert “ligation-free cloning methods using sticky-end hybridization.” Id. at
8—12; Fin. Rej. 7. The Examiner points, in particular, to Church’s disclosure
of 5—8 insert reactions for “multi-insert ligation-free cloning using non-
enzymatic nucleotide removal.” Ans. 8; see Church || 28, 29, Figures 1—10.
Accordingly, the Examiner finds that it would have been obvious to
“to apply the temperatures for cloning with other similar cloning methods
such as Blanusa, and optimize such temperature conditions based on the
teachings of Blanusa; and to use 5-8 inserts with a reasonable expectation of
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success as demonstrated by CHURCH and Blanusa, as evidenced by
ELLEDGE, Li, Quan, Naur-Eldin and Clontech.” Id. at 11—12.
Appellants contend that a person of ordinary skill reading Church and
Blanusa, “would have had no reasonable expectation of success in
performing a hybridization process in order to join more than three nucleic
acid fragments where at last two of the nucleic acid fragments are double
stranded at least in the region of the respective compatible ends, as required
by the claims” because “[i]t is no simple, obvious feat to be able to produce
a new ligation-free technique which requires at least three nucleic acid
segments.” Reply Br. 6. In support, Appellants reference several articles
purporting to show that “[t]he frequency of successful ligation and
transformation events significantly decreases with an increasing number of
fragments to be attached.” Id. at n. 1. To the extent this may be true,
Appellants vigorously argue that the instant claims do not require a ligation
step. See Appeal Br. 10-11. Moreover, the claims, on their face, do not
require transformation events—let alone at any particular frequency. This
is not persuasive because the claims require that “the nucleic acid fragments
are joined by hybridization.” See claim 1. Moreover, Appellants do not
argue, nor do we perceive, that the claims require any particular efficiency
of hybridization. Accordingly, we do not find Appellants’ argument
persuasive.
Appellants also argue that the rejection is in error because claim 1
requires that the method “does not comprise a step of ligation,” whereas
Church “expressly requires a ligation step.” App. Br. 10-13. We note, first,
that claim 1 is directed to “a nucleic acid joining method . . . wherein the
method does not comprise a step of ligation,” and results in “nucleic acids
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joined by hybridization.” Nothing in the claim language, however, prohibits
a ligation step after the nucleic acids are joined by hybridization, i.e.,
subsequent to performance of the recited method.
We further note that the primary reference, Blanusa, does not include
a ligation step (see Ans. 4), whereas the Examiner relies on Church (as well
as Li, Quan, Nour-Eldin, Sleight, and Clontech) as disclosing multi-insert
“ligation-free cloning methods using sticky-end hybridization.” Id. at 8—12;
Fin. Rej. 7. To the extent that Church, in particular, may include a ligation
step, Appellant does not convince us that such a feature would render the
rejection improper. See In re Keller, 642 F.2d 413, 425 (CCPA 1981) (“The
test for obviousness is not whether the features of a secondary reference may
be bodily incorporated into the structure of the primary reference; nor is it
that the claimed invention must be expressly suggested in any one or all of
the references. Rather, the test is what the combined teachings of the
references would have suggested to those of ordinary skill in the art”.); see
also In re Merck & Co., Inc., 800 F.2d 1091, 1097 (Fed. Cir. 1986) (“Non­
obviousness cannot be established by attacking references individually
where the rejection is based upon the teachings of a combination of
references. . . . [The reference] must be read, not in isolation, but for what it
fairly teaches in combination with the prior art as a whole.”).
Appellants further argue that the cited references do not teach or
suggest the time and temperature requirements of claim 1. App. Br. 13—15.
In support, Appellants provide only the conclusory statement that
the specific process steps were found to be critical to the
claimed process, which would not have been expected by a
person of ordinary skill in the art based on the cited
references. In particular, “[b]y adjusting the process to the
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specific conditions being described, it is possible to easily
manipulate nucleic acid sequences in order to mutate proteins
such as enzymes.”
Id. at 14 (citing Rule 1.132 Declaration ofUwe Pressler, dated October 10,
2014,116). In the absence of further argument10 or evidence, we agree with
the Examiner that Appellants have failed to provide objective evidence
demonstrating the criticality of the claimed temperatures and time. Ans. 15-
lb (citing In re Geisler, 116 F.3d 1465, 1469-70 (Fed. Cir. 1997) (“When an
applicant seeks to overcome a prima facie case of obviousness by showing
improved performance in a range that is within or overlaps with a range
disclosed in the prior art, the applicant must ‘show that the claimed range is
critical, generally by showing that the claimed range achieves unexpected
results relative to the prior art range.’”) (Internal brackets removed).
In light of the above, we sustain the rejection.
Claim 14
The Examiner rejects claim 14 under 35 U.S.C. § 102(b) as
anticipated by Church. Claim 14 recites:
14. A kit for joining at least three nucleic acid fragments,
comprising:
10 Appellants also appear to argue that the cited art fails to disclose the
claimed rate of increase and/or decrease in temperature. See App. Br. 13—
14; Reply Br. 7—8. As noted by the Examiner, however, such limitations are
not found in the claim language. See Ans. 16; see also Super Guide Corp. v.
DirecTV Enters., Inc., 358 F.3d 870, 875 (Fed. Cir. 2004). (“Though
understanding the claim language may be aided by the explanations
contained in the written description, it is important not to import into a claim
limitations that are not a part of the claim.”).
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for each nucleic acid fragment, two amplification primers
having nucleotides configured for non-enzymatic removal,
and
one or more non-enzymatic nucleotide removal agents.
In setting forth the rejection, the Examiner relies on paragraphs 174
and 199 of Church. See Ans. 3. Appellants contend that the rejection is
improper for picking and choosing from various disclosures in paragraphs
174 and 199 of the reference. See Reply Br. 1—2 (citing In re Arkley, 455
F.2d 586, 587 (CCPA 1972)). Because we find that Appellants have the
better argument, we reverse the rejection.
Church discloses methods for joining two or more nucleic acid
fragments having complementary single-stranded overhangs. See, e.g.,
Church || 18, 24, 27—29, 35, 90, and 174; Figures 10-12. Paragraph 199 of
the reference discloses kits for joining these nucleic acid fragments
comprising, for each nucleic acid fragment, two amplification primers and
one or more chemical reagents. Paragraph 174, and accompanying Figure
11, disclose that the primers used to create the complementary single-
stranded overhangs may comprise at least one phosphorothioate
intemucleoside linkage, which may be non-enzymatically removed using an
alkylating reagent such as 2-iodoethanol.
In light of Church, it would have been obvious to provide a kit for
joining at least three nucleic acid fragments, comprising, for each nucleic
acid fragment, two amplification primers having nucleotides configured for
non-enzymatic removal, and one or more non-enzymatic nucleotide removal
agents, as set forth in claim 14. One of ordinary skill in the art would have
been motivated by the convenience of having the necessary reagents
collected in a kit, and would have had a reasonable expectation of success in
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arriving at the claimed invention because Church taught that the individual
components were known, as was the assembly of kits comprising
components for joining a multiplicity of nucleic acid fragments.
In view of the above, we enter the new ground of rejection of claim 14
under 35 U.S.C. § 103 in view of Church. 37 C.F.R. § 41.50(b).
SUMMARY
I. We affirm the rejection of claims 1—13 and 16—17 under 35 U.S.C.
§ 103(a) as unpatentable over Blanusa and Church, as evidenced by
Elledge, Li, Quan, Nour-Eldin, and Clontech.
II. We reverse the rejection of claim 14 as unpatentable under 35 U.S.C.
§ 102 in view of Church.
III. We enter a new ground of rejection of claim 14 as unpatentable under
35 U.S.C. § 103 in view of Church pursuant to 37 C.F.R. § 41.50(b).
TIME PERIOD FOR RESPONSE
This decision contains a new ground of rejection pursuant to
37 C.F.R. § 41.50(b) (effective September 13, 2004, 69 Fed. Reg. 49960
(August 12, 2004), 1286 Off. Gaz. Pat. Office 21 (September 7, 2004)).
37 C.F.R. § 41.50(b) provides “[a] new ground of rejection pursuant to this
paragraph shall not be considered final for judicial review.
37 C.F.R. § 41.50(b) also provides that the appellant, WITHIN TWO
MONTHS FROM THE DATE OF THE DECISION, must exercise one of
the following two options with respect to the new ground of rejection to
avoid termination of the appeal as to the rejected claims:
(1) Reopen prosecution. Submit an appropriate
amendment of the claims so rejected or new evidence relating
to the claims so rejected, or both, and have the matter
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reconsidered by the examiner, in which event the proceeding
will be remanded to the examiner . . .
(2) Request rehearing. Request that the proceeding be
reheard under § 41.52 by the Board upon the same record . . .
Further guidance on responding to a new ground of rejection can be
found in the Manual of Patent Examining Procedure§ 1214.01.
AFFIRMED; 37 C.F.R, $ 41.50(b)
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