Ex Parte Xiao et al

Board of Patent Appeals and Interferences

Apr 6, 2010

10835140 (B.P.A.I. Apr. 6, 2010)

UNITED STATES PATENT AND TRADEMARK OFFICE
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BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
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Ex parte HAIGUANG XIAO and
SCOTT HAMILTON
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Appeal 2009-014792
Application 10/835,140
Technology Center 1600
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Decided: April 7, 2010
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Before ERIC GRIMES, FRANCISCO C. PRATS, and STEPHEN WALSH,
Administrative Patent Judges.

GRIMES, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to a nucleic
acid sequencing method, which the Examiner has rejected as obvious. We
have jurisdiction under 35 U.S.C. § 6(b). We affirm.

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Claims 1-3, 9, 11, and 12 are on appeal. The claims have not been
argued separately and therefore stand or fall together. 37 C.F.R.
§ 41.37(c)(1)(vii). Claim 1 is representative and reads as follows:
Claim 1: A method for sequencing nucleic acid molecules,
comprising:
a) forming a mixture containing multiple random sequence
oligonucleotide primers, one or more input DNA molecules, a DNA
polymerase and multiple deoxynucleoside triphosphates
wherein one of the deoxynucleoside triphosphates is a deoxyinosine
triphosphate that upon incorporation changes the melting temperature (Tm)
of amplified DNA products by at least 1°C from the melting temperature
(Tm) of said one or more input DNA molecules;
b) incubating said mixture at substantially constant temperature to
form multiple copies of the input DNA by extension of the primers; and
c) hybridizing the amplified DNA products with a sequencing primer
and sequencing, or cycle sequencing, the amplified DNA products by a
labeled-chain-terminator method.
I.
Issue
The Examiner has rejected claims 1-3 and 9 under 35 U.S.C. § 103(a)
as obvious based on Lasken1 and Dierick2 (Ans. 3). The Examiner has also
rejected claims 11 and 12 under 35 U.S.C. § 103(a) as obvious based on
Lasken, Dierick, and Blanco3 (id. at 5). Since the same issue is dispositive
with respect to both rejections, we will consider them together.

1 Lasken et al., US 6,323,009 B1, Nov. 27, 2001.
2 Herman Dierick, Incorporation of dITP or 7-deaza dGTP during PCR
improves sequencing of the product, 21 NUCLEIC ACIDS RESEARCH no.18,
4427-4428 (1993).
3 Blanco et al., US 5,198,543, Mar. 30, 1993.
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The Examiner finds that Lasken discloses a nucleic acid amplification
and sequencing method meeting all of the limitations of claim 1 except that
“it does not teach the use of one or more of the deoxynucleotide
triphosphates is a modified deoxynucleotide triphosphate which changes the
melting temperature” (Ans. 4). The Examiner finds that “Dierick teaches
that the use of dITP or 7-deaza dGTP in amplification reactions improves
enzymatic activity of the polymerase and reduces enzyme pausing” (id.).
The Examiner concludes that a person of ordinary skill in the art “would
have been motivated to add dITP or 7-deaza dGTP to prevent premature
enzyme pausing and to result in maximal efficiency and completion in
nucleic acid synthesis in the amplification method of Lasken” (id.).
Appellants contend that only a small amount of dITP is incorporated
into the product of Dierick’s reaction and, therefore, it is unclear how much
the melting temperature of the product would change (Appeal Br. 4-5).
Appellants also contend that “[a]lthough Dierick et al. mentions premature
enzyme pausing, they do not offer a solution to prevent this from happening”
(id. at 6) and that the “dye-terminator method used in the current method . . .
[is] immune to problems of polymerase pausing” (id. at 5).
The issue with respect to the rejections on appeal is: Does the
evidence of record support the Examiner’s conclusion that the cited
references would have made it obvious to modify Lasken’s method by
including dITP in the amplification step, and that doing so would decrease
the melting temperature of the amplified DNA by at least 1° C?
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Findings of Fact
1. The Specification states that
[i]n U.S. Patent 6323009 [Lasken] . . . , a means of amplifying
target DNA molecules is described. Some embodiments of this
method feature the use of random-sequence hexamer primers
added in great excess to target DNA, Φ29 DNA polymerase and
the four normal dNTPs (dATP, dCTP, dGTP and dTTP) to
produce multiple copies of all the sequences present in the
original target sample.
(Spec. 19: 30-34.)
2. The Specification states that the “methods of the 6323009 patent
(referred to herein as Multiply Primed Amplification--MPA) . . . improve on
the sensitivity of linear rolling circle amplification by using multiple
primers” (id. at 2: 29-32).
3. The Specification states that “[a]nother advantage of the MPA
method is that the amplification of single-stranded or double-stranded
circular target DNA molecules may be carried out isothermally and/or at
ambient temperatures” (id. at 3: 1-5).
4. The Specification provides a working example in which amplified
DNA is sequenced using “DYEnamic ET terminator premix” and “an ABI
3100 capillary sequencing instrument” (id. at 23: 1-12). The Specification
reports that the sequence obtained using “Standard Multiply-Primed Rolling
Circle Amplification . . . is shown in Figure 1. The sequence obtained was
accurate to about 400 nucleotides.” (Id. at 23: 13-15.)
5. Appellants state that Figure 1 shows the results of dye-terminator
sequencing (Appeal Br. 5-6).
6. Lasken provides a working example in which DNA amplified by
its method “was transferred to a 20 microliter sequencing reaction
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containing . . . DYEnamic ET terminator premix (Amersham Pharmacia
Biotech). This reaction was cycled . . . and ½ of the product was applied to
an ABI 373 sequencing gel apparatus. . . . The sequence obtained was
accurate over more than 400 nucleotides.” (Lasken, col. 20, ll. 11-18.)
7. The Specification states that “Dierick, H. et al., Nucleic Acids
Res[.] 21, 4427-8 (1993) describe PCR amplification of a 560bp sequence
using dGTP analogs dITP or 7-deaza-dGTP. They report that . . . improved
sequences are obtained when PCR is performed using the dGTP analogs,
particularly dITP.” (Spec. 4: 22-26.)
8. Dierick discloses that “[p]remature enzyme pausing due to regions
of complex secondary structure is a common problem in sequencing
reactions” (Dierick 4427, left col.).
9. Dierick discloses that “7-deaza dGTP . . . and dITP (a more
economical alternative), are frequently used in sequencing reactions to
prevent base compressions, caused by stacking of the sequenced fragments,
which lead to abnormal migration patterns” (id.).
10. Dierick concludes that “incorporation of dITP or 7-deaza dGTP
(to a lesser extent) in PCR reactions appears to be an easy and effective
method to avoid premature enzyme pausing during sequencing of PCR
templates” (id. at 4427, right col.).
Principles of Law
“The combination of familiar elements according to known methods
is likely to be obvious when it does no more than yield predictable results.”
KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007). “One of the ways
in which a patent’s subject matter can be proved obvious is by noting that
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there existed at the time of invention a known problem for which there was
an obvious solution encompassed by the patent’s claims.” Id. at 419-420.
Analysis
Lasken discloses a method that meets all of the limitations of claim 1
except that Lasken does not include dITP in its amplification reaction
mixture. Dierick discloses that premature enzyme pausing is a common
problem in sequencing reactions, and including dITP in a PCR amplification
reaction mixture is “an easy and effective method” of avoiding that problem
during sequencing of the amplified DNA. We agree with the Examiner that
it would have been obvious to a person of ordinary skill in the art to include
dITP in Lasken’s amplification reaction mixture in order to avoid the
common problem of enzyme pausing when sequencing the amplified DNA
product.
Appellants argue that the “claimed method, as well as Lasken et al.,
teach isothermal amplification, which is quite different” from the PCR
reaction of Dierick (Appeal Br. 4). Appellants argue that the Fuller
Declaration4 provides evidence that relatively little dITP is incorporated in
the amplified product of Dierick’s PCR reaction, whereas “the current
invention demonstrates incorporation of a large amount of dITP” (id. at 5).
This argument does not persuade us that the Examiner’s rejection is in
error. Dierick discloses that premature enzyme pausing is a common
problem in sequencing reactions and that including dITP in a PCR
amplification reaction avoided that problem. Whether a skilled worker

4 Declaration under 37 C.F.R. § 1.132 of Carl W. Fuller, submitted June 19,
2007.
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would have expected a large amount of dITP to be incorporated in the
amplified DNA is irrelevant; Dierick discloses that including dITP in the
reaction solved the enzyme pausing problem, and Appellants have pointed to
no evidence that a skilled worker would not have expected the same effect
when sequencing the product of Lasken’s amplification reaction.
Appellants also argue that the dye-terminator method recited in the
claims, unlike the dye-primer method used by Dierick, is “immune to
problems of polymerase pausing” (Appeal Br. 5). Appellants go on to say,
however, that although the dye-terminator method does not result in “strong
‘pileups’ of labeled molecules at problem sequences” (id.), “when sequences
are encountered that cause most or all DNA synthesis to pause or stop, the
observed sequence simply ends” (id. at 6). Appellants themselves
characterize this as a “much more severe and difficult situation” than the
result of polymerase pausing in Dierick’s method (id.). Appellants’
assertion is therefore not persuasive of Examiner error.
Finally, Appellants argue that, “[a]s discussed earlier and in Dr.
Fuller’s Rule 132 Declaration, at most only a very small amount of dITP is
incorporated in the PCR product of Dierick et al. It is therefore not clear at
all how much, if any, change in melting temperature is effected in their PCR
product.” (Appeal Br. 5.)
This argument is also unpersuasive. Claim 1 requires that the dITP in
the amplification reaction causes a change in melting temperature of the
amplified DNA of at least 1° C compared to the input DNA. The
amplification reaction recited in claim 1 is that of Lasken, not the PCR
reaction of Dierick. How much dITP was incorporated in the product of
Dierick’s reaction, therefore, is irrelevant to the patentability of the claimed
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process. Appellants have pointed to no evidence that the product of
Lasken’s process, modified by inclusion of dITP as suggested by Dierick,
would not inherently contain enough dITP to change the melting
temperature by at least 1° C.
Conclusion of Law
The evidence of record supports the Examiner’s conclusion that the
cited references would have made it obvious to modify Lasken’s method by
including dITP in the amplification step, and that doing so would decrease
the melting temperature of the amplified DNA by at least 1° C.
SUMMARY
We affirm the rejection of claims 1-3 and 9 as obvious based on
Lasken and Dierick, and the rejection of claims 11 and 12 as obvious based
on Lasken, Dierick, and Blanco.

TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).

Appeal 2009-014792
Application 10/835,140

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AFFIRMED

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