10466580 (B.P.A.I. Apr. 23, 2010)

Ex Parte Knott et al

Board of Patent Appeals and InterferencesApr 23, 2010

10466580 (B.P.A.I. Apr. 23, 2010)

UNITED STATES PATENT AND TRADEMARK OFFICE __________ BEFORE THE BOARD OF PATENT APPEALS AND INTERFERENCES __________ Ex parte TIM KNOTT, CLIFFORD SMITH, JUDITH PICKERING, and TEREK SCHWARZ __________ Appeal 2009-005016 Application 10/466,580 Technology Center 1600 __________ Decided: April 23, 2010 __________ Before TONI R. SCHEINER, MELANIE L. McCOLLUM, and STEPHEN G. WALSH, Administrative Patent Judges. SCHEINER, Administrative Patent Judge. DECISION ON APPEAL This is an appeal under 35 U.S.C. § 134 from the rejection of claims 1, 3, 4, and 6, directed to a method of reducing non-specific nucleic acid amplification in a rolling circle amplification reaction. The claims have been rejected under 35 U.S.C. § 103(a) as obvious. We have jurisdiction under 35 U.S.C. § 6(b). We affirm. Appeal 2009-005016 Application 10/466,580 2 STATEMENT OF THE CASE Many diagnostic assays based on nucleic acid detection “involve exponential amplification of . . . nucleic acid target or probe sequences” (Spec. 1: 13-14), but “[a]mplification of untargeted sequences or nontarget directed amplification impacts severely upon assay reliability” (id. at 1: 25- 26). The present invention is directed to improving the sensitivity and specificity of isothermal rolling-circle amplification (RCA) reactions by modifying the primer(s) used in the reaction in order to impair their ability to serve as effective templates for DNA synthesis, thereby reducing or eliminating non-specific background signals arising from primer-based artifacts (id. at 5: 17-21). Claim 1 is representative of the subject matter on appeal: 1. In a method of reducing non-specific nucleic acid amplification thereby suppressing background signal in an isothermal nucleic acid amplification reaction, the improvement comprising using one or more primers which include a nucleotide analogue, a ribonucleotide, and a fluor or quencher, wherein said isothermal nucleic acid amplification reaction is a rolling circle nucleic acid amplification reaction. The Examiner rejected claims 1, 3, and 4 under 35 U.S.C. § 103(a) as unpatentable over Nardone1 and Tyagi.2 In addition, the Examiner rejected claim 6 under 35 U.S.C. § 103(a) as unpatentable over Nardone, Tyagi, and Ward.3 1 US 6,117,986, issued September 12, 2000 to Nardone et al. 2 US 6,277,607 B1, issued August 21, 2001 to Tyagi et al. 3 US 6,007,994, issued December 28, 1999 to Ward et al. Appeal 2009-005016 Application 10/466,580 3 ISSUE The issue raised by both of the rejections of record is whether the evidence of record supports the Examiner’s conclusion that using a primer including a nucleotide analogue, a ribonucleotide, and a fluor or quencher in an isothermal rolling-circle nucleic acid amplification reaction would have been obvious given the disclosures of Nardone and Tyagi. FINDINGS OF FACT FF1 The present Specification discloses “a method of suppressing background signal in an isothermal nucleic acid amplification reaction wherein at least one of the primers used comprises at least one of a nucleotide analogue a hairpin loop at the 5' end of the primer a ribonucleotide a fluor or quencher” (Spec. 6: 1-6). FF2 The claimed invention is directed to a method of reducing non- specific nucleic acid amplification in an isothermal rolling-circle nucleic acid amplification reaction “comprising using one or more primers which include a nucleotide analogue, a ribonucleotide, and a fluor or quencher” (claim 1). FF3 Tyagi discloses a method of reducing non-specific nucleic acid amplification in thermal cycling and isothermal nucleic acid amplification reactions, including polymerase chain reactions (PCR) and rolling-circle amplifications (RCA), respectively. Tyagi’s method comprises using hairpin primers to minimize the formation of non-specific, non-target Appeal 2009-005016 Application 10/466,580 4 amplification products, such as primer-dimers and mis-primed sequences. (Tyagi, col. 3, ll. 20-25; col. 7, ll. 52-64.) FF4 Tyagi’s hairpin primers “can contain deoxyribonucleotides, ribonucleotides, peptide nucleic acids (PNA), other modified nucleotides, or combinations of these” in order to adjust the strength of the primer’s stem, or to form stronger hybrids (Tyagi, col. 6, ll. 28-43). FF5 In addition, Tyagi’s hairpin primers can be labeled with fluorescent moieties and quenchers in order to monitor the course of the amplification reaction (Tyagi, col. 6, ll. 52-65). FF6 Nardone discloses a hairpin primer which incorporates a nucleotide analogue, a fluorophore and a quencher. The fluorophore acts as a fluorescence indicator when the hairpin is unfolded because the distance between the fluorophore and the quencher is increased. (Nardone, col. 3, ll. 34-46.) Nardone teaches that primers labeled in this manner are useful in following the progress of a PCR reaction. (Id. at cols. 13-14.) PRINCIPLES OF LAW “In determining whether obviousness is established by combining the teachings of the prior art, ‘the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art.’” In re GPAC Inc., 57 F.3d 1573, 1581 (1995) (internal quotations omitted). “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). Appeal 2009-005016 Application 10/466,580 5 ANALYSIS Claims 1, 3, and 4 The evidence of record supports the Examiner’s conclusion that using a primer including a nucleotide analogue, a ribonucleotide, and a fluor or quencher in an isothermal rolling circle nucleic acid amplification reaction would have been obvious over the disclosures of Nardone and Tyagi. As discussed above, Tyagi teaches that using hairpin primers in a rolling circle nucleic acid amplification reaction reduces the formation of false (i.e., non- specific) nucleic acid amplification products (FF3), and that those hairpin primers can include ribonucleotides and modified nucleotides in order to adjust the strength of the stem, or to form stronger hybrids (FF4). Moreover, Tyagi teaches that the hairpin primers can contain fluorescent moieties and quenchers in order to follow the progress of the reaction in real time (FF5). Nardone also teaches that nucleotide analogues, fluorescent moieties and quenchers may be incorporated into hairpin primers in order to label the primers (FF6). Appellants concede that Tyagi “mentions nucleotide analogues and ribonucleotides . . . as possible modifications of the hairpin primers” (App. Br. 4), but contend that Tyagi “does not provide any teaching that a primer without the hairpin structure could still be useful to reduce background in an amplification reaction” (id.). Appellants contend that the present claims “do not have ‘a hairpin’ as a feature of the primer” (id. at 5), and the Examiner’s rejection is improper because the rationale for combining Nardone and Tyagi depends “on a feature not present in the claims” (id.). Moreover, Appellants contend that Nardone is directed to reducing background Appeal 2009-005016 Application 10/466,580 6 fluorescence in real time PCR, rather than reducing non-specific nucleic acid amplification in an isothermal amplification reaction (id. at 3). These arguments are not persuasive. Tyagi is specifically directed to reducing non-specific amplification in thermal cycling and isothermal amplification reactions, including RCA (FF3) using modified hairpin primers. Moreover, the claims use the transitional term “comprising,” and therefore don’t preclude additional modifications to the primer. Nor is there anything in the Specification which would indicate that hairpin primers are precluded by the claims. Indeed, the Specification discloses just such a modification, in combination with a nucleotide analogue, a ribonucleotide, and a fluor or quencher (FF1). Claim 6 Appellants contend that the rejection of claim 6 as unpatentable over Nardone, Tyagi, and Ward is improper for the same reasons discussed in connection with the rejection of claims 1, 3, and 4. We are not persuaded for the same reasons discussed above. CONCLUSIONS OF LAW The evidence of record supports the Examiner’s conclusion that using a primer including a nucleotide analogue, a ribonucleotide, and a fluor or quencher in an isothermal rolling circle nucleic acid amplification reaction would have been obvious over the disclosures of Nardone and Tyagi. The rejection of claims 1, 3, and 4 under 35 U.S.C. § 103(a) as unpatentable over Nardone and Tyagi is affirmed. The rejection of claim 6 under 35 U.S.C. § 103(a) as unpatentable over Nardone, Tyagi, and Ward is affirmed as well. Appeal 2009-005016 Application 10/466,580 7 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)(1)(iv)(2006). AFFIRMED dm GE HEALTHCARE BIO-SCIENCES CORP. PATENT DEPARTMENT 101 CARNEGIE CENTER PRINCETON, NJ 08540