All Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-12 and 14-21 are pending and under examination.
Response to Arguments
Objections Withdrawn
The objection to the specification for missing sequence identifiers is withdrawn.
Rejections Withdrawn
The rejection of claims 1-12 and 14-21 under 35 USC 103 as unpatentable over Raz in view of Sin is withdrawn. Applicant's arguments filed 10/07/2025 have been fully considered and found persuasive.
New Rejections
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-12 and 14-21 are rejected under 35 U.S.C. 103 as being unpatentable over Raz in view of Allawi (“Thermodynamics and NMR of Internal G-T Mismatches in DNA”, Biochemistry Vol 36, Issue 34, 1997).
In regards to claim 1, Raz is in the field of nucleic acid amplification and teach the use of hairpin primers. Specifically, Raz discloses mixing a target nucleic acid with at least two primer pairs – a first target-specific primer pair and a second universal primer pair- and performing a thermocycling amplification protocol. Raz teaches that the target-specific primer comprises a 3’ target-specific sequence with a defined melting temperature (Tm-ts) and a 5’ hairpin region with a higher melting temperature (Tm-as1), where Tm-as1 is at least 8 °C greater than Tm-ts (Figs. 1-4, ¶0006, ¶0010, ¶0050, ¶0053-¶0055, and throughout). Similarly, the universal primer pair also comprises a second hairpin region with at least 90% sequence similarity to the first hairpin and melting temperature, Tm-as2 (¶0073). Raz teaches a two-stage amplification: a first stage using the target specific primer at a low annealing temperature (below Tm-as1) followed by a second stage at a higher annealing temperature where the universal hairpin primer melts into a linear conformation and hybridizes to the first amplicon via complementarity to the 5’ hairpin region (¶0076-¶0079). Further, Raz teaches that the stability of the hairpin stem relative to the annealing temperature is an important design parameter. Raz does not explicitly disclose that the hairpin stem comprises at least one G-T or G-U pairing.
Allawi teaches that G-T wobble base pairs form stable mismatches in DNA duplexes and that such G-T mismatches measurably alter thermodynamic stability and melting temperature of paired nucleic acid regions (see Abstract, Tables 1-6, pg. 1 ¶1-3, pg. 2 ¶5, and throughout). Allawi provides quantitative thermodynamic parameters for G-T mismatches and establishes that such wobble pairing is recognized structural and thermodynamic feature of DNA duplexes.
A person of ordinary skill in the art designing hairpin-containing primers as taught by Raz would have understood that the behavior of the hairpin stem depends upon its thermodynamic stability relative to reaction conditions. It was well established in the art that incorporation of specific base pair interactions, including non-canonical G-T wobble pairings, alters duplex stability in a predictable manner. Accordingly, incorporating at least on G-T pairing in the hairpin stem of Raz’s primers would have represented a routine and predictable means of adjusting stem stability.
The selection of G-T (or G-U) wobble pairing from among known base-pairing interactions to tune thermodynamic properties of a hairpin stem constitutes routine optimization of a known parameter within the skill of the art. Such optimization does not require a specific teaching directed to the identical application, but rather reflects the application of established thermodynamic principles to a known primer structure. Therefore it would have been obvious to a person of ordinary skill in the art to modify the hairpin stems of Raz to include at least one G-T or G-U pairing in order to adjust thermodynamic stability.
In regards to claim 2, Raz teaches that the annealing temperature for the target specific primer is 5 °C or more, lower than the melting temperature of the hairpin loop (¶0076).
In regards to claim 3, a person having ordinary skill in the art would recognize that PCR reactions perform optimally if the annealing temperature is the same as the melting temperature for denaturing the double-stranded region of interest. This is done to reduce off-target primer binding and has been standard practice in the field for over 50 years.
In regards to claim 4, Raz teaches that Tm for the target specific portion of first target specific hairpin primer is 5 °C or more below the Tm of the hairpin region.
In regards to claim 5, Raz teaches that the annealing temperature for the target specific primer pairs occurs between 37 °C and 72 °C (¶0045). Furthermore, one of the examples used by Raz has a melting temperature of 57.8 °C (SEQ ID 1, ¶0072), which falls in the temperature ranges claimed.
Regarding claim 6, as mentioned above, Raz teaches that the hairpin regions of the universal primer and target specific primer maybe identical, furthermore they also teach that the Tm of the two would be within 3 °C of each other (¶0069).
In regards to claim 7, Raz teaches that the Tm of the stem-loop region may between 40 °C - 76 °C or higher (¶0045).
In regards to claim 8, Raz teaches a second annealing step, in which the temperature is greater than the temperature used for the first annealing step (Fig. 1 – 4, ¶0007, ¶0079, and throughout).
In regards to claim 9, Raz teaches that the target-specific primers are present in the amplification reaction at a molar concentration that is 4-fold to 50-fold less than the concentration of the universal primer pairs (¶0069).
In regards to claim 10, Raz teaches the multiplexing capability of this method and envisions using multiple primer sets in a reaction and provides examples of using 50 primer pairs in single reaction (¶0051, ¶0082-¶0085, and throughout).
In regards to claim 11, Raz teaches using individual labels for different primer sets, in which the “5' region of the first target primer pair that forms a hairpin, optionally contains a barcode sequence to allow for indexing of multiple nucleic acid samples (sources) in a single nucleic acid amplification reaction.” (¶0054).
In regards to claim 12, Raz teaches incorporating molecular barcoding into the universal hairpin primers, to be used for indexing of nucleic acid samples during downstream sequencing (¶0054).
In regards to claims 14, Raz teaches all of the components of the kits described and specifically describes a kit containing a first target-specific hairpin primer pair, with all of the properties described previously, and a universal primer pair with the properties described herein.
In regards to claim 15, Raz teaches utilizing their methods and kits in conjunction with materials for other analysis platforms such as next generation sequencing platforms, and provides examples of special adaptations to using those technologies including 454® technology platforms, Solexa platform, Supported Oligonucleotide Ligation and Detection (SOLiD®) platforms or Ion Torrent® platforms. Each of which utilizes their own particularly formatted cartridge. It would have been obvious at the time of filing to include a cartridge compatible with the downstream analysis platform of choice.
In regards to claims 16 and 20, Raz teaches that the annealing temperature may be the same as the target specific melting temperature (¶0045, ¶0076, ¶0079 and throughout).
In regards to claim 17, Raz teaches that the Tm may be between 58-60 °C (¶0045), which would also read on a kit including the same.
In regards to claim 18, 19, and 21, Raz teaches that difference between hairpin melting may be less than 3 °C and may be the same (¶0044-¶0045, ¶0069-¶0071, ¶0079, and throughout), which would also read on a kit including the same.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Matthew H Raymonda whose telephone number is (703)756-5807. The examiner can normally be reached Monday - Friday 10:00 am - 4:00 pm.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/ Primary Examiner, Art Unit 1681