Office Action 2: Notice of Allowance (2025-03-28)

CLAIMS:
Listing of Claims:

1-12. (Cancelled)211

13\. (Previously Presented) A method of amplifying target loci in a nucleic acid sample, the method comprising:tagging a nucleic acid sample comprising cell-free nucleic acids including target loci,
wherein the tagging includes attaching at least one of a plurality of molecular barcodes to a plurality of the cell-free nucleic acids to create tagged nucleic acids;contacting one or more of the tagged nucleic acids with a plurality of primers;
hybridizing the plurality of primers to a plurality of the target loci to produce a reaction mixture;
subjecting the reaction mixture to primer extension reaction conditions and producing amplified products comprising target amplicons;
wherein at least two primers of the plurality of primers that are hybridized to target loci during the hybridizing step each have a melting temperature lower than an annealing temperature for the primer extension reaction conditions, wherein the length of the annealing step of the reaction conditions is greater than 3 minutes, and wherein at least two of the plurality of the target loci are included in the target amplicons; and
performing high-through sequencing on the amplified products.

14\. (Previously Presented) The method of claim 13, wherein melting temperature is determined according to Primer3 program.

15\. (Previously Presented) The method of claim 13, wherein the annealing temperature is at least 3°C greater than a melting temperature of at least two of the primers.

16\. (Previously Presented) The method of claim 13, wherein the annealing temperature is at least 3°C greater than the highest melting temperature of the primers.

17\. (Previously Presented) The method of claim 13, wherein the annealing temperature is at least 8°C greater than the highest melting temperature of the primers.

18\. (Previously Presented) The method of claim 13, wherein the annealing temperature is at least 3°C greater than the average melting temperature of the primers.

19\. (Previously Presented) The method of claim 13, wherein the annealing temperature is at least 8°C greater than the average melting temperature of the primers.

20\. (Previously Presented) The method of claim 13, wherein the range of melting temperatures of the primers is less than 5°C.

21\. (Previously Presented) The method of claim 13, wherein the AG values for each possible combination of two primers in the library are all equal to or greater than -5 kcal/mol.

22\. (Previously Presented) The method of claim 13, wherein 10 to 20,000 target loci are amplified with 10 to 20,000 primers.

23\. (Previously Presented) The method of claim 13, wherein 50 to 2,000 target loci are amplified with 50 to 2,000 primers.

24\. (Previously Presented) The method of claim 13, wherein 100 to 1,000 target loci are amplified with 100 to 1,000 primers.

25\. (Previously Presented) The method of claim 13, wherein at least 90% of the amplified products are target amplicons.

26\. (Previously Presented) The method of claim 13, wherein at least 90% of the target loci are amplified.

27\. (Previously Presented) The method of claim 13, wherein less than 20% of the amplified products are primer dimers.

28\. (Previously Presented) The method of claim 13, wherein the concentration of each primer in the library is less than 20 nM.

29\. (Previously Presented) The method of claim 13, wherein the primers have 2, 1, or 0 guanines or cytosines in the last 5 bases at the 3' end of the primers.

30\. (Previously Presented) The method of claim 13, wherein the length of the annealing step of the reaction conditions is 5-60 minutes.

31\. (Previously Presented) The method of claim 13, wherein the target loci are SNP loci.

32\. (Previously Presented) The method of claim 13, wherein the nucleic acid sample comprises cell-free DNA, and wherein the cell-free DNA comprises tumor DNA.

OFFICE ACTION:
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 .
DETAILED ACTION
Applicant’s amendment filed on August 14, 2025 is acknowledged and has been entered.  Claims 1-12 have been canceled.  Claims 13-32 are pending.
Claims 13-32 are discussed in this Office action.
Terminal Disclaimer
The terminal disclaimer filed on February 14, 2025 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Patent 10,597,708 and 10,597,709 has been reviewed and is accepted.  The terminal disclaimer has been recorded.
Allowable Subject Matter
Claims 13-32 are allowed.
REASONS FOR ALLOWANCE
The following is an examiner’s statement of reasons for allowance: Applicant’s arguments regarding Spier and Ishii are persuasive regarding the claimed feature of selecting an annealing temperature greater than the melting temperature of at least two of the pairs of primers.
The prior art does not teach or suggest each of the features of the claimed method including the selection of an annealing temperature higher than the melting temperature of the primers, the selection of a long annealing time and sequencing of the amplification products. Therefore, the claims are novel and non-obvious over the prior art.
Any comments considered necessary by applicant must be submitted no later than the payment of the issue fee and, to avoid processing delays, should preferably accompany the issue fee.  Such submissions should be clearly labeled “Comments on Statement of Reasons for Allowance.”
Conclusion

APPLICANT REBUTTAL: REMARKS
Applicant respectfully requests reconsideration of the present application in view of the
reasons that follow.
Status of Claims
Claims 13-32 are pending in the subject case. No claim is being amended, cancelled, or
newly added with this submission.
Double Patenting
Claims 13-32 stand provisionally rejected on the ground of nonstatutory obviousness- type double patenting as being unpatentable over claims 1-19 of U.S. Patent No. 10,597,708.
In addition, claims 13-32 stand provisionally rejected on the ground of nonstatutory
obviousness-type double patenting as being unpatentable over claims 1-18 of U.S. Patent No.
10,597,709. Applicant respectfully traverses these rejections.
For the sole purpose of expediting prosecution and without acquiescing to the rejections,
Applicant submits herewith a terminal disclaimer over U.S. Pat. Nos. 10,597,708 and
10,597,709, to overcome the subject rejections.
Claim rejection under 35 USC
§
Claims 13-23 are rejected under 35 USC § 103 as allegedly unpatentable over US
2014/0329245 ("Spier") in view of Ishii et al. Appl. Environ. Micro., 67(8):3753-3755 (2001) ("Ishii"). Claim 32 is rejected under 35 USC § 103 as allegedly obvious over Spier in view of
Ishii and further in view of Pathak et al., Clin. Chem. 52(10):1833-1842 (2006) ("Pathak").
Applicant respectfully traverses this rejection.
Spier does not disclose that "at least two primers of the plurality of primers that are
hybridized to target loci during the hybridizing step each have a melting temperature lower than an annealing temperature for the primer extension reaction conditions" as recited by the present
claims. The Office Action cites to paragraphs 19 and 45-50 of Spier, but those paragraphs do not
disclose the claimed invention. Rather, the cited paragraphs introduce an embodiment that uses
blocked primers cleavable by Endonuclease IV (Endo IV) to reduce primer dimers. However,
there is no evidence that the primers used in Spier (listed in Table 1) have a melting temperature equal to or below 55 0C, which is the annealing temperature used by Spier in the first two cycles where target-specific blocked primers annealed to the target DNA prior to Endo IV cleavage and
subsequent universal amplification. See Spier, paragraph 58 ("we used 10 units in a 10 uL
reaction and long extension times (20 min) during the first 2 cycles followed by fast cycling: 950
C. 5 min; (950 C.-15 s, 550 C.-10 min, 62° C.-10 min)x2 cycles; (950 C.-15 s-620 C.-30 s; 70°
C.-15 s)x40 cycles. After EndoIV cleaves the blocked primers during the first 2 cycles the
reaction is driven by universal primers in subsequent cycles.") Accordingly, Spier fails to teach or suggest using primers having a melting temperature lower than an annealing temperature for
the primer extension reaction conditions.
The Office Action acknowledges at page 8 that Spier does not specifically address
increasing annealing temperature and refers to Ishii for this teaching. However, Ishii does not
teach or suggest using higher annealing temperature than the melting temperature of the primers
hybridizing to the target loci. Indeed, Ishii teaches in the Abstract that "PCRs showed that the bias was reduced at lower temperatures." (Emphasis added.) Moreover, Ishii teaches that "the
bias caused by the difference in primer binding energies was reduced by a lower annealing
temperature.... In any event, the bias caused by the difference in primer binding energies was reduced when the annealing temperature was low." Ishii, page 3754, right column, emphasis
added. Thus, Ishii teaches using a lower annealing temperature, rather than increasing the
annealing temperature to be higher than the melting temperature of the primers as required by the
present claims.
Accordingly, so far as the combination of Spier and Ishii fails to teach or suggest each
and every element of the claimed invention, a prima facie case of obviousness cannot be
sustained.
The rejection of claim 32 relies on the premise that the combination of Spier and Ishii
renders obvious the subject matter of claim 13, which it does not, as explained above. Pathak could not cure the aforementioned deficiencies of Spier and Ishii, nor was Pathak cited for that
purpose. Accordingly, the combination of Spier, Ishii, and Pathak cannot render claim 32
obvious. Reconsideration and withdrawal of the subject rejection are therefore respectfully
requested.
Conc
l
udin
g
Remarks
Applicant believes that the application is in condition for allowance. Favorable
consideration is respectfully requested. The Examiner is invited to contact the undersigned by
telephone if it is felt that a telephone interview would advance prosecution.
The Commissioner is hereby authorized to charge any additional fees which may be
required regarding this application under 37 C.F.R. §§ 1.16-1.17, or credit any overpayment, to
Deposit Account No. 50-6075. If any extensions of time are needed for timely acceptance of
papers submitted herewith, Applicant hereby petitions for such extension under 37 C.F.R. § 1.136
and authorizes payment of any such extension fees to Deposit Account No. 50-6075.
Respectfully submitted,


CITED REFERENCE SUMMARIES: Ref US10597708: Claims:
1. A method of amplifying target loci in a nucleic acid sample, the method comprising:
contacting the nucleic acid sample comprising target loci with a library of at least 2 primers that simultaneously hybridize to at least 2 of the target loci to produce a reaction mixture;
subjecting the reaction mixture to primer extension reaction conditions to produce amplified products comprising target amplicons; wherein the annealing temperature for the reaction conditions is greater than a melting temperature of the at least 2 primers, wherein the length of the annealing step of the reaction conditions is greater than 3 minutes, and wherein the at least 2 of the target loci are simultaneously amplified; and
sequencing the amplified products.
2. The method of claim 1, wherein the annealing temperature is at least 3° C. greater than the melting temperature of the primers.
3. The method of claim 1, wherein the annealing temperature is at least 3° C. greater than the highest melting temperature of the primers.
4. The method of claim 1, wherein the annealing temperature is at least 8° C. greater than the highest melting temperature of the primers.
5. The method of claim 1, wherein the annealing temperature is at least 3° C. greater than the average melting temperature of the primers.
6. The method of claim 1, wherein the annealing temperature is at least 8° C. greater than the average melting temperature of the primers.
7. The method of claim 1, wherein the range of melting temperatures of the primers is less than 5° C.
8. The method of claim 1, wherein the ΔG values for each possible combination of two primers in the library are all equal to or greater than −5 kcal/mol.
9. The method of claim 1, wherein at least 10 target loci are amplified with at least 10 primers.
10. The method of claim 1, wherein at least 50 target loci are amplified with at least 50 primers.
11. The method of claim 1, wherein at least 100 target loci are amplified with at least 100 primers.
12. The method of claim 1, wherein at least 90% of the amplified products are target amplicons.
13. The method of claim 1, wherein at least 90% of the target loci are amplified.
14. The method of claim 1, wherein less than 20% of the amplified products are primer dimers.
15. The method of claim 1, wherein the concentration of each primer in the library is less than 20 nM.
16. The method of claim 1, wherein the primers have 2, 1, or 0 guanines or cytosines in the last 5 bases at the 3′ end of the primers.
17. The method of claim 1, wherein the length of the annealing step of the reaction conditions is 5-60 minutes.
18. The method of claim 1, wherein the target loci are SNP loci.
19. The method of claim 1, wherein the nucleic acid sample comprises cell-free DNA, and wherein the cell-free DNA comprises tumor DNA.