Office Action 1: Non-Final Rejection (2024-12-16)
Rejection types: 103 - Obviousness, Double Patenting

CLAIMS:
Amendments to the Claims:

This listing of claims will replace all prior versions and listing of claims in the application:

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\. (Currently Amended) The method of claim 1 3, wherein 10 to 20,000 target loci are amplified with 10 to 20,000 primers.

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

24\. (Currently Amended) 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.
-1433-9160 1\_4\_ 4855-1433-9160.13 4855-1433-9160.1\_2\_

OFFICE ACTION:
DETAILED 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 .
The preliminary amendment canceling claims 1-12 and amending claims 22-24, filed March 24, 2023 is acknowledged and has been entered.
Claims 13-32 are pending and will be examined.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on September 10, 2024, June 2, 2023 and April 27, 2022 was filed in compliance with the provisions of 37 CFR 1.97.  Accordingly, the information disclosure statement is being considered by the examiner.
The information disclosure statement filed August 6, 2021, February 8, 2022, November 9, 2023 failed to comply with the provisions of 37 CFR 1.97, 1.98 and MPEP § 609 for the reasons put forth in the request for information filed November 22, 2023.  It has been placed in the application file, but the information referred to therein has not been considered as to the merits.  Applicant is advised that the date of any re-submission of any item of information contained in this information disclosure statement or the submission of any missing element(s) will be the date of submission for purposes of determining compliance with the requirements based on the time of filing the statement, including all certification requirements for statements under 37 CFR 1.97(e).  See MPEP § 609.05(a).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA  as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 13-32 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-19 of U.S. Patent No. 10597708 (Patent ‘708 herein). Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are not patentably distinct over the claims of the ‘708 patent.
The differences between the ‘708 patent and the instant claims arises with the scope of the instant claims as compared to the claims of the ‘708 patent.  In the ‘708 patent the claims are focused on the steps of amplification and melting temperature.  The cell free nucleic acid of instant claim 1 is captured in claim 19 of the ‘708 patent.  The dependent claims of the ‘708 patent, encompass very similar subject matter including specific melting temperatures and melt temperature ranges, upon comparison of instant claims 15-20 to claims 2-7 of the ‘708 patent.  Further, limitations including length of annealing step are also captured by the dependent claims of the ‘708 patent, compare instant claim 30-31 to claims 16-18 of the ‘708 patent. Therefore the instant claims are not patentably distinct over the claims of the ‘708 patent.
Claims 13-32 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-18 of U.S. Patent No. 10597709 (Patent ‘709 herein). Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are not patentably distinct over the claims of the ‘708 patent.
The differences between the ‘709 patent and the instant claims arises with the scope of the instant claims as compared to the claims of the ‘709 patent.  In the ‘709 patent the claims are focused on the steps of amplification and melting temperature.  The cell free nucleic acid of instant claim 1 is captured in claim 19 of the ‘709 patent.  The dependent claims of the ‘709 patent, encompass very similar subject matter including specific melting temperatures and melt temperature ranges, upon comparison of instant claims 15-20 to claims 2-7 of the ‘709 patent.  Further, limitations including length of annealing step are also captured by the dependent claims of the ‘709 patent, compare instant claim 30-31 to claims 16-18 of the ‘709 patent. Perhaps most importantly, while instant claim 1 recites a minimum of two primers, dependent claims encompass 2 to 10 and up to 20,000 primers, in the claims of the ’709 patent, the first claim is drawn to amplification with 50 or more primers and as many as 200 primers.  The dependent claims of the ‘709 patent and the instant claims encompass higher level of multiplex, compare instant claims 22-24 to claims 10-11 of the ‘709 patent.  Therefore the instant claims are not patentably distinct over the claims of the ‘709 patent.
Claim Rejections - 35 USC § 103
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary.  Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 13-23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Spier et al. (US PgPub 20140329245; November 2014) in view of Ishii et al. (Appl Environ Micro, 2001, 67(8):3753-3755).
With regard to claim 13, Spier teaches 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 ((paragraph 19, where the annealing time is relatively long; paragraph 19, paragraph 45-50, where high multiplex is taught).
With regard to claim 14, Spier teaches a method of claim 13, wherein melting temperature is determined according to Primer3 program (paragraph 19).
With regard to claim 21, Spier teaches a 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 (paragraph 19).
With regard to claim 22, Spier teaches a method of claim 13, wherein 10 to 20,000 target loci are amplified with 10 to 20,000 primers (paragraph 45-50, where high multiplex is taught).
With regard to claim 23, Spier teaches a method of claim 13, wherein 50 to 2,000 target loci are amplified with 50 to 2,000 primers (paragraph 45-50, where high multiplex is taught).
With regard to claim 24, Spier teaches a method of claim 13, wherein 100 to 1,000 target loci are amplified with 100 to 1,000 primers (paragraph 45-50, where high multiplex is taught).
With regard to claim 25, Spier teaches a method of claim 13, wherein at least 90% of the amplified products are target amplicons (paragraph 45-50, where high multiplex is taught).
With regard to claim 26, Spier teaches a method of claim 13, wherein at least 90% of the target loci are amplified (paragraph 45-50, where high multiplex is taught).
With regard to claim 27, Spier teaches a method of claim 13, wherein less than 20% of the amplified products are primer dimers (paragraph 45-50, where high multiplex is taught).
With regard to claim 28, Spier teaches a method of claim 13, wherein the concentration of each primer in the library is less than 20 nM (paragraph 19).
With regard to claim 29, Spier teaches a 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 (paragraph 19).
With regard to claim 30, Spier teaches a method of claim 13, wherein the length of the annealing step of the reaction conditions is 5-60 minutes (paragraph 19, where the annealing time is relatively long).
With regard to claim 31, Spier teaches a method of claim 13, wherein the target loci are SNP loci (paragraph 61).
Regarding claims 15-21, while Spier teaches varying the melting temperatures of the primers, Spier does not specifically address increased annealing temperature.
With regard to claim 15, Ishii teaches a 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 (Table 2, Abstract).
With regard to claim 16, Ishii teaches a method of claim 13, wherein the annealing temperature is at least 3°C greater than the highest melting temperature of the primers (Table 2, Abstract).
With regard to claim 17, Ishii teaches a method of claim 13, wherein the annealing temperature is at least 8°C greater than the highest melting temperature of the primers (Table 2, Abstract).
With regard to claim 18, Ishii teaches a method of claim 13, wherein the annealing temperature is at least 3°C greater than the average melting temperature of the primers (Table 2, Abstract).
With regard to claim 19, Ishii teaches a method of claim 13, wherein the annealing temperature is at least 8°C greater than the average melting temperature of the primers (Table 2, Abstract).
With regard to claim 20, Ishii teaches a method of claim 13, wherein the range of melting temperatures of the primers is less than 5°C (Table 2, Abstract).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have applied the teachings of multiplex amplification as taught by Spier to the amplification using increased annealing temperature as taught by Ishii to arrive at the claimed invention with a reasonable expectation for success.  While Spier teaches multiplex amplification, Spier does not teach that it includes increased annealing temperatures.  As taught by Ishii, “To reduce PCR bias derived from a primer mismatch, the effect of the annealing temperature on the product ratio was investigated by denaturing gradient gel electrophoresis analysis of PCR products from a mixture of perfect-match and one-mismatch templates” (Abstract).  Therefore, one of ordinary skill in the art at the time the invention was made would have been motivated to have applied the teachings of multiplex amplification as taught by Spier to the amplification using increased annealing temperature as taught by Ishii to arrive at the claimed invention with a reasonable expectation for success.
Claim 32 is/are rejected under 35 U.S.C. 103 as being unpatentable over Spier et al. (US PgPub 20140329245; November 2014) in view of Ishii et al. (Appl Environ Micro, 2001, 67(8):3753-3755) as applied over claims 2-19 above and further in view of Pathak et al. (Clin Chem, 2006, 52:10, p. 1833-1842).
With regard to claim 32, Pathak teaches a method of claim 13, wherein the nucleic acid sample comprises cell-free DNA, and wherein the cell-free DNA comprises tumor DNA (Table 1, where extracellular DNA is studied in lung cancer patients, as an example).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have adjusted the teachings of Spier in view of Ishii to include cell free DNA in tumor DNA as taught by Pathak to arrive at the claimed invention with a reasonable expectation for success.  Pathak teaches “The findings from the studies on serum/ plasma DNA suggest that it would be possible to develop a simple blood test with high sensitivity and specificity that has potential for screening of high-risk individuals, for prognostic or staging purposes, and to be used as intermediate end-points of efficacy in chemoprevention and therapeutic trials” (Abstract).  Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Spier in view of Ishii to include cell free DNA in tumor DNA as taught by Pathak to arrive at the claimed invention with a reasonable expectation for success.
Conclusion
No claims are allowed.  All claims stand rejected.

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,

NEXT 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

CITED REFERENCE SUMMARIES: Ref US20140329245A1: Claims:
A PCR reaction mixture comprising: (a) at least one pair of target-specific primers comprising universal 5′ tags, where at least one target-specific primer cannot be extended by polymerase (3′-blocked primer); (b) at least one pair of universal primers that can prime on the universal 5′ tags; (c) an enzyme that unblocks the 3′-blocked primer after it anneals to the target DNA generating a 3′-OH end; and (d) a polymerase that extends unblocked primers during one or more initial PCR cycles; and subsequently extends universal primers driving amplification in a closed tube reaction.
(a) at least one pair of target-specific primers comprising universal 5′ tags, where at least one target-specific primer cannot be extended by polymerase (3′-blocked primer);
(b) at least one pair of universal primers that can prime on the universal 5′ tags;
(c) an enzyme that unblocks the 3′-blocked primer after it anneals to the target DNA generating a 3′-OH end; and
(d) a polymerase that extends unblocked primers during one or more initial PCR cycles; and subsequently extends universal primers driving amplification in a closed tube reaction.
The PCR reaction mixture of claim 1 , wherein multiple blocked target-specific primers amplify multiple targets (multiplex PCR) and target-specific primers for targets that are to be detected together comprise the same universal tags, and optionally blocked target-specific primers for targets to be detected separately comprise different universal tags.
The PCR reaction mixture of claim 1 , wherein the concentration of the target-specific primers is at least 40-fold less than the concentration of the universal primers.
The reaction mixture of claim 2 , wherein universal primers and optionally universal probes are labeled and different universal primers and optionally probes are used to detect targets by priming on the different universal tags of the amplicons generated by the blocked primers.
A method to detect or enrich target nucleic acids in a sample in a closed tube reaction, comprising the use of PCR with one or more 5′ tagged target-specific primer(s) with a blocked 3′-end and universal primers that optionally comprise blocked 3′ ends; that can prime on these universal tags and optionally probes; such that during initial PCR cycles, the target-specific primer anneal, get unblocked and extend on target DNA, and in subsequent PCR cycles, the universal primers drive PCR.
The method of claim 5 , wherein the read-out for the detection of nucleic acids is real-time or end-point PCR for sequencing, NGS enrichment, fragment sizing using electrophoresis, surface hybridization, amplicon melting, molecular weight determination via electrophoresis or mass spectrometry.
The reaction mixture of claim 1 or the method of claim 5 , wherein the one or more 3′ blocked primer(s) comprise(s) a C3 spacer(s) or another 3′-blocking modification.
The reaction mixture or method of claim 7 , comprising, or comprising the use of, a DNA polymerase with 3′-exonuclease activity or an endonuclease to unblock the primers.
The reaction mixture or method of claim 7 , comprising, or comprising the use of, a blocked primer comprising an abasic site to anneal to the target on both sides of the abasic site, and an endonuclease IV to cleave 5′ from the abasic site, unblocking the primer.
The reaction mixture of claim 1 or the method of claim 5 , wherein the primer comprises at least one RNA base, and further comprising, or comprising the use of, RNase H enzyme for unblocking the primer.
A blocked PCR primer and an enhancer 3′-blocked oligo that anneals downstream from the blocked primer, forming no gap or a single base gap between the primer and the oligo when both are annealed to the target in a PCR or isothermal amplification mixture comprising an endonuclease that cleaves off a cleavable group at the single base gap and enables primer extension.
The reaction mixture of claim 2 or the method of claim 5 used for PCR enrichment for sequencing, wherein at least one of the universal primers comprises a sample bar-code that enables sample pooling for sequencing; and optionally wherein the blocked target-specific primers comprise random molecular barcodes allowing identification of individual DNA strands amplified in PCR, such that the first two PCR cycles incorporate random molecular bar-codes for each DNA strand present in the sample.
The method of claim 5 , wherein the PCR reaction is normalized by slowing down cooling from melting to annealing temperature during late PCR cycles, such that the majority of abundant target amplicons reanneal and do not amplify, but rare ones continue their amplification.
A multiplex allele (mutation) specific PCR reaction mixture used to detect two or more closely spaced mutations, comprising: (a) mutation (allele) specific primers for each mutation to be detected, wherein the primers comprise the same target-specific 3′-regions, except for different 3′-ends; and (b) the primers of (a) further comprising different non-target-specific 5′ tags; such that after the first two PCR cycles the first allele-specific primer that was extended in the first PCR cycle continues PCR by annealing to the amplicons that are 100% complementary to the whole first primer, but other allele-specific primers with 5′-tags not matching the amplicons generated by the first primer do not anneal at the annealing temperature used in PCR.
(a) mutation (allele) specific primers for each mutation to be detected, wherein the primers comprise the same target-specific 3′-regions, except for different 3′-ends; and
(b) the primers of (a) further comprising different non-target-specific 5′ tags; such that after the first two PCR cycles the first allele-specific primer that was extended in the first PCR cycle continues PCR by annealing to the amplicons that are 100% complementary to the whole first primer, but other allele-specific primers with 5′-tags not matching the amplicons generated by the first primer do not anneal at the annealing temperature used in PCR.
The PCR reaction mixture of claim 14 , wherein the allele (mutation) and target-specific primers of (a) and (b) further comprise universal 5′-tags and the different non-target specific tags of (b) are situated between the 3′-target-specific region and the universal 5′ tags of the allele specific primers; and wherein the reaction mixture further comprises (c) at least one pair of universal primers that can prime on the universal 5′ tags.
the allele (mutation) and target-specific primers of (a) and (b) further comprise universal 5′-tags and the different non-target specific tags of (b) are situated between the 3′-target-specific region and the universal 5′ tags of the allele specific primers; and wherein the reaction mixture further comprises (c) at least one pair of universal primers that can prime on the universal 5′ tags.
The reaction mixture of claim 14 or 15 , where at least one of the allele or locus specific primers is blocked.
A method of using PCR to detect two or more closely-spaced mutations with increased sensitivity, comprising the use of mutation-specific primers for each mutation with 5′-non target tags different for each primer, such that after first two PCR cycles, an increased annealing temperature is used to decrease completion between different mutation-specific primers, thus increasing assay sensitivity.
A PCR amplification method comprising the use of at least one blocked PCR primer with a 3′-end complementary to the template, such that unblocking is performed by a 3′-exonuclease activity and extension by a polymerase activity, respectively, and wherein the two enzymatic activities are performed by a single enzyme or two separate enzymes.
A PCR reaction mixture comprising at least one 3′-blocked primer comprising a 3′-end having a nucleotide sequence 100% complementary to a template; and either a polymerase with 3′-exonuclease activity, or a separate 3′-exonuclease and a polymerase; such that after the 3′-blocked primer anneals to the template the exonuclease unblocks the primer(s) and the polymerase extends the unblocked primer(s) during PCR.
Ref US10597708B2: [content unavailable]
Ref US10597709B2: [content unavailable]
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.
Ref US10597709: Claims:
1. A method of amplifying target loci in a nucleic acid sample, the method comprising:
contacting the nucleic acid sample comprising at least 50 target loci with a library of at least 50 different primers 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 50 primers, and wherein the at least 50 of the target loci are simultaneously amplified; and
sequencing the amplified products.
2. The method of claim 1, wherein the target loci are SNP loci.
3. The method of claim 1, wherein the annealing temperature is at least 3° C. greater than the melting temperature of the primers.
4. The method of claim 1, wherein the annealing temperature is at least 3° C. greater than the highest melting temperature of the primers.
5. The method of claim 1, wherein the annealing temperature is at least 8° C. greater than the highest melting temperature of the primers.
6. The method of claim 1, wherein the annealing temperature is at least 3° C. greater than the average melting temperature of the primers.
7. The method of claim 1, wherein the annealing temperature is at least 8° C. greater than the average melting temperature of the primers.
8. The method of claim 1, wherein the range of melting temperatures of the primers is less than 5° C.
9. 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.
10. The method of claim 1, wherein at least 100 target loci are amplified in a single reaction mixture.
11. The method of claim 1, wherein at least 200 target loci are amplified in a single reaction mixture.
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 3-60 minutes.
18. The method of claim 1, wherein the target loci are SNP loci, and wherein the nucleic acid sample comprises cell-free DNA of mixed origin.
19. The method of claim 18, wherein the nucleic acid sample comprises cell-free DNA from a transplant.
20. The method of claim 18, wherein the nucleic acid sample is from a transplant recipient.
Ref US20140329245: Claims:
What is claimed is: 1. A PCR reaction mixture comprising: (a) at least one pair of target-specific primers comprising universal 5′ tags, where at least one target-specific primer cannot be extended by polymerase (3′-blocked primer); (b) at least one pair of universal primers that can prime on the universal 5′ tags; (c) an enzyme that unblocks the 3′-blocked primer after it anneals to the target DNA generating a 3′-OH end; and (d) a polymerase that extends unblocked primers during one or more initial PCR cycles; and subsequently extends universal primers driving amplification in a closed tube reaction. 2. The PCR reaction mixture of claim 1, wherein multiple blocked target-specific primers amplify multiple targets (multiplex PCR) and target-specific primers for targets that are to be detected together comprise the same universal tags, and optionally blocked target-specific primers for targets to be detected separately comprise different universal tags. 3. The PCR reaction mixture of claim 1, wherein the concentration of the target-specific primers is at least 40-fold less than the concentration of the universal primers. 4. The reaction mixture of claim 2, wherein universal primers and optionally universal probes are labeled and different universal primers and optionally probes are used to detect targets by priming on the different universal tags of the amplicons generated by the blocked primers. 5. A method to detect or enrich target nucleic acids in a sample in a closed tube reaction, comprising the use of PCR with one or more 5′ tagged target-specific primer(s) with a blocked 3′-end and universal primers that optionally comprise blocked 3′ ends; that can prime on these universal tags and optionally probes; such that during initial PCR cycles, the target-specific primer anneal, get unblocked and extend on target DNA, and in subsequent PCR cycles, the universal primers drive PCR. 6. The method of claim 5, wherein the read-out for the detection of nucleic acids is real-time or end-point PCR for sequencing, NGS enrichment, fragment sizing using electrophoresis, surface hybridization, amplicon melting, molecular weight determination via electrophoresis or mass spectrometry. 7. The reaction mixture of claim 1 or the method of claim 5, wherein the one or more 3′ blocked primer(s) comprise(s) a C3 spacer(s) or another 3′-blocking modification. 8. The reaction mixture or method of claim 7, comprising, or comprising the use of, a DNA polymerase with 3′-exonuclease activity or an endonuclease to unblock the primers. 9. The reaction mixture or method of claim 7, comprising, or comprising the use of, a blocked primer comprising an abasic site to anneal to the target on both sides of the abasic site, and an endonuclease IV to cleave 5′ from the abasic site, unblocking the primer. 10. The reaction mixture of claim 1 or the method of claim 5, wherein the primer comprises at least one RNA base, and further comprising, or comprising the use of, RNase H enzyme for unblocking the primer. 11. A blocked PCR primer and an enhancer 3′-blocked oligo that anneals downstream from the blocked primer, forming no gap or a single base gap between the primer and the oligo when both are annealed to the target in a PCR or isothermal amplification mixture comprising an endonuclease that cleaves off a cleavable group at the single base gap and enables primer extension. 12. The reaction mixture of claim 2 or the method of claim 5 used for PCR enrichment for sequencing, wherein at least one of the universal primers comprises a sample bar-code that enables sample pooling for sequencing; and optionally wherein the blocked target-specific primers comprise random molecular barcodes allowing identification of individual DNA strands amplified in PCR, such that the first two PCR cycles incorporate random molecular bar-codes for each DNA strand present in the sample. 13. The method of claim 5, wherein the PCR reaction is normalized by slowing down cooling from melting to annealing temperature during late PCR cycles, such that the majority of abundant target amplicons reanneal and do not amplify, but rare ones continue their amplification. 14. A multiplex allele (mutation) specific PCR reaction mixture used to detect two or more closely spaced mutations, comprising: (a) mutation (allele) specific primers for each mutation to be detected, wherein the primers comprise the same target-specific 3′-regions, except for different 3′-ends; and (b) the primers of (a) further comprising different non-target-specific 5′ tags; such that after the first two PCR cycles the first allele-specific primer that was extended in the first PCR cycle continues PCR by annealing to the amplicons that are 100% complementary to the whole first primer, but other allele-specific primers with 5′-tags not matching the amplicons generated by the first primer do not anneal at the annealing temperature used in PCR. 15. The PCR reaction mixture of claim 14, wherein the allele (mutation) and target-specific primers of (a) and (b) further comprise universal 5′-tags and the different non-target specific tags of (b) are situated between the 3′-target-specific region and the universal 5′ tags of the allele specific primers; and wherein the reaction mixture further comprises (c) at least one pair of universal primers that can prime on the universal 5′ tags. 16. The reaction mixture of claim 14 or 15, where at least one of the allele or locus specific primers is blocked. 17. A method of using PCR to detect two or more closely-spaced mutations with increased sensitivity, comprising the use of mutation-specific primers for each mutation with 5′-non target tags different for each primer, such that after first two PCR cycles, an increased annealing temperature is used to decrease completion between different mutation-specific primers, thus increasing assay sensitivity. 18. A PCR amplification method comprising the use of at least one blocked PCR primer with a 3′-end complementary to the template, such that unblocking is performed by a 3′-exonuclease 
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