View source for Patent Application 14281212 - SAMPLE MULTIPLEXING - Rejection

= Patent Application 14281212 - SAMPLE MULTIPLEXING =

'''Title:''' SAMPLE MULTIPLEXING

== Application Information ==
* '''Invention Title:''' SAMPLE MULTIPLEXING
* '''Application Number:''' 14281212
* '''Submission Date:''' 2025-05-21T00:00:00.000Z
* '''Effective Filing Date:''' 2014-05-19T00:00:00.000Z
* '''Filing Date:''' 2014-05-19T00:00:00.000Z
* '''National Class:''' 435
* '''National Sub-Class:''' 006100
* '''Examiner Employee Number:''' 77475
* '''Art Unit:''' 1683
* '''Tech Center:''' 1600

== Rejection Summary ==
* 102 Rejections: 0
* 103 Rejections: 2

== Cited Patents ==
The following patents were cited in the rejection:

* [https://wikipatents.org/index.php?title=Special:Search&search=0242560 US 0242560]&#x1F517;
* [https://wikipatents.org/index.php?title=Special:Search&search=0031857 US 0031857]&#x1F517;
* [https://wikipatents.org/index.php?title=Special:Search&search=0053798 US 0053798]&#x1F517;
* [https://wikipatents.org/index.php?title=Special:Search&search=0233802 US 0233802]&#x1F517;
* [https://wikipatents.org/index.php?title=Special:Search&search=0166793 US 0166793]&#x1F517;

== Office Action Text ==
<pre>


    DETAILED ACTION
CONTINUED EXAMINATION UNDER 37 CFR 1.114 AFTER FINAL REJECTION

A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection.  Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.  Applicant’s submission of RCE and the amendment filed on February 12, 2025 have been entered.  The claims pending in this application are claims 47, 48, 55, 57-59, 61, 62, and 64-71. The rejections not reiterated from the previous office action are hereby withdrawn in view of applicant’s amendments filed on February 12, 2025. Claims 47, 48, 55, 57-59, 61, 62, and 64-71 will be examined. 

Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA  35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter 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 under pre-AIA  35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary.  Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA  35 U.S.C. 103(c) and potential pre-AIA  35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA  35 U.S.C. 103(a).
Claims 47, 55, 65, and 66-71 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Bignell et al., (US 2009/0233802 A1, published on September 17, 2009) in view of Makarov et al., (US 2007/0031857 A1, published on February 8, 2007), Beer et al., (US 2008/0166793 A1, published on July 10, 2008) and Hindson et al., (US 2011/0053798 A1, priority date: September 2, 2009). 
Regarding claims 47 and 55, Bignell et al., teach a sample preparation method, the method comprising: producing adaptor ligated fragments by ligating adaptors to fragments of a target nucleic acid from a sample wherein each of the adaptors comprises a sample barcode unique for the sample (ie., a tag sequence to code or track the identity of the samples) and a universal primer site; generating a mixture of one or more adaptor ligated fragments and a first primer pair comprising (i) a universal primer that hybridizes to the universal primer site of the one or more adaptor ligated fragments, and (ii) a locus-specific primer (ie., a sample specific primer) comprising a 3’ portion that hybridizes to a target locus in the one or more adaptor ligated fragments; conducting a first amplification reaction using the first primer pair in the mixture to yield amplicons comprising a copy of the sample barcode; immobilizing the amplicons on a solid support (eg., an array); sequencing the amplicons to obtain sequence data of the target nucleic acid from the sample; and assigning, using the sample barcode, the sequence data to the sample as recited in claim 47 wherein the target nucleic acid and a portion of the adaptor are double stranded as recited in claim 55 (see paragraphs [0011], [0012], [0014], [0015], [0048], [0052], [0070] to [0073], and [0305]). 
Regarding claim 65, Bignell et al., teach a method for sample preparation for multiplex sequencing, the method comprising: attaching adaptors and a barcode (ie., a tag sequence to code or track the identity of the samples) to genomic DNA fragments from a sample wherein the barcode is a unique for the sample; amplifying the genomic DNA fragments attached to the adaptors and barcode using a first primer that anneals to a universal site in the adaptors and a second primer (ie., a sample specific primer) that anneals to a target locus in the genomic DNA fragments to yield amplified fragments; conducting a secondary amplification on the amplified fragments to yield amplification products; and sequencing the amplification product (see paragraphs [0011], [0012], [0014], [0015], [0019], [0038], [0048], [0052], [0070] to [0073], and [0305]). 
Regarding claim 66, Bignell et al., teach a sample preparation method, the method comprising: attaching barcodes (ie., a tag sequence to code or track the identity of the samples) and copies of an adaptor to nucleic acid fragments, wherein the adaptor comprises a universal site, to yield adaptor-ligated fragments; conducting a first amplification on the adaptor ligated fragments using a first primer that anneals to the universal site and a second primer (ie., a sample specific primer) that annels to a target site in the nucleic acid fragments to yield amplified target; and conducting a second amplification on the amplified target (ie., amplified again prior to sequencing to generating an amplified array) (see paragraphs [0011], [0012], [0014], [0015], [0019], [0048], [0052], [0070] to [0073], and [0305]). 
Bignell et al., do not disclose generating droplets comprising one or more adaptor ligated fragments and a first primer pair comprising (i) a universal primer that hybridizes to the universal primer site on the one or more adaptor ligated fragments, and (ii) a locus-specific primer comprising a 3’ portion that hybridizes only to a target locus in one or more of the adaptor ligated fragments and not to the universal primer site; conducting a first amplification reaction in the droplets to yield amplicons comprising a copy of the sample barcode and releasing the amplicons from the droplets as recited in claim 47, attaching adaptors and a barcode to genomic DNA fragments from a sample within the droplets, amplifying the genomic DNA fragment attached to the adaptors and barcode within the droplets using a first primer that anneal to a universal site in the adaptors and a second primer that anneals only to a target locus in the genomic DNA fragments, wherein the second primer does not anneal to the universal site in the adaptors, to yield amplified fragments, and releasing the amplified fragments from the droplets as recited in claim 65, and conducting a first amplification in droplets on the adaptor ligated fragments using a first primer that anneals to the universal site and a second primer that anneals only to a target site in the nucleic acid fragments to yield amplified target and releasing the amplified target from the droplets as recited in claim 66. 
Makarov et al., teach to ligate an adaptor to the 5’ end, the 3’ end, or both strands of DNA and perform a ligation-mediated PCR amplification by using a locus-specific primer (or several nested primers) and a universal primer complementary to the adaptor sequence (see paragraph [0006]). 
	Beer et al., teach to make droplets having a sample, amplify nucleic acids from the sample in the droplets, release amplified nucleic acids from the droplets, and further analyze amplified nucleic acid released from the droplets (see paragraphs [0034] to [0038], [0041], [0042], [0059], and [0076], and Figure 1). 
Hindson et al., teach to form fused droplets that combine a nucleic acid sample and PCR reagents by fusing a sample-containing droplet and a reagent-containing droplet and performing a PCR assay in the fused droplets and advantages of performing a PCR in a droplet (see paragraphs [0006] to [0008], [0031], [0081], [0091], [0099], [0105] and [0111] and claims 1-5). 
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the methods recited in claims 47, 65, and  66 by generating droplets comprising the adaptor ligated fragments and a first primer pair comprising (i) a universal primer that hybridizes to the universal primer site, and (ii) a locus- specific primer comprising a 5’ priming site and a 3’ portion that hybridizes to a locus in the target nucleic acid, conducting a first amplification reaction in the droplets to yield amplicons comprising a copy of the sample barcode and releasing the amplicons from the droplets, attaching adaptors and a barcode to genomic DNA fragments from a sample within the droplets, amplifying the fragments within the  droplets using a first primer that anneals to a universal site in the adaptors and a second primer that anneals to a target locus in the genomic DNA fragments to yield amplified fragments, and releasing the amplified fragments from the droplets, and conducting a first amplification in droplets on the adaptor ligated fragments using a first primer that anneals to the universal site and a second primer that hybridizes to a target site in the nucleic acid fragments to yield amplified target and releasing the amplified target from the droplets in view of the prior arts of Bignell et al., Makarov et al., Beer et al., and Hindson et al.. One having ordinary skill in the art would have been motivated to do so because Makarov et al., teach to ligate an adaptor to the 5’ end, the 3’ end, or both strands of DNA and perform a ligation-mediated PCR amplification by using a locus-specific primer (or several nested primers) and a universal primer complementary to the adaptor sequence (see paragraph [0006]), Beer et al., teach to make droplets having a sample, amplify nucleic acids from the sample in the droplets, release amplified nucleic acids from the droplets, and further analyze amplified nucleic acid released from the droplets (see paragraphs [0034] to [0038], [0041], [0042], [0059], and [0076], and Figure 1), and Hindson et al., have successfully formed fused droplets that combine a nucleic acid sample and PCR reagents by fusing a sample-containing droplet and a reagent-containing droplet and performed a PCR assay in the fused droplets (see paragraphs [0081], [0091], [0099], [0105] and [0111] and claims 1-5) and teach advantages of performing a PCR in droplets such as “[S]plitting a sample into droplets offers numerous advantages. Small reaction volumes (picoliters to nanoliters) can be utilized, allowing earlier detection by increasing reaction rates and forming more concentrated products. Also, a much greater number of independent measurements (thousands to millions) can be made on the sample, when compared to conventional bulk volume reactions performed on a micoliter scale. Thus, the sample can be analyzed more accurately (i.e., more repetitions of the same test) and in greater depth (i.e., a greater number of different tests). In addition, small reaction volumes use less reagent, thereby lowering the cost per test of consumables. Furthermore, microfluidic technology can provide control over processes used for generation, mixing, incubation, splitting, sorting, and detection of droplets, to attain repeatable droplet-based measurements”, “[A]queous droplets can be suspended in oil to create a water-in-oil emulsion (W/O). The emulsion can be stabilized with a surfactant to reduce or prevent coalescence of droplets during heating, cooling, and transport, thereby enabling thermal cycling to be performed. Accordingly, emulsions have been used to perform single-copy amplification of nuclei acid target molecules in droplets using the polymerase chain reaction (PCR)”, “[C]ompartmentalization of single molecules of a nucleic acid target in droplets of an emulsion alleviates problems encountered in amplification of larger sample volumes. In particular, droplets can promote more efficient and uniform amplification of targets from samples containing complex heterogeneous nucleic acid populations, because sample complexity in each droplet is reduced”, and “[T]he systems disclosed herein may offer substantial advantages over other approaches to mixing small volumes of fluid. These advantages may include any combination of the following: (1) an ability to mix small volumes of reactants with a sample on-demand; (2) scalable methods and apparatus to accomplish mixing of a large number of reagents with a sample in small-volume reaction vessels (e.g., femtoliter, picoliter, or nanoliter), a large numbers of samples with a reagent in small-volume reaction vessels, and/or reagents with samples in small volumes using reproducible processes on low-cost instrumentation; (3) high-throughput fluid mixing that uses minimum amounts of reagents to reduce assay costs; (4) an ability to screen a sample for the presence of one or up to thousands or more targets on the same instrument; (5) an activation step that can initiate mixing of small volumes and that does not require complex timing of droplet streams using precision instrumentation; (6) an ability to perform more complex mixing steps at a centralized facility, to permit an end user's instrument to have less complexity, thereby making tests easier to perform; (7) a high-throughput assay platform that reduces the number of consumables per test; and/or (8) accommodation of many test reagents and samples with a simplified instrument architecture with minimized fluidic complexity (such as by reducing the number of fluidic connections, valves, etc.)” (see paragraphs [0006] to [0008] and [0031]). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the methods recited in claims 47, 65, and 66 by generating droplets comprising the adaptor ligated fragments and a first primer pair comprising (i) a universal primer that hybridizes to the universal primer site, and (ii) a locus- specific primer comprising a 5’ priming site and a 3’ portion that hybridizes to a locus in the target nucleic acid, conducting a first amplification reaction in the droplets to yield amplicons comprising a copy of the sample barcode and releasing the amplicons from the droplets, attaching adaptors and a barcode to genomic DNA fragments from a sample within the droplets, amplifying the fragments within the  droplets using a first primer that anneals to a universal site in the adaptors and a second primer that anneals to a target locus in the genomic DNA fragments to yield amplified fragments, and releasing the amplified fragments from the droplets, and conducting a first amplification in droplets on the adaptor ligated fragments using a first primer that anneals to the universal site and a second primer that hybridizes to a target site in the nucleic acid fragments to yield amplified target and releasing the amplified target from the droplets in view of the prior arts of Bignell et al., Makarov et al., Beer et al., and Hindson et al., in order to take above advantages of performing a PCR in droplets and further analyze amplified nucleic acid released from the droplets. 

Claims 48, 57-59, 61, 62, and 64 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Bignell et al., in view of Makarov et al., Beer et al., and Hindson et al., as applied to claims 47, 55, 65, and 66-71 above, and further in view of Gunderson et al., (US 2008/0242560 A1, published on October 2, 2008) and Technology Spotlight: Illumina® Sequencing (published on October 11, 2010). 
The teaching of Bignell et al., Makarov et al., Beer et al., and Hindson et al., have been summarized previously, supra. 
Bignell et al., Makarov et al., Beer et al., and Hindson et al., do not disclose that the immobilizing step comprises attaching the amplicons to single-stranded oligonucleotides bound to the surface of a flow cell, wherein the amplicons comprise sequences from the ligated adapter and the universal priming site that hybridize to the single-stranded oligonucleotides, and performing a bridge polymerase chain reaction (PCR) on the attached amplicons to form a nucleic acid cluster on the flow cell as recited in claim 48 wherein the first A adaptor sequence and the second B adaptor sequence are complementary to the single-stranded oligonucleotides bound to the surface of the flow cell as recited in claim 64. However, Bignell et al., teach that the sequencing step creates at least a first read from a genomic region of the target nucleic acid and at least a second read from the sample barcode as recited in claim 57, assigning the first read from the genomic region of the target nucleic acid to the sample using the second read from the barcode as recited in claim 58 wherein the barcode comprises about 4 to 20 nucleotides as recited in claim 59, the barcode comprises no homopolymer regions as recited in claim 61, and the adaptors comprise a first A adaptor sequence (eg., Seq 1) and a second B adaptor sequence (ie., Seq 2) as recited in claim 62 (see paragraphs [0011], [0012], [0014], [0015], [0019], [0048], [0052], [0070] to [0073], and [0305], and Figures 1 and 8). 
Since Technology Spotlight: Illumina® Sequencing teaches to immobilize adaptor-ligated nucleic acid fragments to a flow cell surface of an array by hybridizing the adaptor-ligated nucleic acid fragments to single-stranded oligonucleotides bound to the surface of the flow cell surface during a process of performing a bridge amplification (see page 1, left column and Figures 1-13) and Gunderson et al., teach to attach adaptor-ligated nucleic acid fragments to an array platform commercially available from Illumina Inc. and perform a bridge amplification (see paragraphs [0077], [0086] to [0088] and [0097]), Gunderson et al., in view of Technology Spotlight: Illumina® Sequencing disclose attaching a nucleic acid (eg., an amplicon) to single-stranded oligonucleotides bound to the surface of a flow cell, wherein the nucleic acid comprise sequences from the ligated adapter that hybridize to the single-stranded oligonucleotides, and the method further comprises performing a bridge polymerase chain reaction (PCR) on the attached nucleic acid to form a nucleic acid cluster on the flow cell as recited in claim 48 wherein the first A adaptor sequence and the second B adaptor sequence are complementary to the single-stranded oligonucleotides bound to the surface of the flow cell as recited in claim 64.
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the methods recited in claims 48 and 64 by 
attaching the amplicons released from the droplets to single-stranded oligonucleotides bound to the surface of a flow cell and performing a bridge polymerase chain reaction (PCR) on the attached amplicons to form a nucleic acid cluster on the flow cell wherein the amplicons comprise sequences from the ligated adapter and the universal priming site that hybridize to the single-stranded oligonucleotides and the first A adaptor sequence and the second B adaptor sequence are complementary to the single-stranded oligonucleotides bound to the surface of the flow cell in view of the prior arts of Bignell et al., Makarov et al., Beer et al., Hindson et al., Gunderson et al., and Technology Spotlight: Illumina® Sequencing. One having ordinary skill in the art would have been motivated to do so because Gunderson et al., in view of Technology Spotlight: Illumina® Sequencing disclose attaching a nucleic acid (eg., an amplicon) to single-stranded oligonucleotides bound to the surface of a flow cell, wherein the nucleic acid comprise sequences from the ligated adapter that hybridize to the single-stranded oligonucleotides, and the method further comprises performing a bridge polymerase chain reaction (PCR) on the attached nucleic acid to form a nucleic acid cluster on the flow cell as recited in claim 48 wherein the first A adaptor sequence and the second B adaptor sequence are complementary to the single-stranded oligonucleotides bound to the surface of the flow cell as recited in claim 64 and Gunderson et al., have suggested advantages of a bridge amplification such as a bridge amplification can be performed isothermally by physically exposing the surface to alternating cycles of denaturation and extension and generate colonies or polonies for sequencing applications (see paragraphs [0045] and [0097). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the methods recited in claims 48 and 64 in view of the prior arts of Bignell et al., Makarov et al., Beer et al., Hindson et al., Gunderson et al., and Technology Spotlight: Illumina® Sequencing in order to form nucleic acid clusters or colonies or polonies on flow cells for sequencing applications.

Response to Arguments
Applicant’s arguments with respect to claims 47, 48, 55, 57-59, 61, 62, and 64-66 have been considered but are moot because the new ground of rejection does not rely on any teaching or matter specifically challenged in the argument.

Conclusion
6.	No claim is allowed. 
7.	Papers related to this application may be submitted to Group 1600 by facsimile transmission.  Papers should be faxed to Group 1600 via the PTO Fax Center.  The faxing of such papers must conform with the notices published in the Official Gazette, 1096 OG 30 (November 15, 1988), 1156 OG 61 (November 16, 1993), and 1157 OG 94 (December 28, 1993)(See 37 CAR § 1.6(d)).  The CM Fax Center number is (571)273-8300. 
	Any inquiry concerning this communication or earlier communications from the examiner should be directed to Frank Lu, Ph.D., whose telephone number is (571)272-0746.  The examiner can normally be reached on Monday-Friday from 9 A.M. to 5 P.M.
 	If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Dr. Anne Gussow, Ph.D., can be reached on (571)272-6047.   
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system.  Status information for published applications may be obtained from either Private PAIR or Public PAIR.  Status information for unpublished applications is available through Private PAIR only.  For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/FRANK W LU/Primary Examiner, Art Unit 1683                                                                                                                                                                                                      
May 16, 2025











    
        
            
        
            
        
            
        
            
        
            
        
            
        
            
        
            
        
            
        
            
        
            
        
            
    

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[[Category:103_Rejections]]
[[Category:Rejection_Report_04th Jun 2025]]

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