DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
The previous Office Action, the Final Rejection mailed 12/10/2025, is vacated in favor of the present Non-Final Rejection.
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 filed on 10/29/2025 has been entered.
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claims 3, 5, and 6 have been canceled. Claims 14-24 remain withdrawn. Claims 2 and 7-13 are pending and are examined on the merits herein.
Response to Applicant’s Amendments
Claim Objections
Claim 2 was rejected due to a minor informality. In light of Applicant’s amendments to the claims submitted 10/29/2025, this objection has been withdrawn. See also new grounds of objection below.
35 USC 103 Rejections
Claims 2-3 and 5-13 were rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo et al. (US 2009/0029377 A1), in view of Mir et al. (US 2010/0120038 A1), and in view of Chan et al. (Clinical Chemistry, 2004). In light of Applicant’s amendments to the claims, these rejections have been withdrawn for all currently pending claims. Claims 3, 5, and 6 are canceled, and so these rejections have been rendered moot. However, see “Response to Applicant’s Arguments” and new grounds of rejection below.
Duplicate Claims
Claims 3 and 5 were found to be substantial duplicates of one another. These claims have been canceled, and so this finding is rendered moot.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that the amplification taught by Mir, which is cited to read on the amplification described by the instant claims, does not meet the limitations of the instant claims, particularly with regard to the first set of primers (which in Mir, would be the pre-preamplification primers). Specifically, Applicant states that the claimed first primers, which have now been amended to distinguish one sample from another, can be considered to have an index sequence with which to do so, which is not taught by Mir at the pre-preamplification step. Additionally, Applicant states that Mir does not meet the limitation of (ii) of claim 2, which requires that that the first primers contain a sequence common to which the second primers hybridize to. Allegedly, the nested primer design of Mir does not meet this limitation due to the explanation of nested primer design in Mir. Applicant also states that the references, alone or in combination, do not meet the limitations described by the final wherein clause of the claim (Remarks, pages 8-10).
As to Applicant’s arguments against Mir not teaching a common sequence on the first primers that the second primers can hybridize to, in the Final Rejection mailed 7/31/2025, regarding the pre-preamplification step, paras. 22-24 state that it would be prima facie that the pre-preamplification primers can also be tagged, and that the pre-preamplification primer tags can be a common sequence that the preamplification primers can hybridize to. Specifically, the tags of Mir can be common to a type of primer, and thus these tags would be common among all pre-preamplification primer sequences (see paras. 10 and 94-95 for example). Applicant does not appear to address a majority of these teachings or rationale in their arguments. Applicant mainly discusses nested primers, which are detailed in para. 23 of the Final Rejection to mainly point out that Mir generally teaches that the primers for the different amplification reactions may overlap in sequence and hybridize to one another, and not to suggest that nested primers imply a common sequence that is specific among all of a particular type of primer.
As to a sample-specific tag on the first amplification primers to distinguish between maternal nucleic acids and other nucleic acids in the sample, Mir does teach the use of sample-specific tags, as noted in para. 23 of the Final Rejection. These sample-specific tags used in the Final Rejection are taught by Mir as being on the preamplification and amplification primers, and not necessarily on the pre-preamplification primers. However, Mir does teach the use of multiple tags on primers, and given the teachings of the reference, along with ordinary skill, knowledge, and creativity that the ordinary artisan would possess, these teachings can also be used to read on the currently amended claims. See the new grounds of rejection below. See also MPEP 2141.03, which discusses the capabilities of the ordinary artisan.
In the newly amended claims, it is noted that although the 300 first primers must now comprise a sequence that distinguishes polynucleotides in the maternal blood sample from polynucleotides in another sample, this sequence does not have to be a different sequence than the common sequence for the first amplification primers.
Finally, regarding Applicant’s arguments concerning the final wherein clause of the claim, Applicant does not provide any substantive reason for believing the cited references do not read on this limitation. Mir teaches that the tagged and amplified sequences may be analyzed by DNA sequencing, where the tags may be used (para. 160). This use of tags is presented as an alternative to the ligation of sequencing adapters, which would hybridize to sequencing primers. Thus, Mir does teach that their tag sequences may be used to hybridize to sequencing primers, and so the common tag sequences on the preamplification and/or the amplification primers could be used for such purposes.
Thus, Applicant’s arguments are overall not considered persuasive, and new grounds of rejection are provided below in tandem with previously presented teachings/rationale solely in order to address Applicant’s new claim amendments.
Claim Objections
Claim 7 is objected to because of the following informality: in line 2, “sequences comprises” should read “sequences comprise.” Appropriate correction is required.
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.
The factual inquiries for establishing a background for determining obviousness under pre-AIA 35 U.S.C. 103(a) are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 2 and 7-13 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo et al. (US 2009/0029377 A1), in view of Mir et al. (US 2010/0120038 A1), and in view of Chan et al. (Clinical Chemistry, 2004).
Lo teaches methods for determining whether fetal chromosomal aneuploidy exists via analysis of a maternal sample (Abstract). Said maternal sample can be blood (para. 3). Cell-free fetal DNA in maternal plasma can specifically be analyzed (para. 14). When detecting chromosomal abnormalities, sequences can be analyzed from a first chromosome and a second chromosome and then compared (paras. 16 and 18). N fetal sequences can be analyzed, where N is chosen based on user needs, and so could be at least 300 (paras. 17 and 81). While Lo shows measurements of the amounts of all chromosomes in fetal samples are possible (e.g. Figure 4A-B), this reference specially discusses examples related to trisomy 21 (para. 77; instant claim 13). When analyzing aneuploidies for chromosome 21, “only a portion of the human genome needs to be sequenced to differentiate trisomy 21 from euploid cases. Thus, it would be possible and cost-effective to enrich the pool of nucleic acids to be sequenced prior to random sequencing of a fraction of the enriched pool,” (para. 77). Lo teaches that such enrichment techniques can involve hybridization (para. 79). After said enrichment, sequencing can occur (para. 79). In a particular trisomy 21 example, Lo teaches that sequencing can occur via the Illumina Genome Analyzer (para. 92). Compilation of all sequencing reads can then occur (para. 94). Paras. 95 through 104 then explain how this information is used to determine if trisomy 21 was present.
However, though Lo does generally teach enrichment (e.g. paras. 47, 72, 77-79), they do not teach specific enrichment methods, such as those described in instant claim 2. Additionally, Lo does not specifically teach primers with common sequences. It is noted however that Lo does teach that random sequencing, “may be preceded by procedures to enrich a biological sample with particular populations of nucleic acid molecules sharing certain common features,” (para. 47), though the reference does not provide examples of what these features may be.
Mir teaches assay methods to increase the number of target nucleic acids that can be analyzed in a single assay (Abstract). Specifically, Mir teaches a preamplification encoding reaction with specific forward and reverse primers, where either the forward or reverse primers contain a common sample-specific nucleotide tag (paras. 8-10). The number of primers that can be used is represented by T, where T is simply an integer greater than 1 (para. 13). This preamplification is then followed by amplification with primer pairs, where the forward or reverse primers may anneal to the sample-specific tag (paras. 10 and 12). The produced tagged sequences may then be analyzed by DNA sequencing (para. 160). This use of tags is presented as an alternative to the ligation of sequencing adapters, which would hybridize to sequencing primers. Thus, Mir teaches that their tag sequences may be used to hybridize to sequencing primers, and so the common tag sequences on the preamplification and/or the amplification primers could be used for such purposes. Prior to the encoding reaction, Mir teaches that a pre-preamplification step may occur (para. 119). This step can be done in multiplex with target-specific primers. Mir teaches targeting 9216 different nucleic acids, and so at least 300 target-specific primers would be involved, as required by instant claim 2 (and at least 500, as required by instant claim 7; para. 119). The amplification step can include performing PCR or means of linear amplification (para. 69; instant claims 10-11). In Mir, a “common sample-specific nucleotide tag” refers to a tag that is “linked to all target nucleotide sequences produced during an encoding reaction, such that all tagged target nucleotide sequences produced from a given sample are each identified by a tag having the same sequence,” (para. 66). In para. 94, Mir states, “All tagged target nucleotide sequences produced from a given sample can be tagged with a common sample-specific nucleotide tag, i.e., one that has the same nucleotide sequence.” As for the tag composition, the tag can be “a predetermined nucleotide sequence that is added to a target nucleotide sequence,” (para. 61).
Regarding the pre-preamplification step, Mir teaches that, “To increase specificity, the primers employed for preamplification can be nested relative to primers employed for pre-preamplification,” (para. 119), and so the preamplification primers can hybridize to sequences on the pre-preamplification primers. The pre-preamplification primers are target-specific (and so target specific loci), but Mir does not explicitly mention that these sequences can have tags. However, the ordinary artisan would recognize that these pre-preamplification primers could also be tagged, as many of the other primers described by Mir are tagged. Additionally, Mir teaches embodiments in which multiple tags are employed, and are in common among each of a particular type of primer (i.e. forward or reverse primers) for a given amplification reaction. Paras. 13-15, 16-18, and 19-20 describe using at least two different tags, and para. 21 describes an embodiment in which a single primer can have two different types of tags – a sample specific-tag and a set-specific tag. In this embodiment, the preamplification primers contain the two tags, and the amplification primers anneal to the set-specific tags (paras. 22-23). Figure 5 of Mir also shows that when multiple tags are employed in the preamplification step when using multiple samples, during the amplification step, a tag primer can be used that will ensure amplification only occurs when the correct tag is present. A similar process is also noted in para. 33. Para. 101 also notes the use of two tags that are not limited to sample and set specific tags.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the enrichment method of Mir in the overall method of Lo to arrive at the method of instant claim 2. This would entail utilizing the three amplification steps (pre-preamplification, preamplification, and amplification) as taught by Mir in the method of Lo. It would be prima facie obvious to add a common tag to the pre-preamplification primers of Mir that the preamplification primers can then hybridize to, similar to the structure of the preamplification and amplification primers of Mir. This common tag could be the sample-specific tag taught by Mir that could then distinguish particular maternal samples from others in the method of Lo in view of Mir, which would be useful in multiplex amplification settings, as taught by the reference. By incorporating a sample-specific tag on the first amplification primers used (i.e. the pre-preamplification primers), this would allow for amplification products to immediately be tagged with sample information, as opposed to waiting until later amplification reactions. This would increase overall reaction accuracy and would allow for deeper analysis of each amplification step to determine amplification efficiencies for particular samples. As Mir teaches that these tags can be placed on primers, there would be no increased difficulty with utilizing these sequences on the pre-preamplification primers compared to using them on preamplification or amplification primers. These sample-specific tags would also act as common sequences among all of the pre-preamplification primers. Additionally, as Mir teaches that two tags (sample-specific and set-specific tags) may be placed on primer sequences, and specifically on the preamplification primers, where the amplification primers anneal to the set-specific tags (paras. 22-23), these specific amplification methods can also be used in the method of Lo in view of Mir while still reading on the instant claims (i.e. the set-specific tags would act as common sequences for the preamplification primers that the amplification primers hybridize to).
By utilizing two common tag sequences, the ordinary artisan would recognize that this would allow for better sorting of sequence reads in the method of Lo in view of Mir. Specifically, this would ensure that only reads which have gone through all three rounds of amplification are considered, and would not include reads that may have resulted from improper hybridization or incomplete amplification. Mir also notes that by nesting primers, this can increase specificity (paras. 55, 58, 119), and by performing nesting over three rounds of amplification, this will increase specificity even further. These rationale would motivate the ordinary artisan. There would be a reasonable expectation of success because Mir already teaches designing primers with common sequences that can be hybridized to by other primer sequences, and also generally teaches that methods of primer design are commercially available and/or are well-known to the ordinary artisan (paras. 126-129). This would be adding a “cost-effective enrichment” method described by Lo (para. 77) to specifically enrich the desired target sequences in order to determine if trisomy 21 exists in a fetal sample, and would also result in enriched sequences containing sequencing primer hybridization regions. Mir also teaches that their enrichment methods minimize increases in assay costs (para. 37), making it appealing to the ordinary artisan. Lo also teaches that hybridization enrichment techniques would reduce sequencing costs (para. 79). Mir’s methods are shown to work on human genomic DNA (para. 35 and Figure 8), and as noted above, are compatible with sequencing methods, so there would be a reasonable expectation of success.
However, neither Lo nor Mir teach that the non-random polynucleotide sequences are 10-500 nucleotides in length. Lo describes choosing fragments of a particular size to sequence, but does not teach the overall size range of the sequences that are enriched (e.g. paras. 61 and 93).
Chan teaches the size distribution of maternal and fetal DNA in plasma, specifically focusing on circulating DNA (Abstract, page 88, column 2, para. 2, and page 91, column 1, para. 3). This was analyzed via PCR (pages 89-90, “Size Distribution Analysis of Fetal DNA in Maternal Plasma”). The results are shown in Figure 1B, where fetal plasma is shown in blue. Chan teaches that less than 1% of fetal amplicons were longer than 313 bp, but also shows that the highest concentration of fetal plasma had a length of about 130 bp to about 190 bp (page 90, “Size Distribution of Fetal-Derived DNA in Maternal Plasma” and Figure 1B).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Chan to enrich and sequence the most common fetal DNA fragment sizes in the method of Lo in view of Mir. In this case, that would mean a length of about 130 bp to about 190 bp. Choosing this relatively small size range would allow for primer lengths for each non-random polynucleotide sequence to remain of similar sizes, and would also ensure that outlier fetal DNA fragments with atypical sizes (such as those above 300 bp) are not analyzed. The 130 bp to 190 bp range overlaps with those ranges described by the instant claims (10-500 bp in claim 2, 10-260 bp in instant claim 8, and 50-150 bp in instant claim 9). MPEP 2144.05 I states that “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).” In the absence of critical or unexpected results utilizing these ranges disclosed by Applicant, using the most common fetal DNA size range disclosed by Chan would therefore render the claimed ranges obvious.
Thus, claims 2, 7-11, and 13 are prima facie obvious over Lo, in view of Mir, and in view of Chan.
Regarding claim 12, Lo teaches that the same pool of nucleic acids can be sequenced multiple times to achieve several folds of coverage. Specifically, “In such situations, the number of times a particular nucleic acid species have been sequenced relative to that of another nucleic acid species correlate with their relative concentrations in the original sample,” (para. 68). Thus, if a nucleic acid appears more often in a sample relative to another nucleic acid, it will have more sequencing coverage. Mir teaches that their encoding reaction and final amplification reaction can be one or multiple cycles, up to 40 for each reaction (paras. 62, 70, and 159). This reference also teaches that “one can simply monitor the amount of amplification product after a predetermined number of cycles sufficient to indicate the presence of the target nucleic acid sequence in the sample. One skilled in the art can easily determine, for any given sample type, primer sequence, and reaction condition, how many cycles are sufficient to determine the presence of a given target nucleic acid,” (para. 156). Thus, Lo, in view of Mir, and in view of Chan teaches that the ordinary artisan can gauge how much amplification product will be produced after a certain number of amplification cycles. The ordinary artisan can thus ensure that desired sequences appear greater than 5x more often than undesired sequences in an amplification product pre-sequencing. Additionally, although the sequencing methods of Lo encompass random sequencing (para. 80), Lo’s teachings about performing multiple sequencing runs can ensure that target sequences are present in the final product in desired amounts before analysis.
Thus, claim 12 is prima facie obvious over Lo, in view of Mir, and in view of Chan.
Conclusion
No claims are currently allowable.
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/GARY BENZION/Supervisory Patent Examiner, Art Unit 1681
/F.F.G./Examiner, Art Unit 1681