Office Action 1: Non-Final Rejection (2024-10-09)
Rejection types: 103 - Obviousness, 112 - Written Description/Enablement, Double Patenting

CLAIMS:
CLAIMS
1\. A method of characterizing polynucleotide molecules, said method comprising the steps of: (a) providing a solid support comprising a surface; (b) providing a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of one or more target sequences, wherein each polynucleotide molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface, wherein the polynucleotide molecules are arranged on the surface such that at least thirty percent of the polynucleotide molecules are optically resolvable; and then (c) detecting complete or partial nucleotide sequences of said one or more target sequences from said polynucleotide molecules; wherein the solid support is not a bead.
2\. The method of claim 1, wherein density of said polynucleotide molecules of the surface is at least 1,000 per μm2.
3\. The method of claim 1, wherein said concatemers have a diameter of from 100 to 300 nm.
4\. The method of claim 1, wherein said concatemers are generated by rolling circle replication.
5\. The method of claim 1 wherein each of said concatemers contains at least 10 copies of its respective target sequences.
6\. The method of claim 1 wherein said nucleotide sequences are detected by probe hybridization, probe-probe ligation, or sequencing by synthesis.
7\. The method of claim 6 wherein said nucleotide sequences are detected by scanning electron microscopy, near field scanning optical microscopy (NSOM), or total internal reflection fluorescence microscopy (TIRFM).

OFFICE ACTION:
DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
1.	The present application is being examined under the pre-AIA  first to invent provisions.
Specification
2.	The use of trade names or marks used in commerce (including but not necessarily limited to Alexa-Fluor and Cascade Blue), has been noted in this application. The term should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM, or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
3.	Claim  2 is objected to because of the following informalities: the phrase molecules of the surface should read molecules on the surface.  Appropriate correction is required.
Priority
4.	Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 112 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA  35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed provisional applications, Application Nos. 60/690,771, 60/725,116, and 60/776,415, fail to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA  35 U.S.C. 112, first paragraph for one or more claims of this application.
Claim 1 is drawn to “polynucleotide molecules are arranged on the surface such that at least thirty percent of the polynucleotide molecules are optically resolvable.” These provisional applications do not describe or illustrate or suggest “thirty percent of the polynucleotide molecules are optically resolvable,” and therefore do not provide adequate support under 35 USC §112 for the invention of claim 1 and those that depend from claim 1. Therefore the effective filing date for claims 1-7 is 06/13/2006.
Claim Rejections - 35 USC § 112
Claim Rejections - 35 USC § 112 1st
5.	The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA  35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
6.	Claim 2 is rejected  under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement.  The claim contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Claim 2 states “wherein the density of said polynucleotide molecules of the surface is at least 1,000 per µm2.” However, applicant’s specification states that the diameter of the polynucleotides is 100-300 nm ([0036], lines 1-2, [0065] line 6). If the diameter of these polynucleotides was 100 nm, then only 100 of them could fit in a 1 µm x 1 µm square (10 rows of 10). Applicant’s specification does not describe how one would reduce the size of said polynucleotides to satisfy this requirement.
Additionally, applicant’s specification states that each discrete region on the surface has an area that permits the capture of no more than a single molecule ([0009], lines 13-14) and that each region has a linear dimension of between 125 nm to 250 nm ([0042], line 3). No guidance is provided as to how discrete regions with a linear dimension of 125 nm, comprising a single polynucleotide molecule, could fit 1,000 polynucleotide molecules in an area of only 1 µm2.
Applicant’s claimed polynucleotide/feature density also stands out from the state of the prior art at the time of the earliest available priority date. For example, Steemers et al (US Patent Application No. US 20050181394 A1, published 2005-08-18) describes a “very high density” molecular array with a feature density of about 2,000,000,000 array locations/cm2, or about 20 array locations/µm2. Applicant’s specification does not adequately address the 50-fold difference between the polynucleotide density provided in claim 2 with the state of the prior art at the time.
In order to make the claimed invention as described, one would have to develop techniques to reproducibly prepare smaller concatemers while maintaining the claimed properties of the array (i.e., polynucleotide molecules that have a random coil state, and that are arranged on the surface such that at least thirty percent of the polynucleotide molecules are optically resolvable). Further experimentation is required to prepare smaller concatemers, possibly by careful timing of the rolling circle replication (RCR) reaction, or by altering the conditions of the RCR reaction (i.e., adjusting enzyme/template/primer quantity, altering salt concentration or temperature, addition of reaction additives to increase molecular crowding or alter the melting temperatures of nucleic acid species). Alternatively, the existing concatemers of the invention could be shrunk in order to achieve an appropriate size, possibly by adjusting the salt or pH of the buffer, although the applicant’s specification suggests the diameter of the claimed polynucleotides is as described under “conventional conditions” ([0036]).
The applicant’s specification provides no guidance on how concatemers of this size would be achieved, and it would not be immediately understood by one of ordinary skill in the art how (and to what extent) the above alterations would affect the diameter of the claimed polynucleotides. Following all of this, it is concluded that it would require undue experimentation to make and use the claimed invention.
Claim Rejections - 35 USC § 112 2nd
7.	The following is a quotation of 35 U.S.C. 112(b):
(b)  CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
8.	Claim 2 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA  35 U.S.C. 112, the applicant), regards as the invention.
Claim 2 is indefinite in its recitation of “wherein the density of said polynucleotide molecules of the surface is at least 1,000 per µm2” as the said polynucleotide molecules from claim 1 comprise concatemers, and concatemers are disclosed in applicant’s specification as having a diameter of 100-300 nm ([0036] lines 1-2). With a diameter of even 100 nm, only 100 polynucleotides could be disposed on the surface in an area of 1 µm2, and so it is unclear what is intended when the claim recites the stated density in combination with the recitation that the polynucleotides are concatemers.
Claim Rejections - 35 USC § 103
9.	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.
10.	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).
11.	Claims 1 and 3-6 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Church et al (International Patent Application No. WO 2007120208 A2, filed 2006-11-14 with effective priority to 2005-11-14, hereafter referred to as Church).
Regarding claim 1, Church teaches a method comprising steps of providing support-attached nucleic acids (i.e., polynucleotides) bound to a surface ([Abstract]). Church also teaches that these nucleic acids are amplified via rolling circle amplification (RCA) from an original template to generate many copies that are covalently attached to each other (i.e., a concatemer [045]). Long, ssDNA products from RCA reactions inherently have a random coil state.
It is noted that In re Best (195 USPQ 430) and In re Fitzgerald (205 USPQ 594) discuss the support of rejections wherein the prior art discloses subject matter which there is reason to believe includes functions that are newly cited or is identical to a product instantly claimed.  In such a situation the burden is shifted to the applicants to “prove that subject matter shown to be in the prior art does not possess the characteristic relied on” (205 USPQ 594, second column, first full paragraph).
Church teaches that these concatemers have a diameter of greater than 300 nm, and are immobilized on an array within a region having a diameter of less than 300 nm ([011]).
Church does not teach that each polynucleotide is attached to the surface within “a region substantially equivalent to a projection of the random coil on the surface.” The term “a region substantially equivalent” is defined in the application’s specification as either “a substantially circular region with a diameter equal to the root mean square of the end-to-end distance of such linear polymer” (i.e., a concatemer) or “a substantially circular region having a diameter that is one half or less that the total length of the polymer, or in another embodiment one tenth less; or in another embodiment, one hundredth less ([0010]).” The applicant’s specification also defines the root mean square of the end-to-end distance as roughly the diameter of the randomly coiled structure ([0036]). Thus, “a region substantially equivalent to a projection of the random coil” describes a substantially circular region having a diameter roughly equivalent to the diameter of the randomly coiled structure (i.e., the concatemer).
Given the breadth of the phrase “substantially equivalent”, the disclosure of Church clearly sets forth situation(s) where the claimed ranges of sized molecules relative to region size overlap with the ranges taught by the reference. In the case where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima facie case of obviousness exists. See MPEP 2144.05.    Church teaches the spot sizes (diameter of less than 300 nm) and the concatemer sizes (diameter greater than 300 nm) which encompass embodiments wherein these diameters would be considered substantially equivalent (e.g., a 290 nm spot and a 310 nm concatemer). Additionally, an ordinary artisan would have been motivated to have the spot size be slightly smaller than the diameter of the concatemer in order to ensure that only one concatemer molecule occupied any single spot on an array ([045]).
Church teaches that these concatemers were hybridized to a slide (i.e., a surface) and “almost every spot on the slide” was identifiably fluorescent (i.e., using optical microscopy) without any overlapping signal ([078]).  “Almost every spot on the slide” is much more than the required “at least 30 percent” of instant claim 1.
Church teaches that the concatemers on the surface are sequenced (i.e., all or a portion of their sequence is determined [006]).
Church teaches bead-free amplicons (i.e., concatemers) on the array ([044]).
With regard to claim 3, the method of claim 1 is described above. Church teaches that concatemers have a diameter of at least 50, 75, 100, 150, 200, 300, 400, or 500 nm ([007]).
It is noted that the courts have stated where the claimed ranges “overlap or lie inside the ranges disclosed by the prior art” and even when the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have similar properties, a prima facie case of obviousness exists (see 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); Titanium Metals Corp. of America v. Banner, 778 F2d 775. 227 USPQ 773 (Fed. Cir. 1985) (see MPEP 2144.05.I).
The courts have also found that “where the general conditions of a claim
are disclosed in the prior art, it is not inventive to discover the optimum or
workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105
USPQ 233, 235 (CCPA 1955). See MPEP 2144.05.II.
Therefore, the claimed ranges merely represent an obvious variant and/or
routine optimization of the values of the cited prior art.
With regard to claim 4, Church teaches that the concatemer is a product of rolling circle amplification ([029]). Rolling Circle Replication (RCR) and RCA are used interchangeably in the art, see ([029]) in Church and ([0118], [0120], [0121], and [0161]) in the applicant’s specification.
With regard to claim 5, Church teaches that the concatemer product of an RCA of a 135 base pair template yields a single stranded nucleic acid product of approximately 70,000 base pair ([045]). This clearly demonstrates that an RCA product will have at least 10 copies of its respective sequencing template (i.e., target sequence).
With regard to claim 6, Church teaches that the RCA product is detected by the hybridization of fluorescently labeled probes ([022]).
12.	Claim 7 is rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Church as applied to claim 6 above, and further in view of Mir et al (US Patent Application No. US 20040248144A1, published 2004-12-09, hereafter referred to as Mir).
With regard to claim 7, the method of claim 6 is described above and fully incorporated here. Church teaches that concatemers are visualized by hybridization to fluorescent probes ([022]) and imaged using an epifluorescence microscope ([063]).
Church does not teach the detection of the concatemers (i.e., nucleotide sequences) by either scanning electron microscopy, near field scanning optical microscopy (NSOM), or total internal reflection fluorescence microscopy (TIRFM).
However, Mir teaches additional methods for the interrogation of molecular arrays, such as electron microscopy ([0210]), NSOM, and TIRFM ([0216]). Mir teaches that higher resolution detection systems (i.e., NSOM) provide improved spatial resolution ([0218]). It would have been obvious to one having ordinary skill in the art to have replaced the epifluorescence microscope used by Church with any of the higher resolution interrogation techniques taught by Mir.  One of ordinary skill in the art would have been motivated to make this modification in order to enable the use of higher density microarrays that would provide more sequencing information. In addition, it would have been obvious to the ordinary artisan that the known techniques in the cited references could have been combined with predictable results because the known techniques of the cited references predictably result in techniques useful for imaging DNA arrays.
Double Patenting
13.	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.
14.	Claims 1, 5, and 6 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-19 and 70 of copending Application No. 17522708.
Regarding claim 1 of the instant application, the copending application discloses:
A.	A support having a surface (Claims 1, 9 and 70 of the copending application);
B.	Providing a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of one or more target sequences (Claims 1 and 9 of the copending application);
C.	Wherein each polynucleotide molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface (Claims 1 and 9 of the copending application);
D.	Wherein the polynucleotide molecules are arranged on the surface such that at least thirty percent of the polynucleotide molecules are optically resolvable (Claims 1, 9 and 70 of the copending application);
E.	Detecting complete or partial nucleotide sequences of said one or more target sequences from said polynucleotide molecules (Claim 70 of the copending application) and;
F.	Wherein the solid support is not a bead (Claim 3 “planar surface” of the copending application).
The copending application does not teach all of the limitations instant claim 1 in a single claim.  However, it would have been obvious to one having ordinary skill in the art to have modified the copending claims to combine these disclosed elements because they are all disclosed in the copending claims as preferred embodiments of the invention.
Regarding claim 5 of the instant application, claim 18 of the copending application discloses that the concatemers have more than at least 10 copies of the target sequence.
Regarding claim 6 of the instant application, claim 70 of the copending application discloses that the concatemers are sequenced by probe-probe ligation.
This is a provisional nonstatutory double patenting rejection.
Claims 3 and 4 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-19 and 70 of copending Application No. 17522708 in view of Church et al (International Patent Application No. WO 2007120208 A2, filed 2006-11-14 with effective priority to 2005-11-14, hereafter referred to as Church).
The copending claims do not teach the limitations of claims 3 and 4.
Claims 3 and 4 were well-known in the art as taught by Church who specifically teaches concatemer diameters ([007]) and that concatemers are generated by rolling circle amplification ([029]), as fully discussed previously in this Office action and incorporated here.
It would have been obvious to have modified the copending claims so as to have included the limitations of instant claims 3 (diameter of the concatemers) and 4 (concatemers are generated by rolling circle amplification). An ordinary artisan would have been motivated to make this modification because rolling circle amplification was well-known in the art to result in concatemers containing multiple copies of a target sequence and a defined diameter. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques of the cited reference could have been combined with the claims of the copending application with predictable results, because both techniques relate to the production of concatemers on arrays.
This is a provisional nonstatutory double patenting rejection.
Claim 7 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-19 and 70 of copending Application No. 17522708 in view of Mir et al (US Patent Application No. US 20040248144A1, published 2004-12-09, hereafter referred to as Mir).
The copending claims do not teach the limitations of claim 7.
Claim 7 was well-known in the art as taught by Mir who specifically teaches that electron microscopy ([0210]), NSOM and TIRF ([0216]) can be used as interrogation/detection methods in nucleic acid sequencing applications, as fully discussed previously in the office action and incorporated here.
It would have been obvious to have modified the claims of the copending application to include the limitations of claim 7 (related to the detection elements of the invention). An ordinary artisan would have been motivated to make this modification in order to enable the use of higher density microarrays that would provide more sequencing information. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques in the cited reference could be combined with the techniques of the copending application with predictable results, because both techniques relate to the acquisition of sequencing information from microarrays.
This is a provisional nonstatutory double patenting rejection.
15.	Claims 1 and 3-6 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-36 of U.S. Patent No. US 9650673 B2 in view of Church et al (International Patent Application No. WO 2007120208 A2, filed 2006-11-14 with effective priority to 2005-11-14, hereafter referred to as Church).
With regard to claim 1 of the instant application, the claims of the patent disclose:
A.	A solid substrate comprising a surface (claim 29 of the patent);
B.	Providing a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of one or more target sequences (Claim 29 of the patent, having a random coil state is an inherent property of polynucleotide concatemers) and;
C.	Wherein each polynucleotide molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface (Claim 29 of the patent).
Regarding claim 3, the patent discloses that the concatemers have a diameter approximately equal to that of the discrete regions, and that the discrete regions have a diameter of 100-300 nm (claim 29 of the patent).
However, the patent does not teach the remaining limitations of claim 1: polynucleotide molecules arranged on the surface such that at least thirty percent are optically resolvable, detecting complete or partial sequences of the polynucleotides, or wherein the solid support is not a bead. Nor does it teach the limitations of claims 4-6.
Additional aspects of claim 1, as well as claims 4-6, were well-known in the art as taught by Church and fully discussed previously in this Office Action and incorporated here.
It would have been obvious to have modified the patent claims so as to have included the remaining limitations of claim 1, as well as the limitations of claims 5 and 6. An ordinary artisan would have been motivated to make this modification because Church’s teachings have the additional advantage of providing methods for the imaging and sequencing of concatemers on arrays such as those disclosed in the patent. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques of the cited reference could have been combined with the claims of the patent because both elements relate to the preparation of arrays of concatemers on a surface.
Claim 7 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-36 of US Patent No. 9650673 in view of Mir et al (US Patent Application No. US 20040248144A1, published 2004-12-09, hereafter referred to as Mir).
The patent claims do not teach the limitations of claim 7.
Claim 7 was well-known in the art as taught by Mir who specifically teaches that electron microscopy ([0210]), NSOM and TIRF ([0216]) can be used as interrogation/detection methods in nucleic acid sequencing applications, as fully discussed previously in the office action and incorporated here.
It would have been obvious to have modified the claims of the patent to include the limitations of claim 7 (related to the detection elements of the invention). An ordinary artisan would have been motivated to make this modification in order to enable the use of higher density microarrays that would provide more sequencing information. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques in the cited reference could be combined with the techniques of the patent with predictable results, because both techniques relate to the acquisition of sequencing information from microarrays.
16.	Claims 1 and 3-6 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-15 of U.S. Patent No. US 8609335 B2 in view of Church et al (International Patent Application No. WO 2007120208 A2, filed 2006-11-14 with effective priority to 2005-11-14, hereafter referred to as Church).
With regard to claim 1 of the instant application, the claims of the patent disclose:
A.	A solid substrate comprising a surface (claim 7 of the patent);
B.	Providing a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of one or more target sequences (Claim 7 of the patent, having a random coil state is an inherent property of polynucleotide concatemers) and;
C.	Wherein each polynucleotide molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface (this limitation is met by Church as fully addressed above and incorporated here).
D. 	Wherein the polynucleotide molecules are arranged on the surface such that at least thirty percent of the polynucleotide molecules are optically resolvable (Claim 7 of the patent);
E.	Detecting complete or partial nucleotide sequences of said one or more target sequences from said polynucleotide molecules (Claim 2 of the patent) and;
F.	Wherein the solid support is not a bead (this limitation is met by Church as fully addressed above and incorporated here).
The patent does not teach all of the limitations instant claim 1 in a single claim.  However, it would have been obvious to one having ordinary skill in the art to have modified the patent claims to combine these disclosed elements because they are all disclosed in the patent claims as preferred embodiments of the invention.
Regarding claim 6, the patent discloses that the target polynucleotide is sequenced by probe-probe ligation (claim 3).
The patent does not teach limitations C or F of claim 1, nor does it teach the limitations of claims 3-5.
Additional aspects of claim 1, as well as claims 3-5, were well-known in the art as taught by Church and fully discussed previously in this Office Action and incorporated here.
It would have been obvious to have modified the patent claims so as to have included limitation C and F of claim 1, as well as the limitations of claims 3-5. An ordinary artisan would have been motivated to make this modification because Church’s teachings have the additional advantage of providing methods for the imaging and sequencing of concatemers on arrays such as those disclosed in the patent. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques of the cited reference could have been combined with the claims of the patent because both elements relate to the preparation of arrays of concatemers on a surface.
Claim 7 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-15 of US Patent No. 8609335 in view of Mir et al (US Patent Application No. US 20040248144A1, published 2004-12-09, hereafter referred to as Mir).
The patent claims do not teach the limitations of claim 7.
Claim 7 was well-known in the art as taught by Mir who specifically teaches that electron microscopy ([0210]), NSOM and TIRF ([0216]) can be used as interrogation/detection methods in nucleic acid sequencing applications, as fully discussed previously in the office action and incorporated here.
It would have been obvious to have modified the claims of the patent to include the limitations of claim 7 (related to the detection elements of the invention). An ordinary artisan would have been motivated to make this modification in order to enable the use of higher density microarrays that would provide more sequencing information. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques in the cited reference could be combined with the techniques of the patent with predictable results, because both techniques relate to the acquisition of sequencing information from microarrays.
17.	Claims 1 and 3-6 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-21 of U.S. Patent No. US 8445196 B2 in view of Church et al (International Patent Application No. WO 2007120208 A2, filed 2006-11-14 with effective priority to 2005-11-14, hereafter referred to as Church).
With regard to claim 1 of the instant application, the claims of the patent disclose:
A.	A solid substrate comprising a surface (claim 16 of the patent) and;
B.	Providing a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of one or more target sequences (Claim 16 of the patent);
The patent does not teach the remaining limitations of claim 1, nor does it teach the limitations of claims 3-6.
Additional aspects of claim 1, as well as claims 3-6, were well-known in the art as taught by Church and fully discussed previously in this Office Action and incorporated here.
It would have been obvious to have modified the patent claims so as to have included the additional limitations of claim 1, as well as the limitations of claims 3, 5 and 6. An ordinary artisan would have been motivated to make this modification because Church’s teachings have the additional advantage of providing methods for the imaging and sequencing of concatemers on arrays such as those disclosed in the patent. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques of the cited reference could have been combined with the claims of the patent because both elements relate to the preparation of arrays of concatemers on a surface.
Claim 7 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-21 of US Patent No. 8445196 in view of Mir et al (US Patent Application No. US 20040248144A1, published 2004-12-09, hereafter referred to as Mir).
The patent claims do not teach the limitations of claim 7.
Claim 7 was well-known in the art as taught by Mir who specifically teaches that electron microscopy ([0210]), NSOM and TIRF ([0216]) can be used as interrogation/detection methods in nucleic acid sequencing applications, as fully discussed previously in the office action and incorporated here.
It would have been obvious to have modified the claims of the patent to include the limitations of claim 7 (related to the detection elements of the invention). An ordinary artisan would have been motivated to make this modification in order to enable the use of higher density microarrays that would provide more sequencing information. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques in the cited reference could be combined with the techniques of the patent with predictable results, because both techniques relate to the acquisition of sequencing information from microarrays.
18.	Claims 1 and 3-6 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-29 of U.S. Patent No. US 8133719 B2 in view of Church et al (International Patent Application No. WO 2007120208 A2, filed 2006-11-14 with effective priority to 2005-11-14, hereafter referred to as Church).
With regard to claim 1 of the instant application, the claims of the patent disclose:
A.	A solid substrate comprising a surface (claim 12 of the patent);
B.	Providing a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of one or more target sequences (Claim 12 of the patent, having a random coil state is an inherent property of polynucleotide concatemers) and;
C.	Wherein each polynucleotide molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface (Claim 13 of the patent).
D. 	Wherein the polynucleotide molecules are arranged on the surface such that at least thirty percent of the polynucleotide molecules are optically resolvable (this limitation is met by Church as fully addressed above and incorporated here);
E.	Detecting complete or partial nucleotide sequences of said one or more target sequences from said polynucleotide molecules (Claim 15 of the patent) and;
F.	Wherein the solid support is not a bead (this limitation is met by Church as fully addressed above and incorporated here).
The patent does not teach all of the limitations instant claim 1 in a single claim.  However, it would have been obvious to one having ordinary skill in the art to have modified the patent claims to combine these disclosed elements because they are all disclosed in the patent claims as preferred embodiments of the invention.
Regarding claim 5, the patent discloses that the concatemers contain at least 10 copies of their respective target sequences (claim 25).
The patent does not teach limitations D and F of claim 1, nor does it teach the limitations of claims 3, 4 and 6.
Additional aspects of claim 1, as well as claims 3, 4 and 6, were well-known in the art as taught by Church and fully discussed previously in this Office Action and incorporated here.
It would have been obvious to have modified the patent claims so as to have included limitations D and F of claim 1, as well as the limitations of claims 3, 4 and 6. An ordinary artisan would have been motivated to make this modification because Church’s teachings have the additional advantage of providing methods for the imaging and sequencing of concatemers on arrays such as those disclosed in the patent. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques of the cited reference could have been combined with the claims of the patent because both elements relate to the preparation of arrays of concatemers on a surface.
Claim 7 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-29 of US Patent No. 8133719 in view of Mir et al (US Patent Application No. US 20040248144A1, published 2004-12-09, hereafter referred to as Mir).
The patent claims do not teach the limitations of claim 7.
Claim 7 was well-known in the art as taught by Mir who specifically teaches that electron microscopy ([0210]), NSOM and TIRF ([0216]) can be used as interrogation/detection methods in nucleic acid sequencing applications, as fully discussed previously in the office action and incorporated here.
It would have been obvious to have modified the claims of the patent to include the limitations of claim 7 (related to the detection elements of the invention). An ordinary artisan would have been motivated to make this modification in order to enable the use of higher density microarrays that would provide more sequencing information. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques in the cited reference could be combined with the techniques of the patent with predictable results, because both techniques relate to the acquisition of sequencing information from microarrays.
19.	Claims 1 and 3-6 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 8445194 in view of Church et al (International Patent Application No. WO 2007120208 A2, filed 2006-11-14 with effective priority to 2005-11-14, hereafter referred to as Church).
Regarding claim 1 of the instant application, the patent discloses:
A.	A support having a surface (Claims 1, 11, 12 and 18 of the patent);
B.	Providing a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of one or more target sequences (Claims 1, 8 [concatemers produced by RCR will inherently have a random coil state], 11, 12 and 18 of the patent);
C.	Wherein each polynucleotide molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface (Claim 16 of the patent);
D.	Wherein the polynucleotide molecules are arranged on the surface such that at least thirty percent of the polynucleotide molecules are optically resolvable (Claims 1, 11 and 18 of the patent); and
E.	Detecting complete or partial nucleotide sequences of said one or more target sequences from said polynucleotide molecules (Claims 1, 11 and 18 of the patent);
The patent does not teach all of the limitations instant claim 1 in a single claim.  However, it would have been obvious to one having ordinary skill in the art to have modified the patent claims to combine these disclosed elements because they are all disclosed in the patent claims as preferred embodiments of the invention.
Regarding claim 4 of the instant application, claim 8 of the patent discloses that the concatemers were generated by rolling circle amplification.
Regarding claim 6 of the instant application, claims 1-6, 11 and 18 of the patent disclose that the concatemers are sequenced by probe-probe ligation.
However, the claims of the patent do not teach that the solid support is not a bead, nor do they teach the limitations of claims 3 or 5 in the instant application.
Church teaches solid supports that are not beads ([044]). It would have been obvious to one of ordinary skill in the art to modify the claims of the patent to use the bead-free substrates taught by Church. The ordinary artisan would have been motivated to make this modification in order to have arrays comprising attachment regions that clonally bind a single concatemer due to volume exclusion effects as taught by Church ([045]). It would have been obvious to one of ordinary skill in the art the known elements of the cited reference could be used to modify the claims of the patent with predictable results, because both elements related to the binding of concatemers to specific regions on the surface.
Regarding claim 3 of the instant application, Church teaches concatemer diameters ([007]).
Regarding claim 5 of the instant application, Church teaches that concatemers produced by rolling circle amplification have at least 10 copies of its target sequence ([045])
Claim 7 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 8445194 in view of Mir et al (US Patent Application No. US 20040248144A1, published 2004-12-09, hereafter referred to as Mir).
Claim 7 was well-known in the art as taught by Mir who specifically teaches that electron microscopy ([0210]), NSOM and TIRF ([0216]) can be used as interrogation/detection methods in nucleic acid sequencing applications.
It would have been obvious to have modified the claims of the patent to include the limitations of claim 7 (related to the detection elements of the invention). An ordinary artisan would have been motivated to make this modification in order to enable the use of higher density microarrays that would provide more sequencing information. In addition, it would have been obvious to one of ordinary skill in the art that the known techniques in the cited reference could be combined with the techniques of the patent with predictable results, because both techniques relate to the acquisition of sequencing information from microarrays.
Therefore, the inventions are not patentably distinct.
Conclusion
20.	No claims are allowed
21.


NEXT OFFICE ACTION: Notice of Pre-AIA  or AIA  Status
1.	The present application is being examined under the pre-AIA  first to invent provisions.
Status of the Claims
2.	This action is in response to papers filed 08 January 2025 in which claims 1, 2, 5, and 6 were amended and claims 8-14 were added.
The rejections in the Office action dated 09 October 2024 under U.S.C. § 112 1st and 2nd are withdrawn in view of the amendments.
The previous rejections under 103 are withdrawn in view of the amendments and Applicant’s Remarks submitted 08 January 2025.
The previous rejections for double patenting were overcome by the filing of appropriate terminal disclaimers.
Priority
3.	Applicant’s comments regarding priority documents and support for claim 1 have been found persuasive in view of the amendments.
EXAMINER’S AMENDMENT
4.	An examiner’s amendment to the record appears below. Should the changes and/or additions be unacceptable to applicant, an amendment may be filed as provided by 37 CFR 1.312. To ensure consideration of such an amendment, it MUST be submitted no later than the payment of the issue fee.
5.	Authorization for this examiner’s amendment was given in an interview with Michael Schiff on 15 January 2025.
6.	The application has been amended as follows:
In claim 1, line 7, the word ---binding--- was inserted between the word “a” and the word “region” so the claim now reads “…molecule is attached to the surface within a binding region substantially…”
In claim 12, line 7, the word ---binding--- was inserted between the word “a” and the word “region” so the claim now reads “…molecule is attached to the surface within a binding region substantially…”
Allowable Subject Matter
7.	The following is an examiner’s statement of reasons for allowance:
The instantly claimed invention is drawn to a method of characterizing polynucleotide molecules on a surface; wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of one or more target sequences, is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface, and wherein the surface is not a bead. Applicant’s amendments to claim 1 provide basis in the 60/725,116 provisional application, and overcome the earliest filing date of the cited prior art. The prior art does not teach or reasonably suggest the instantly claimed method of characterizing polynucleotides as defined by the claim.  The closest prior art is now Sabanayagam (US Patent Application No. US 20020076716 A1, published 2002-06-20). The reference teaches a surface comprising the polynucleotide molecules of the claim but does not teach that these polynucleotides molecules are attached to the surface within a binding region substantially equivalent to a projection of the random coil (of the claimed polynucleotide molecule) on the surface. The prior art does not provide a reason to modify the closest prior art to arrive at the claimed invention.
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
8.	Claims 1-14 are allowed.
9.

CITED REFERENCE SUMMARIES: Ref US20040248144A1: Claims:
A method for producing a molecular array which method comprises immobilising to a solid phase a plurality of molecules at a density which allows individual immobilised molecules to be individually resolved, wherein each molecule in the array is spatially addressable and the identity of each molecule is known or determined prior to immobilisation.
A method according to claim 1 wherein the molecules are applied to the solid phase by a method selected from printing, electronic addressing, in situ light-directed synthesis, ink jet synthesis or physical masking.
A method according to claim 2 wherein the molecules are applied to the solid phase by printing of dilute solutions.
A method for producing a molecular array which method comprises: (i) providing a molecular array comprising a plurality of molecules immobilised to a solid phase at a density such that individual immobilised molecules are not capable of being individually resolved; and (ii) reducing the density of functional immobilised molecules in the array such that remaining individual functional immobilised molecules are capable of being individually resolved; wherein each individual functional molecule in the resulting array is spatially addressable and the identity of each molecule is known or determined prior to the density reduction step.
(i) providing a molecular array comprising a plurality of molecules immobilised to a solid phase at a density such that individual immobilised molecules are not capable of being individually resolved; and
(ii) reducing the density of functional immobilised molecules in the array such that remaining individual functional immobilised molecules are capable of being individually resolved;
wherein each individual functional molecule in the resulting array is spatially addressable and the identity of each molecule is known or determined prior to the density reduction step.
A method according to claim 4 wherein the density of functional molecules is reduced by cleaving all or part of the molecules from the solid phase.
A method according to claim 4 wherein the density of functional molecules is reduced by functionally inactivating the molecules in situ.
A method according to claim 4 wherein the density of functional molecules is reduced by labelling some of the plurality of molecules such that individual immobilised labelled molecules are capable of being individually resolved.
A method according to any one of the preceding claims wherein the immobilised molecules are present within discrete spatially addressable elements.
A method according to claim 8 wherein the structure of molecules present in each discrete spatially addressable element is known and unintended structures are substantially absent.
A method according to claim 8 wherein a plurality of molecular species are present within one or more elements and each molecular species in an element can be distinguished from other molecular species in the element by means of a label.
A method according to any one of the preceding claims wherein the plurality of molecules which are capable of being individually resolved are capable of being resolved by optical means.
A method according to any one of the preceding claims wherein the plurality of scanning probe microscopy.
A method according to any one of claims 1 to 12 wherein the molecules are attached to the solid phase at a single defined point.
A method according to any one of claims 1 to 12 wherein the molecules are attached to the solid phase at two or more points.
A method according to any preceding claim, wherein the molecules comprise a detectable label.
A method according to claim 15 wherein the label can be read by optical methods.
A method according to claim 15 or claim 16 wherein the label is a single fluorescent molecule, nanoparticle or nanorod, or a plurality of fluorescent molecules, nanoparticles or nanorods.
A method according to claim 15 where the label can be read by SPM.
A method according to claim 18 wherein the label is a non-fluorescent molecule, nanoparticle or nanorod.
A method according to any one of claims 1 to 19 wherein the molecules are selected from defined chemical entities, oligonucleotides, polynucleotides, peptides, polypeptides, conjugated polymers, small organic molecules or analogues, mimetics or conjugates thereof.
A method according to claim 20 wherein the molecules are cDNAs and/or genomic DNA.
A method according to any one of the preceding claims wherein the immobilised molecules are present within discrete spatially addressable elements and each element comprises a distinct spatially addressable microelectrode or nanoelectrode.
A method according to claim 22 wherein said electrodes are formed of conducting polymers.
A method according to claim 23 wherein said electrodes are produced by a method selected from inkjet printing, soft lithography, nanoimprint lithography/lithographically induced self assembly, VLSI methods and electron beam writing.
A method according to any one of claims 1 to 24 wherein the immobilised molecules are immobilised onto a single electrode.
A method according to any one of claims 22 to 25 wherein the electrode(s) transduce a signal when a target molecule binds to an immobilised molecule present in the same element as an electrode.
A molecular array obtained by the method of any one of the preceding claims.
Use of a molecular array in a method of identifying one or more target molecules in a sample, which molecular array comprises a plurality of molecules immobilised to a solid phase at a density which allows individual immobilised molecules to be individually resolved, wherein each individual immobilised molecule in the array is spatially addressable and the identity of each immobilised molecule is known or encoded.
Use according to claim 28 wherein said method comprises contacting the array with the sample and interrogating one or more individual immobilised molecules to determine whether a target molecule has bound.
Use according to claim 29 wherein substantially all of the immobilised molecules are interrogated.
Use according to any one of claims 28 to 30 wherein interrogation is by an optical method.
Use according to claim 35 wherein the optical method is selected from far-field optical methods, near-field optical methods, epi-fluorescence spectroscopy, scanning confocal microscopy, two-photon microscopy, total internal reflection microscopy,
Use according to claim 36 where pulsed laser excitation illumination is coupled with Time-correlated single molecule counting (TCSPC) or synchronised time gating.
Use according to any one of claims 28 to 30 wherein interrogation is by scanning probe microscopy or electron microscopy.
Use according to any one of claims 28 to 34 wherein a physicochemical property of the immobilised molecules is determined, such as shape, size, mass, hydrophobicity or charge.
Use according to any one of claims 28 to 34 wherein an electromagnetic, electrical, optoelectronic and/or electrochemical property of the immobilised molecules is determined.
Use according to any one of claims 29 to 34 wherein a characteristic of a complex between an immobilised molecule and a target molecule is determined.
Use according to any one of claims 28 to 37 wherein the immobilised molecules are of the same chemical class as the target molecules.
Use according to any one of claims 28 to 37 wherein the immobilised molecules are of a different chemical class to the target molecules.
Use according to any one of claims 28 to 37 wherein the target molecules are genomic DNA or reduced complexity representations thereof.
Use according to claim 40 wherein complexity is reduced by fragmenting the target and pre-hybridising it to C 0 t=1 DNA
Use according to claim 40 or claim 41 wherein the genomic DNA undergoes whole genome amplification prior to analysis.
Use according to any one of claims 28 to 37 wherein the target molecules are mRNA or cDNA.
Use of a molecular array as defined in claim 28 in genetic analysis, gene expression studies, identifying one or more molecules in the array which interact with a molecular target or in the detection or typing of single nucleotide polymorphisms in a sample of nucleic acids, haplotyping or sequencing.
Use of a molecular array as defined in claim 32 wherein the immobilised molecules of the array and the target molecules are nucleic acids and the contacting step takes place under conditions which allow hybridisation of the immobilised molecules to the target molecules.
Use according to claim 45 wherein hybridisation of a target nucleic acid to an immobilised nucleic acid is detected by means of primer extension from the resulting complex.
Use according to claim 45 wherein observation of successive tagged monomer base additions enables sequencing by synthesis.
Use according to claim 46 or claim 47 , wherein the enzyme Apyrase is used to reduce incorporation 3′ end mismatch bases.
Use according to claim 45 wherein hybridisation of a target nucleic acid to an immobilised nucleic acid is detected by means of hybridisation of nucleic acid probes to the target nucleic acid/immobilised nucleic acid complex.
Use according to claim 49 wherein the probes are differentially labelled.
Use according to claim 47 wherein hybridisation of a target nucleic acid to an immobilised nucleic acid is detected by means of ligation of nucleic acid probes to the target nucleic acid/immobilised nucleic acid complex.
Use according to claim 47 wherein observation of successive ligations with tagged oligonucleotides leads enables sequencing by synthesis.
Use according to any one of claims 28 to 52 wherein the array is contacted with two or more populations of target molecules.
Use according to claim 53 wherein each population of target molecules is differentially labelled.
A method for typing single nucleotide polymorphisms (SNPs) and mutations in nucleic acids, comprising the steps of: a) providing a repertoire of probes complementary to one or more nucleic acids present in a sample, which nucleic acids may possess one or more polymorphisms, said repertoire being presented such that molecules in said repertoire may be individually resolved; b) exposing the sample to the repertoire and allowing nucleic acids present in the sample to hybridise to the probes at a desired stringency and optionally to be processed by enzymes; c) detecting individual reacted nucleic acid molecules after optionally eluting the unreacted nucleic acids from the repertoire.
a) providing a repertoire of probes complementary to one or more nucleic acids present in a sample, which nucleic acids may possess one or more polymorphisms, said repertoire being presented such that molecules in said repertoire may be individually resolved;
b) exposing the sample to the repertoire and allowing nucleic acids present in the sample to hybridise to the probes at a desired stringency and optionally to be processed by enzymes;
c) detecting individual reacted nucleic acid molecules after optionally eluting the unreacted nucleic acids from the repertoire.
A method according to claim 55 , wherein the repertoire is arrayed on a solid phase.
A method according to claim 56 , wherein said array is an array according to claim 27 .
A method according to any one of claims 55 to 57 , wherein the sample is exposed to a second repertoire of probes, which probes bind to one or more molecules of the sample at a different position to the probes of the first repertoire.
A method according to claim 58 , wherein said first and second repertoires are differentially labelled.
A method for determining the complete or partial sequence of a target nucleic acid, comprising the steps of: a) providing a first set of probes complementary to one or more nucleic acids present in a sample, said first set of probes being presented such that arrayed molecules may be individually resolved; b) hybridising a sample comprising a target nucleic acid to the first set of probes; c) hybridising one or more further probes of defined sequence to the target nucleic acid; and d) detecting the binding of individual further probes to the target nucleic acid. e) and detecting the approximate distance separating each probe or the order of each probe
a) providing a first set of probes complementary to one or more nucleic acids present in a sample, said first set of probes being presented such that arrayed molecules may be individually resolved;
b) hybridising a sample comprising a target nucleic acid to the first set of probes;
c) hybridising one or more further probes of defined sequence to the target nucleic acid; and
d) detecting the binding of individual further probes to the target nucleic acid.
e) and detecting the approximate distance separating each probe or the order of each probe
A method according to claim 60 , wherein the first set of probes is a repertoire of probes.
A method according to claim 61 , wherein the repertoire is arrayed on a solid phase.
A method according to claim 62 , wherein the target nucleic acids are captured to the solid phase at one or more points.
A method according to any one of claims 60 to 63 , wherein the repertoire is arrayed at a density which allows molecules in said repertoire to be individually resolved.
A method according to claim 64 , wherein said array is an array according to claim 27 .
A method according to any one of claims 60 to 65 , wherein the probes are differentially labelled.
A method for determining the number of sequence repeats in a sample of nucleic acid, comprising the steps of: a) providing one or more probes complementary to one or more nucleic acids present in a sample, which nucleic acids may possess one or more sequence repeats, said probes being complementary to a sequence flanking one end of the repeats, said probes being presented such that molecules may be individually resolved; b) contacting the nucleic acids with labelled probes complementary to units of said sequence repeats and a differentially labelled probe complementary to the flanking sequence at the other end of the targeted repeats; c) contacting the complex formed in b) with probes in a); and d) determining the number of repeats present on each sample nucleic acid by individual assessment of the number of labels incorporated into each molecule and only counting those molecules to which the differentially labelled probe complementary to the flanking sequence is also associated with.
a) providing one or more probes complementary to one or more nucleic acids present in a sample, which nucleic acids may possess one or more sequence repeats, said probes being complementary to a sequence flanking one end of the repeats, said probes being presented such that molecules may be individually resolved;
b) contacting the nucleic acids with labelled probes complementary to units of said sequence repeats and a differentially labelled probe complementary to the flanking sequence at the other end of the targeted repeats;
c) contacting the complex formed in b) with probes in a); and
d) determining the number of repeats present on each sample nucleic acid by individual assessment of the number of labels incorporated into each molecule and only counting those molecules to which the differentially labelled probe complementary to the flanking sequence is also associated with.
A method according to claim 67 , wherein the repertoire is arrayed on a solid phase.
A method according to claim 67 or claim 68 , wherein the repertoire is arrayed at a density which allows molecules in said repertoire to be individually resolved.
A method according to claim 69 , wherein said array is an array according to claim 27 .
A method for analysing the expression of one or more genes in a sample, comprising the steps of: a) providing a repertoire of probes complementary to one or more nucleic acids present in a sample, said repertoire being presented such that molecules may be individually resolved; b) hybridising a sample comprising said nucleic acids to the probes; and c) determining the nature and quantity of individual nucleic acid species present in the sample by counting single molecules which are hybridised to the probes.
a) providing a repertoire of probes complementary to one or more nucleic acids present in a sample, said repertoire being presented such that molecules may be individually resolved;
b) hybridising a sample comprising said nucleic acids to the probes; and
c) determining the nature and quantity of individual nucleic acid species present in the sample by counting single molecules which are hybridised to the probes.
A method according to claim 71 , wherein the repertoire is arrayed on a solid phase.
A method according to claim 71 or claim 72 , wherein the repertoire is arrayed at a density which allows molecules in said repertoire to be individually resolved.
A method according to claim 73 , wherein said array is an array according to claim 27 .
A method according to any one of claims 71 to 74 , wherein the repertoire comprises a plurality of probes of each given specificity.
A method for typing single nucleotide polymorphisms (SNPs) and mutations in nucleic acids, comprising the steps of: a) providing a repertoire of probes complementary to one or more nucleic acids present in a sample, which nucleic acids may possess one or more polymorphisms; b) arraying said repertoire such that each probe in the repertoire is resolvable individually; c) exposing the sample to the repertoire and allowing nucleic acids present in the sample to hybridise to the probes at a desired stringency and optionally be processed by enzymes such that hybridised/processed nucleic acid/probe pairs are detectable; d) eluting the unhybridised nucleic acids from the repertoire and detecting individual hybridised/processed nucleic acid/probe pairs; e) analysing the signal derived from step (d) and computing the confidence in each detection event to generate a PASS table of high-confidence results; and f) displaying results from the PASS table to assign base calls and type polymorphisms present in the nucleic acid sample.
a) providing a repertoire of probes complementary to one or more nucleic acids present in a sample, which nucleic acids may possess one or more polymorphisms;
b) arraying said repertoire such that each probe in the repertoire is resolvable individually;
c) exposing the sample to the repertoire and allowing nucleic acids present in the sample to hybridise to the probes at a desired stringency and optionally be processed by enzymes such that hybridised/processed nucleic acid/probe pairs are detectable;
d) eluting the unhybridised nucleic acids from the repertoire and detecting individual hybridised/processed nucleic acid/probe pairs;
e) analysing the signal derived from step (d) and computing the confidence in each detection event to generate a PASS table of high-confidence results; and
f) displaying results from the PASS table to assign base calls and type polymorphisms present in the nucleic acid sample.
A method according to 76 wherein step (e) involves analysing the signal from step (d) and computing in each detection event a FAIL table of low confidence results and using this table to inform primer and assay design.
A method according to claim 76 or claim 77 where the process is iterated for sequencing by synthesis.
A method according to claim 76 , wherein confidence in each detection event is computed in accordance with Table 1.
A method according to claim 76 or claim 77 , wherein detection events are generated by labelling the sample nucleic acids and/or the probe molecules, and imaging said labels on the array using a detector.
A method according to any one of claims 55 and 76 - 80 where the SNPs that are probed are tags for a haplotype block or a region of linkage disequilibrium.
A method of obtaining allele frequencies by single molecule counting of pooled DNA.
A method according to claim 82 wherein obtained allele freq
[Truncated to input limit]