(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2016/0017392 A1
`Arnold et al.
`(43) Pub. Date:
`Jan. 21, 2016
`
`US 201600l7392A1
`
`(54)
`
`METHODS FOR AMPLIFICATION OF
`NUCLEIC ACIDS ON SOLID SUPPORT
`
`(71)
`
`Applicants:
`
`Lyle J. ARNOLD, Poway, CA (US);
`Norman C. NELSON, San Diego, CA
`(US)
`
`Inventors:
`
`(72)
`
`Lyle J. Arnold, Poway, CA (US);
`Norman C. Nelson, San Diego, CA
`(US)
`
`(21)
`
`Appl. No .:
`
`14/773,362
`
`(22)
`
`(86)
`
`PCT Filed:
`
`Mar. 14, 2014
`
`PCT No.:
`
`PCT/US2014/029817
`
`§ 371 (C)(1),
`(2) Date:
`
`Sep. 7, 2015
`
`Related U.S. Application Data
`
`(60)
`
`Provisional application No. 61/781,356, filed on Mar.
`14, 2013.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`C12P 19/34
`
`(52) U.S.Cl.
`CPC .................................... .. C12P 19/34 (2013.01)
`
`ABSTRACT
`(57)
`The present invention provides methods for amplifying a
`nucleic acid from a sample containing a mixture of nucleic
`acids utilizing a solid support. Methods are provided utilizing
`user-defined primer oligonucleotides for directional amplifi-
`cation that assists in further manipulation ofthe target nucleic
`acid, such as sequencing. Methods are also provided utilizing
`blocker and displacer oligonucleotides for generating ampli-
`fied target nucleic acids of defined length. One of these meth-
`ods provides a first oligonucleotide and a second oligonucle-
`otide affixed to a solid support or separate solid supports. The
`first oligonucleotide is blocked to prevent extension from the
`3'-terminus and has a sequence complementary to a first por-
`tion of a target nucleic acid. The second oligonucleotide has
`a sequence that is identical to a second portion of the target
`nucleic acid. In this method, a sample is applied to the solid
`support and the target nucleic acid within the sample binds
`said first oligonucleotide. The solid support is then washed to
`remove unbound nucleic acids. A primer sequence containing
`a target binding region and a polymerase promoter sequence
`is then annealed to the bound target nucleic acid and extended
`producing a first duplex nucleic acid. The target sequence is
`then removed leaving a first nucleic acid that can now bind the
`second oligonucleotide. The second oligonucleotide is
`extended to produce a second duplex nucleic acid that con-
`tains a second nucleic acid. The second nucleic acid is then
`
`(2006.01)
`
`amplified by adding a polymerase.
`
`Exhibit 2019 Page 1
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`Enzo Exhibit 2019
`BD v. Enzo
`Case IPRZO17-00172
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`Enzo Exhibit 2019
`BD v. Enzo
`Case IPR2017-00172
`
`Exhibit 2019 Page 1
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`

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`Patent Application Publication
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`Jan. 21, 2016 Sheet 1 of 15
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`US 2016/0017392 A1
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`Patent Application Publication
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`Jan. 21, 2016 Sheet 2 of 15
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`US 2016/0017392 A1
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`Jan. 21, 2016 Sheet 5 of 15
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`Jan. 21, 2016 Sheet 6 of 15
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`Jan. 21, 2016 Sheet 7 of 15
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`US 2016/0017392 A1
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`Jan. 21, 2016 Sheet 8 of 15
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`US 2016/0017392 A1
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`Patent Application Publication
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`Jan. 21, 2016 Sheet 9 of 15
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`US 2016/0017392 A1
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`Patent Application Publication
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`Jan. 21, 2016 Sheet 10 of 15
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`US 2016/0017392 A1
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`Patent Application Publication
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`Exhibit 2019 Page 13
`
`Exhibit 2019 Page 13
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 13 of 15
`
`US 2016/0017392 A1
`
`J1’ GUR“. 6
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`Exhibit 2019 Page 14
`
`Exhibit 2019 Page 14
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 14 of 15
`
`US 2016/0017392 A1
`
`J: GUR*.
`
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`Exhibit 2019 Page 15
`
`Exhibit 2019 Page 15
`
`

`

`Patent Application Publication
`
`Jan. 21, 2016 Sheet 15 of 15
`
`US 2016/0017392 A1
`
`J1’ GUR“.
`
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`Exhibit 2019 Page 16
`
`Exhibit 2019 Page 16
`
`

`

`US 2016/0017392 A1
`
`Jan. 21,2016
`
`METHODS FOR AMPLIFICATION OF
`NUCLEIC ACIDS ON SOLID SUPPORT
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application is a non-provisional patent appli-
`cation of provisional patent application Ser. No. 61/781,356
`filed Mar. 14, 2013 and claims the benefit of the filing date of
`PCT/US2014/029817 filed 14 Mar. 2014 under 35 U.S.C.
`
`§371 from which the PCT application claims priority.
`
`STATEMENT REGARDING FEDERALLY
`SPONSORED RESEARCH OR DEVELOPMENT
`
`[0002] None
`
`INCORPORATION-BY-REFERENCE OF
`MATERIAL SUBMITTED ON COMPACT DISC
`
`[0003] None
`
`BACKGROUND OF THE INVENTION
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0008] The present invention provides methods of immo-
`bilizing and amplifying nucleic acids on solid support. More
`specifically, it is a method of immobilizing and amplifying a
`desired nucleic acid or a number of nucleic acids (multiplex-
`ing) from a sample containing a mixture of nucleic acids.
`[0009]
`In one aspect of the present invention, the method
`provides first and second oligonucleotides afiixed to a solid
`support or alternatively separate solid supports. The first oli-
`gonucleotide is blocked to prevent extension from the 3'-ter-
`minus and has a sequence complementary to a first portion of
`a target nucleic acid. The second oligonucleotide has a
`sequence that is identical to a second portion of the target
`nucleic acid. A sample is applied to the solid support and
`target sequences in the sample bind the first oligonucleotide.
`The solid support is then washed to remove unbound nucleic
`acids and other components from the sample. A primer con-
`taining a target-binding region and a tag region comprising
`polymerase promoter sequence is armealed to the bound tar-
`get nucleic acid. The primer is extended by polymerase to
`produce a first duplex nucleic acid. The target sequence is
`removed from the first duplex nucleic acid to produce an
`unbound first nucleic acid. The first nucleic acid is annealed to
`
`(1) Field of the Invention
`[0004]
`[0005] The present invention relates to methods of purifi-
`cation, immobilization and amplification of nucleic acids.
`Specifically, nucleic acid amplification using a solid support.
`[0006]
`(2) Description of RelatedArt
`[0007] There are a variety of methods for the purification
`and amplification of nucleic acids known to those skilled in
`the art. However, these methods can be slow, tedious, expen-
`sive and difficult to automate and manufacture. They also lack
`the performance in regards to sensitivity, specificity, preci-
`sion, accuracy and other features that would otherwise allow
`them to be used successfully in a number of diverse applica-
`tions. These methods are often complicated, requiring isola-
`tion of nucleic acid targets from samples, preparing the tar-
`gets
`for
`amplification, performing amplification and
`detecting the amplification product. In some applications, this
`may also include preparing templates for sequencing includ-
`ing next generation sequencing and sequencing the target
`nucleic acids. In many of these methods one or more purifi-
`cation steps may also be required. All of these steps increase
`performance time, cost, complexity and labor. Providing
`methods with versatility as to specificity, sensitivity, selectiv-
`ity, precision and accuracy is important. As are methods that
`are rapid, efficient and easy to use with minimal assay steps
`and reagents for readily obtaining a desired isolated and
`amplified product from a raw sample. These products may be
`amplified nucleic acids or set of amplified nucleic acids (i.e.,
`multiplexing, including high level multiplexing) that may be
`utilized as templates, free in solution or on solid supports, for
`immediate sequencing and next generation sequencing. In
`addition, methods for directional amplification that preserve
`strand orientation provide proper alignment of sequences
`during mapping and more accurate quantitative determina-
`tions (e.g. of gene expression levels). In addition, these meth-
`ods can be utilized with a wide variety of nucleic acid ampli-
`fication methods that are known in the art. In addition these
`
`methods can be utilized with tag sequences. The present
`invention provides methods for purifying, immobilizing and
`amplifying specific nucleic acids from a sample that over-
`come many of the current limitations in the art.
`
`the second oligonucleotide and the second oligonucleotide is
`extended by polymerase to produce a second duplex nucleic
`acid containing a first nucleic acid and a second nucleic acid.
`Multiple copies of a third nucleic acid are then generated by
`adding polymerase specific to the promoter sequence to the
`solid support thereby amplifying the target nucleic acid.
`[0010]
`In a second embodiment the primer may be com-
`bined with the sample and solid support such that the primer
`hybridizes to the target and the target hybridizes to the first
`oligonucleotide concurrently. The solid support
`is then
`washed to remove unbound nucleic acids and primer as well
`as other components from the sample.
`[001 1]
`In a third embodiment, the method may further com-
`prise steps that allow for additional amplification of the
`desired target nucleic acid. In these steps, the multiple copies
`ofthe third nucleic acid bind to the second oligonucleotide on
`the solid support. The second oligonucleotide is extended by
`polymerase to produce a third duplex nucleic acid. The third
`nucleic acid is removed from the third duplex nucleic acid to
`produce a fourth nucleic acid bound to the solid support. The
`primer sequence is annealed to the fourth nucleic acid and
`both the primer and the fourth nucleic acid are extended by
`polymerase to produce a fourth-duplex nucleic acid contain-
`ing a fifth nucleic acid and additional second nucleic acid. The
`fourth duplex nucleic acid is incubated with a polymerase
`being specific to a promoter on the fourth duplex nucleic acid
`to produce additional multiple copies ofthe third nucleic acid.
`According to this embodiment the third nucleic acid may then
`be amplified further.
`[0012]
`In a fourth embodiment, the primer comprises a tag
`sequence containing one or more non-natural nucleotides,
`such as isocytosine (isoC) and/or isoguanine (isoG)
`to
`increase specificity for binding the target nucleic acid and
`decreases amplification of other nucleic acids.
`[0013]
`In a fifth embodiment, four different second oligo-
`nucleotides may be bound to the solid support each having a
`different nucleotide at the 3'-terminus and sequence that
`binds the first nucleic acid at a specific single nucleotide
`polymorphism (SNP) site. In this configuration, amplifica-
`tion may be utilized to identify all four possible SNPs in a
`single assay.
`Exhibit 2019 Page 17
`
`Exhibit 2019 Page 17
`
`

`

`US 2016/0017392 A1
`
`Jan. 21,2016
`
`oligonucleotides and the target nucleic acid are applied to the
`solid support. The displacer and blocker oligonucleotides are
`annealed to the target nucleic acid and the annealed target is
`immobilized onto the solid support by hybridization to the
`first primer.
`[0020] The solid support is washed to remove unbound
`nucleic acids and other components of the sample. The first
`primer is extended by polymerase. Extension terminates at
`the blocker oligonucleotide to produce a first duplex nucleic
`acid of defined length. The displacer oligonucleotide is also
`extended by polymerase to displace the target strand from the
`first duplex nucleic acid. Extension terminates at the blocker
`oligonucleotide to produce a second duplex nucleic acid con-
`taining a second nucleic acid of defined length unbound to the
`solid support.
`[0021] A second primer is annealed to the first nucleic acid
`and extended by polymerase to produce a third duplex con-
`taining the first nucleic acid and a third nucleic acid. The
`second and third duplex nucleic acids are then dissociated,
`e.g., heat denaturation or other appropriate means known in
`the art. The second primer is armealed to the first nucleic acid
`and extended to produce more of the third duplex nucleic
`acid.
`
`In a second aspect, directional amplification of a
`[0014]
`fragmented nucleic acid target is provided for subsequent
`manipulation, such as sequencing. In one embodiment, a
`sample and a first primer are applied to a solid support. The
`first primer comprises a random sequence of about 6 to about
`9 nucleotides on the 3'-terminus and a first tag sequence on the
`5'-terminus, which is used to determine the orientation of the
`fragmented nucleic acid target. First and second oligonucle-
`otides bound to solid support are provided that may be bound
`to the same solid support or alternatively on different solid
`supports. The first oligonucleotide is optionally blocked to
`prevent potential exonuclease digestion, for example, at the
`5'-terminus and has a sequence complementary to a portion of
`the fragmented nucleic acid target. Alternatively, the first
`oligonucleotide comprises a sequence (such as for example, a
`random or semi-random sequence) that allows for immobili-
`zation of a number of fragments from the fragmented nucleic
`acid target. The second oligonucleotide sequence is comple-
`mentary to at least a portion of said tag sequence of the first
`primer.
`[0015] The first primer is annealed to the fragmented
`nucleic acid target and the bound fragmented nucleic acid
`target is armealed to the first oligonucleotide. The solid sup-
`port is washed to remove unbound sample and first primer.
`[0022] The third nucleic acid is immobilized onto the solid
`The first primer sequence is extended by polymerase to pro-
`support by hybridization to unoccupied first primer. The first
`duce a first duplex nucleic acid containing the fragmented
`primer is extended by polymerase to produce more third
`nucleic acid target and a first nucleic acid.
`duplex nucleic acid. The original target nucleic acid, with
`[0016] The fragmented nucleic acid target is removed from
`displacer and blocker oligonucleotides bound thereto,
`is
`the first duplex nucleic acid to produce a first nucleic acid.
`annealed to unoccupied first primer on the solid support. The
`The first nucleic acid is armealed to the second oligonucle-
`first primer is extended by polymerase to produce more first
`otide and a second primer is added to the solid support. The
`duplex nucleic acid. The displacer oligonucleotide is also
`second primer comprises a random sequence of about 6 to
`extended, displacing the target nucleic acid from the first
`about 9 nucleotides on the 3'-terminus and a second tag
`duplex nucleic acid. Extension terminates at the blocker oli-
`sequence on the 5'-terminus, which may or may not contain
`gonucleotide to produce a second duplex nucleic acid con-
`elements the same as or similar to those found in the first tag
`taining a second nucleic acid of defined length that is not
`sequence of the first primer. The second primer is annealed to
`bound to the support.
`the first nucleic acid and extended by polymerase to produce
`[0023] The second primer is annealed to the second nucleic
`a second duplex containing the first nucleic acid and a second
`acid by hybridization and is extended by polymerase to pro-
`nucleic acid having a first tag sequence on the 3'-teminus and
`duce a fourth duplex nucleic acid containing the second
`a second tag sequence on the 5'-terminus.
`nucleic acid and a fourth nucleic acid. All duplexes are then
`[0017] This second nucleic acid is further amplified to pro-
`denatured, and the cycle is repeated as desired to amplify the
`duce a target nucleic acid containing tag sequences that deter-
`target nucleic acid.
`mine the orientation of the nucleic acid target.
`[0024]
`In one embodiment of this aspect, the nucleic acid
`[0018]
`In one embodiment of this aspect, the first primer
`target is double stranded and both strands are immobilized
`may further comprise a barcode sequence as part of the tag
`and amplified. A primer and optionally a displacer and/or a
`sequence to further assist in identification and orientation.
`blocker are prepared for each strand according to the method
`described above. The primer for each target nucleic acid
`[0019]
`In third aspect, a method is provided wherein the
`strand is afiixed to a solid support ifa single support is utilized
`first primer having a sequence is complementary to a first
`or one primer on each support if two supports are utilized.
`portion of the target nucleic acid is immobilized on a solid
`Each target strand is immobilized by hybridization to the
`support. In one embodiment, displacer and blocker oligo-
`primer having a complementary sequence for that target
`nucleotides are utilized. The displacer oligonucleotide
`nucleic acid strand. The primer is extended by polymerase to
`hybridizes to the nucleic acid target at a location in the target
`produce a cDNA and the target nucleic acid strands are then
`sequence that is 3' of another bound oligonucleotide or oli-
`removed. Removal may be by displacement if a displacer is
`gonucleotides on the same nucleic acid target. When the
`used, by heat denaturation if a displacer is not used, or by
`displacer is extended with polymerase, the growing extension
`some other appropriate methodknown in the art. At this point,
`product encounters the other bound oligonucleotide(s) and
`the primer designed for one of the target nucleic acid strands
`displaces it/them, along with any extension product(s) initi-
`can serve as the second primer for the cDNA produced from
`ated by the bound oligonucleotide(s), from the target nucleic
`the other target nucleic acid strand, and vice versa. The
`acid strand. In this embodiment, the displacer oligonucleotide
`cDNAs bind to their respective second primers (i.e., the first
`has a sequence complementary to a second portion of the
`primers of the opposite target nucleic acid strands), the sec-
`target nucleic acid that is located to the 3' side of the first
`ond primers are extended by polymerase and the resulting
`portion. The blocker has an oligonucleotide sequence
`duplexes are disassociated. This process is then repeated as
`complementary to a third portion of the target nucleic acid
`desired thereby amplifying the target nucleic acid.
`sequence that is located to the 5' side ofthe first portion. These
`Exhibit 2019 Page 18
`
`Exhibit 2019 Page 18
`
`

`

`US 2016/0017392 A1
`
`Jan. 21,2016
`
`[0025] Other aspects of the invention are found throughout
`the specification.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`the primer
`interspersed into the primer, provided that
`sequence has suflicient complementarity with the sequence
`of the target nucleic acid to permit hybridization and exten-
`sion.
`
`In addition, primers may be nuclease resistant and
`[0037]
`include primers that have been modified to prevent degrada-
`tion by exonucleases. In some embodiments, the primers have
`been modified to protect against 3' or 5' exonuclease activity.
`Such modifications can include but are not limited to 2'-O-
`
`ribonucleotide modifications, phosphorothioate
`methyl
`backbone modifications,
`phosphorodithioate backbone
`modifications, phosphoramidate backbone modifications,
`methylphosphonate backbone modifications, 3'
`terminal
`phosphate modifications and 3' alkyl substitutions. In some
`embodiments, the primer(s) and/or probe(s) employed in an
`amplification reaction are protected against 3' and/or 5' exo-
`nuclease activity by one or more modifications.
`[0038] The skilled artisan is capable of designing and pre-
`paring primers that are appropriate for extension of a target
`sequence. The length of primers for use in the methods and
`compositions provided herein depends on several factors
`including the nucleotide sequence identity and the tempera-
`ture at which these nucleic acids are hybridized or used during
`in vitro nucleic acid extension. The considerations necessary
`to determine a preferred length for the primer of a particular
`sequence identity are well known to the person of ordinary
`skill.
`
`FIG. 1: (A) is a schematic diagram ofone method of
`[0026]
`the present invention and (B) shows additional steps that may
`be incorporated into the method in FIG. 1A.
`[0027]
`FIG. 2: is a schematic diagram of another method of
`the present invention utilizing a primer containing non-natu-
`ral nucleotides to increase selectivity and decrease amplifi-
`cation of undesired nucleic acid sequences.
`[0028]
`FIG. 3: is a schematic diagram of another method of
`the present invention utilizing a primer with a tag sequence to
`establish the orientation of the target oligonucleotide after
`amplification.
`[0029]
`FIG. 4: is a schematic diagram of another method of
`the present invention utilizing blocker and displacer to pre-
`pare amplified nucleic acid target of defined length.
`[0030]
`FIG. 5: is a schematic diagram of another method of
`the present invention utilizing first and second immobiliza-
`tion oligonucleotides that bind the first and second strand
`cDNAs of the target nucleic acid in a bridge configuration.
`[0031]
`FIG. 6: is a schematic diagram of another method of
`the present invention that incorporates PCR.
`[0032]
`FIG. 7: is a schematic diagram of another method of
`the present invention wherein the first immobilization oligo-
`nucleotide and blocker oligonucleotide are bound to the target
`nucleic acid concurrently at the initiation of the method.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0033] Unless defined otherwise, all terms used herein have
`the same meaning as are commonly understoodby one of skill
`in the art to which this invention belongs. All patents, patent
`applications and publications referred to throughout the dis-
`closure herein are incorporated by reference in their entirety.
`In the event that there is a plurality of definitions for a term
`herein, those in this section prevail.
`[0034] The term “oligonucleotide” as used herein refers to
`a polymeric form of nucleotides, either ribonucleotides or
`deoxyribonucleotides, incorporating natural and non-natural
`nucleotides of a length ranging from at least 2, or generally
`about 5 to about 200, or more commonly to about 100. Thus,
`this term includes double- and single-stranded DNA and
`RNA. In addition, oligonucleotides may be nuclease resistant
`and include but are not limited to 2'-O-methyl ribonucle-
`otides, phosphorothioate nucleotides, phosphorodithioate
`nucleotides, phosphoramidate nucleotides, and methylphos-
`99
`:4
`phonate nucleotides.
`target sequence,” or “target
`[0035] The term “target,
`nucleic acid” as used herein refers to a nucleic acid that
`
`[0039] The term “support” or “solid support” refers to con-
`ventional supports that include, for example, polymers such
`as microtiter wells, beads, particles or fibers, and silane or
`silicate supports such as glass slides or tubes to which capture
`m