`USOO9562263B2
`
`(12) United States Patent
`(10) Patent No.:
`US 9,562,263 B2
`(45) Date of Patent:
`*Feb. 7, 2017
`Maples et al.
`
`(54)
`
`(71)
`
`(72)
`
`NICKING AND EXTENSION
`AMPLIFICA’I‘ION REACTION FOR THE
`EXPONENTIAI, ANTPIAIFICATION 0F
`NUCLEIC ACIDS
`
`Applicant: Ionian Technologies, Inc., San Diego,
`CA (US)
`
`Inventors: Brian K Maples, Lake Forest, CA
`(US); Rebecca C. Holmberg, San
`Diego, CA (US): Andrew P. Miller,
`San Diego, CA (US); Jarrod Provins,
`Dana Point, CA (US); Richard Roth,
`Carlsbad, CA (US); Jcfl'rcy Mandcll,
`San Diego. CA (US)
`
`(73)
`
`Assignee:
`
`Ionian Technologies, Inc., San Diego,
`CA (US)
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(1)) by 0 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21)
`
`Appl. No.: 14/067,620
`
`(22)
`
`(65)
`
`(63)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`Filed:
`
`Oct. 30, 2013
`
`Prior Publication Data
`
`US 2014/0093883 A1
`
`Apr. 3, 2014
`
`Related U.S. Application Data
`
`Continuation of application No. 11/778,018, filed on
`Jul. 14, 2007.
`
`Int. Cl.
`C12Q 1/68
`U.S. Cl.
`
`CPC .......... C12Q 1/686 (2013.01); C12Q 1/6844
`(2013.01)
`
`(2006.01)
`
`Field of Classification Search
`None
`See application file for complete search history.
`
`References Cited
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`WO
`WO
`W0
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`1850981
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`, on Mar. 27,
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`
`(Continued)
`
`Primary Examiner Angela M Bertagna
`(74) Attorney, Agent, or Firm 7 Fish & Richardson PC.
`
`(57)
`
`ABSTRACT
`
`The invention is in general directed to the rapid exponential
`amplification of short DNA or RNA sequences at a constant
`temperature.
`
`35 Claims, 19 Drawing Sheets
`
`ELIX 100 1
`
`ELIX 1001
`
`1
`
`

`

`US 9,562,263 B2
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`Page 2
`
`(56)
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`* cited by examiner
`
`3
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`

`

`U.S. Patent
`
`Feb. 7, 2017
`
`Sheet 1 of 19
`
`US 9,562,263 B2
`
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`

`

`U.S. Patent
`
`Feb. 7, 2017
`
`Sheet 2 of 19
`
`US 9,562,263 B2
`
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`

`

`U.S. Patent
`
`Feb. 7, 2017
`
`Sheet 3 of 19
`
`US 9,562,263 B2
`
`Polymerase > nicking
`
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`
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`
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`
`FIG. 1C
`
`Recognition region to
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`
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`,
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`antisense strand of target
`
`FIG. JD
`
`6
`
`

`

`U.S. Patent
`
`Feb. 7, 2017
`
`Sheet 4 of 19
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`US 9,562,263 B2
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`Feb. 7, 2017
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`Sheet 5 of 19
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`US 9,562,263 B2
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`Feb. 7, 2017
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`Feb. 7, 2017
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`Feb. 7, 2017
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`Sheet 10 of 19
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`U.S. Patent
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`Feb. 7, 2017
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`U.S. Patent
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`Feb. 7, 2017
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`U.S. Patent
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`Feb. 7, 2017
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`Sheet 14 of 19
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`US 9,562,263 B2
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`U.S. Patent
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`Feb. 7, 2017
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`Sheet 15 of 19
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`US 9,562,263 B2
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`U.S. Patent
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`Feb. 7, 2017
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`Sheet 16 of 19
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`US 9,562,263 B2
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`U.S. Patent
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`Feb. 7, 2017
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`Sheet 17 of 19
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`US 9,562,263 B2
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`U.S. Patent
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`Feb.7,2017
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`U.S. Patent
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`Feb. 7, 2017
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`Sheet 19 of 19
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`US 9,562,263 B2
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`

`US 9,562,263 B2
`
`1
`NICKING AND EXTENSION
`AMPLIFICATION REACTION FOR THE
`EXPONENTIAL AMPLIFICATION OF
`NUCLEIC ACIDS
`
`RELATED APPLICATIONS
`
`This application is a continuation of US. application Ser.
`No. 11/778,018, filed Jul. 14, 2007, the entire contents of
`which are hereby incorporated.
`
`FIELD OF THE INVENTION
`
`The invention is in general directed to the rapid exponen-
`tial amplification of short DNA or RNA sequences at a
`constant temperature.
`
`BACKGROUND
`
`The field of in vitro diagnostics is quickly expanding as
`the need for systems that can rapidly detect the presence of
`harmful species or determine the genetic sequence of a
`region of interest
`is
`increasing exponentially. Current
`molecular diagnostics focus on the detection of biomarkers
`and include small molecule detection,
`immuno-based
`assays, and nucleic acid tests. The built-in specificity
`between two complementary nucleic acid strands allows for
`fast and specific recognition using unique DNA or RNA
`sequences, the simplicity of which makes a nucleic acid test
`an attractive prospect. Identification of bacterial and viral
`threat agents, genetically modified food products, and single
`nucleotide polymorphisms for disease management are only
`a few areas where the advancement of these molecular
`
`diagnostic tools becomes extremely advantageous. To meet
`these growing needs, nucleic acid amplification technologies
`have been developed and tailored to these needs of speci-
`ficity and sensitivity.
`Historically, the most common amplification technique is
`the polymerase chain reaction (PCR), which has in many
`cases become the gold standard for detection methods
`because of its reliability and specificity. This technique
`requires the cycling of temperatures to proceed through the
`steps of denaturation of the dsDNA, annealing of short
`oligonucleotide primers, and extension of the primer along
`the template by a thermostable polymerase. Though many
`new advances in engineering have successfully shortened
`these reaction times to 20-30 minutes, there is still a steep
`power requirement to meet the needs of these thermocycling
`units.
`
`Various isothermal amplification techniques have been
`developed to circumvent the need for temperature cycling.
`From this demand, both DNA and RNA isothermal ampli-
`fication technologies have emerged.
`Transcription-Mediated Amplification (TMA) employs a
`reverse transcriptase with RNase activity, an RNA poly-
`merase, and primers with a promoter sequence at the 5' end.
`The reverse transcriptase synthesizes cDNA from the
`primer, degrades the RNA target, and synthesizes the second
`strand after the reverse primer binds. RNA polymerase then
`binds to the promoter region of the dsDNA and transcribes
`new RNA transcripts which can serve as templates for
`further reverse transcription. The reaction can produce a
`billion fold amplification in 20-30 minutes. This system is
`not as robust as other DNA amplification techniques and is
`therefore, not a field-deployable test due to the ubiquitous
`presence of RNAases outside of a sterile laboratory. This
`amplification technique is very similar to Self-Sustained
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`
`Sequence Replication (38R) and Nucleic Acid Sequence
`Based Amplification (NASBA), but varies in the enzymes
`employed.
`Isothermal Amplification (SPIA) also
`Single Primer
`involves multiple polymerases and RNaseH. First, a reverse
`transcriptase extends a chimeric primer along an RNA
`target. RNaseH degrades the RNA target and allows a DNA
`polymerase to synthesize the second strand of cDNA.
`RNaseH then degrades a portion of the chimeric primer to
`release a portion of the cDNA and open a binding site for the
`next chimeric primer to bind and the amplification process
`proceeds through the cycle again. The linear amplification
`system can amplify very low levels of RNA target in roughly
`3.5 hrs.
`
`The Q-Beta replicase system is a probe amplification
`method. A probe region complementary to the target of
`choice is inserted into MDV—1 RNA, a naturally occurring
`template for Q-Beta replicase. Q-Beta replicates the MDV—l
`plasmid so that the synthesized product is itself a template
`for Q-Beta replicase, resulting in exponential amplification
`as long as the there is excess replicase to template. Because
`the Q-Beta replication process is
`so sensitive and can
`amplify whether the target is present or not, multiple wash
`steps are required to purge the sample of non-specifically
`bound replication plasmids. The exponential amplification
`takes approximately 30 minutes; however, the total time
`including all wash steps is approximately 4 hours.
`Numerous isothermal DNA amplification technologies
`have been developed as well. Rolling circle amplification
`(RCA) was developed based on the natural replication of
`plasmids and viruses. A primer extends along a circular
`template resulting in the synthesis of a single-stranded
`tandem repeat. Capture, washing, and ligation steps are
`necessary to preferentially circularize the template in the
`presence of target and reduce background amplification.
`Ramification amplification (RAM) adds cascading primers
`for additional geometric amplification. This
`technique
`involves amplification of non-specifically sized strands that
`are either double or single-stranded.
`Helicase-dependent amplification (HDA) takes advantage
`of a thermostable helicase (Tte-Uer) to unwind dsDNA to
`create single-strands that are then available for hybridization
`and extension of primers by polymerase. The thermostable
`HDA method does not require the accessory proteins that the
`non-thermostable HDA requires. The reaction can be per-
`formed at a single temperature, though an initial heat dena-
`turation to bind the primers generates more product. Reac-
`tion times are reported to be over 1 hour to amplify products
`70-120 base pairs in length.
`Loop mediated amplification (LAMP) is a sensitive and
`specific isothermal amplification method that employs a
`thermostable polymerase with strand displacement capabili-
`ties and four or more primers. The primers are designed to
`anneal consecutively along the target in the forward and
`reverse direction. Extension of the outer primers displaces
`the extended inner primers to release single strands. Each
`primer is designed to have hairpin ends that, once displaced,
`snap into a hairpin to facilitate self-priming and further
`polymerase extension. Additional loop primers can decrease
`the amplification time, but complicates the reaction mixture.
`Overall, LAMP is a difficult amplification method to mul-
`tiplex, that is, to amplify more than one target sequence at
`a time, although it is reported to be extremely specific due
`to the multiple primers that must anneal to the target to
`further the amplification process. Though the reaction pro-
`ceeds under isothermal conditions, an initial heat denatur-
`ation step is required for double-stranded targets. Amplifi-
`
`23
`
`23
`
`

`

`US 9,562,263 B2
`
`3
`cation proceeds in 25 to 50 minutes and yields a ladder
`pattern of various length products.
`Strand displacement amplification (SDA) was developed
`by Walker et. al. in 1992. This amplification method uses
`two sets of primers, a strand displacing polymerase, and a
`restriction endonuclease. The bumper primers serve to dis-
`place the initially extended primers to create a single-strand
`for the next primer to bind. A restriction site is present in the
`5' region of the primer. Thiol-modified nucleotides are
`incorporated into the synthesized products to inhibit cleav-
`age of the synthesized strand. This modification creates a
`nick site on the primer side of the strand, which the
`polymerase can extend. This approach requires an initial
`heat denaturation step for double-stranded targets. The reac-
`tion is then run at a temperature below the melting tempera-
`ture of the double-stranded target region. Products 60 to 100
`bases in length are usually amplified in 30-45 minutes using
`this method.
`
`These and other amplification methods are discussed in,
`for example, VanNess, J, et al., PNAS 2003. Vol. 100, no 8,
`p 4504-4509; Tan, E., et al., Anal. Chem. 2005, 77, 7984-
`7992; Lizard, P., et al., Nature Biotech. 1998, 6, 1197-1202;
`Notomi, T., et al., NAR 2000, 28, 12, e63; and Kurn, N., et
`al., Clin. Chem. 2005, 51:10, 1973-1981. Other references
`for these general amplification techniques include,
`for
`example, U.S. Pat. Nos. 7,112,423; 5,455,166; 5,712,124;
`5,744,311; 5,916,779; 5,556,751; 5,733,733; 5,834,202;
`5,354,668; 5,591,609; 5,614,389; 5,942,391; and U.S. patent
`publication numbers US20030082590; US20030138800;
`US20040058378; and US20060154286.
`There is a need for a quicker method of amplification of
`single-stranded and double-stranded nucleic acid target
`sequences that can be performed without
`temperature
`cycling and that is suitable for shorter target sequences.
`
`SUMMARY
`
`Provided herein are methods of amplifying nucleic acid
`target sequences that rely on nicking and extension reactions
`and amplify shorter sequences in a quicker timeframe than
`traditional amplification reactions, such as, for example,
`strand displacement amplification reactions. Embodiments
`of the invention include, for example, reactions that use only
`two templates to prime, one or two nicking enzymes, and a
`polymerase, under
`isothermal conditions.
`In exemplary
`embodiments, the polymerase and the nicking enzyme are
`thermophilic, and the reaction temperature is significantly
`above the melting temperature of the hybridized target
`region. The nicking enzyme nicks only one strand in a
`double-stranded duplex, so that incorporation of modified
`nucleotides is not necessary as it is in strand displacement.
`An initial heat denaturation step is not required for the
`methods of the present invention. Due to the simplicity of
`the reaction, in exemplary embodiments, the reaction is very
`easy to perform and can amplify 20-30mer products 108 to
`1010 fold from genomic DNA in 2.5 to 10 minutes. Further-
`more, in other exemplary embodiments, the method is able
`to amplify RNA without a separate reverse transcription
`step.
`Thus, provided in a first embodiment of the present
`invention is a method for amplifying a double-stranded
`nucleic acid target sequence, comprising contacting a target
`DNA molecule
`comprising a double-stranded target
`sequence having a sense strand and an antisense strand, with
`a forward template and a reverse template, wherein said
`forward template comprises a nucleic acid sequence com-
`prising a recognition region at the 3' end that is comple-
`
`10
`
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`
`20
`
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`
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`
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`
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`mentary to the 3' end of the target sequence antisense strand;
`a nicking enzyme site upstream of said recognition region,
`and a stabilizing region upstream of said nicking enzyme
`site; said reverse template comprises a nucleotide sequence
`comprising a recognition region at the 3' end that is comple-
`mentary to the 3' end of the target sequence sense strand, a
`nicking enzyme site upstream of said recognition region,
`and a stabilizing region upstream of said nicking enzyme
`site; providing a first nicking enzyme that is capable of
`nicking at the nicking enzyme site of said forward template,
`and does not nick within said target sequence; providing a
`second nicking enzyme that is capable of nicking at the
`nicking enzyme site of said reverse template and does not
`nick within said target sequence; and providing a DNA
`polymerase; under conditions wherein amplification is per-
`formed by multiple cycles of said polymerase extending said
`forward and reverse templates along said target sequence
`producing a double-stranded nicking enzyme site, and said
`nicking enzymes nicking at said nicking enzyme sites,
`producing an amplification product.
`In certain embodiments of the invention, the DNA poly-
`merase is a thermophilic polymerase. In other examples of
`the invention, the polymerase and said nicking enzymes are
`stable at temperatures up to 37° C., 42° C., 60° C., 65° C.,
`70° C., 75° C., 80° C., or 85° C. In certain embodiments, the
`polymerase is stable up to 60° C. The polymerase may, for
`example, be selected from the group consisting of Bst (large
`fragment), 9° N, VentR® (exo-) DNA Polymerase, Thermi-
`nator, and Therminator II.
`The nicking enzyme may, for example, nick upstream of
`the nicking enzyme binding site, or, in exemplary embodi-
`ments, the nicking enzyme may nick downstream of the
`nicking enzyme binding site. In certain embodiments, the
`forward and reverse templates comprise nicking enzyme
`sites recognized by the same nicking enzyme and said first
`and said second nicking enzyme are the same. The nicking
`enzyme may,
`for example, be selected from the group
`consisting of Nt.BspQI, Nb.Bvai, Nb.BsmI, Nb.BerI,
`Nb.BtsI, Nt.AlwI, Nt.BvaI, Nt.BstNBI, Nt.CviPII,
`Nb.BpulOI, and Nt.BpulOI.
`the target
`invention,
`In certain aspects of the present
`sequence comprises from 1 to 5 nucleotides more than the
`sum of the nucleotides of said forward template recognit