United States Patent
`
`[19]
`
`[11] Patent Number:
`
`5,422,252
`
`Walker et al.
`
`[45] Date of Patent:
`
`Jun. 6, 1995
`
`l|||||IIIIIIIIllllllllIIIIIIIIIIIIIIIHIIIlllllllllllllllllllllllllllllllll
`USOOS422252A
`
`[54] SINIULTANEOUS AMPLIFICATION OF
`MULTIPLE TARGETS
`
`[75]
`
`Inventors:
`
`George T. Walker; James G. Nadeau,
`both of Chapel Hill; Michael C.
`Little, Raleigh, all of NC.
`
`[73] Assignee:
`
`Becton, Dickinson and Company,
`Franklin Lakes, NJ.
`
`[21] Appl. No.: 73,197
`
`[22] Filed:
`
`Jun. 4, 1993
`
`Int. 01.6 ......................... C12P 19/34; C12Q 1/70
`[51]
`[52] US. (:1. ....................................... 435/912; 435/6;
`935/17; 935/77, 935/78
`[58] Field of Search ................ 435/6, 5, 91.2; 935/77,
`935/78,17
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`7/1987 Mullis ..................................... 435/6
`4,683,195
`4,683,202 7/1987 Mullis ................. 435/91
`
`4,800,159
`1/1989 Mullis et al.
`.. 435/1723
`5,104,792 4/ 1992 Silver ...................................... 43 5/6
`
`FOREIGN PATENT DOCUMENTS
`
`2/1990 European Pat. Off.
`0356021
`0364255 4/1990 European Pat. Off.
`0379369
`7/1990 European Pat. Off.
`0469755
`2/1992 European Pat. Off.
`WO90/01064 2/1990 WIPO .
`WO90/09457
`8/1990 WIPO .
`
`.
`.
`.
`.
`
`OTHER PUBLICATIONS
`
`in Biochem. Molec. Biol.
`
`Bej, Critical Reviews
`26:301—334 (1991).
`Fries et a1 Molec. and Cell Probes (1990) 4:87—105.
`G. T. Walker, et al. “Isothermal in vitro amplification of
`DNA by a restriction enzyme/DNA polymerase sys-
`tem” Proc. Acad. Sci USA89z392—396 (1992).
`G. T. Walker, et a1. “Strand displacement amplifica-
`tion—an isothermal, in vitro DNA amplification tech-
`nique” Nuc. Acids Res. 20:1691—1696 (1992).
`P. R. Mueller and B. Wold “In Vivo Footprinting of a
`
`Muscle Specific Enhancer by Ligation Mediated PCR”
`Science 246:780—786 (1989).
`V. Shyamala and G. F.—L. Ames “Genome walking by
`single—specific-primer
`polymerase
`chain
`reaction:
`SSP—PCR” Gene 84:1—8 (1989).
`A. R. Shuldiner, et a1. “RNA template-specific poly-
`merase chain reaction (RS—PCR): a novel strategy to
`reduce dramatically false positives” Gene 91:139—142
`(1990).
`D. H. Jones and S, C. Winistorfer “Sequence specific
`generation of a DNA panhandle permits PCR amplifi-
`cation of unknown flanking DNA” Nuc. Acids Res.
`20:595—600 (1992).
`K. D. Eisenach, et al. “Detection of Mycobacterium
`tuberculosis in Sputum Samples Using a Polymerase
`Chain Reaction” Amer. Rev. Resp. Dis. 14421160—1163
`(1991).
`
`Primary Examiner—Margaret Parr
`Assistant Examiner—Carla Myers
`Attorney, Agent, or Firm—~Donna R. Fugit
`
`[57]
`
`ABSTRACT
`
`Methods for multiplex amplification of target nucleic
`acid sequences using a single pair of primers. Defined
`sequences are appended to the ends of multiple target
`sequences as part of the amplification reaction so that
`no steps in addition to amplification are required. The
`target sequences with the appended defined sequences
`need not be isolated prior to amplification. In one em-
`bodiment for coamplification of two target sequences, a
`sequence corresponding to a terminal segment of the
`first target sequence is appended to one end of the sec-
`ond target sequence and a sequence corresponding to a
`terminal segment of the second target sequence is ap-
`pended to one end of the first target sequence. Amplifi-
`cation of the two targets then requires only a single pair
`of primers. Alternatively, a single defined sequence may
`be appended to the 5’ and 3’ ends of any number of
`selected targets. All such modified target sequences
`may then be amplified using a single pair of primers
`which hybridize to the defined end-sequences.
`
`17 Claims, 5 Drawing Sheets
`
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`
`Ariosa Exhibit 1019, pg. 1
`|PR2013-00276
`
`Ariosa Exhibit 1019, pg. 1
`IPR2013-00276
`
`

`

`US. Patent
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`Ariosa Exhibit 1019, pg. 2
`|PR2013-00276
`
`Ariosa Exhibit 1019, pg. 2
`IPR2013-00276
`
`
`
`

`

`US. Patent
`
`June 6, 1995
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`Ariosa Exhibit 1019, pg. 3
`|PR2013-00276
`
`Ariosa Exhibit 1019, pg. 3
`IPR2013-00276
`
`

`

`US. Patent
`
`June 6, 1995
`
`Sheet 3 of 5
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`Ariosa Exhibit 1019, pg. 4
`|PR2013-00276
`
`Ariosa Exhibit 1019, pg. 4
`IPR2013-00276
`
`

`

`US. Patent
`
`June 6, 1995
`
`Sheet 4 of 5
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`5,422,252
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`Ariosa Exhibit 1019, pg. 5
`|PR2013-00276
`
`Ariosa Exhibit 1019, pg. 5
`IPR2013-00276
`
`

`

`US. Patent
`
`June 6, 1995
`
`Sheet 5 of 5
`
`5,422,252
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`Ariosa Exhibit 1019, pg. 6
`|PR2013-00276
`
`Ariosa Exhibit 1019, pg. 6
`IPR2013-00276
`
`

`

`1
`
`SIlVIULTANEOUS AMPLIFICATION OF
`MULTIPLE TARGETS
`
`5,422,252
`
`FIELD OF THE INVENTION
`
`The present invention relates to isothermal amplifica-
`tion of nucleic acid target sequences, in particular to
`simultaneous amplification of multiple target sequences.
`BACKGROUND OF THE INVENTION
`
`In vitro nucleic acid amplification techniques have
`provided powerful tools for detection and analysis of
`small amounts of nucleic acids. The extreme sensitivity
`of such methods has lead to attempts to develop them
`for diagnosis of infectious and genetic diseases, isolation
`of genes for analysis, and detection of specific nucleic
`acids as in forensic medicine. Nucleic acid amplification
`techniques can be grouped according to the tempera—
`ture requirements of the procedure. The polymerase
`chain reaction (PCR; R. K. Saiki, et a1. 1985. Science
`230, 1350—1354), ligase chain reaction (LCR; D. Y. Wu,
`et a1. 1989. Genomics 4, 560—569; K. Barringer, et a1.
`1990. Gene 89, 117—122; F. Barany. 1991. Proc. Natl.
`Acad. Sci. USA 88, 189—193) and transcription-based
`amplification (D. Y. Kwoh, et a1. 1989. Proc. Natl. Acad.
`Sci. USA 86, 1173—1177) require temperature cycling. In
`contrast, methods such as strand displacement amplifi-
`cation (SDA; G. T. Walker, et al. 1992. Proc. Natl.
`Acad. Sci. USA 89, 392—396; G. T. Walker, et al. 1992.
`Nuc. Acids. Res. 20, 1691-1696), selfsustained sequence
`replication (38R; J. C. Guatelli, et al. 1990. Proc. Natl.
`Acad. Sci. USA 87, 1874—1878) and the QB replicase
`system (P. M. Lizardi, et a1. 1988. BioTechnology 6,
`1197—1202) are isothermal reactions. In addition, W0
`90/ 10064 and WO 91/03573 describe use of the bactefi-
`ophage phi29 replication origin for isothermal replica-
`tion of nucleic acids.
`In general, diagnosis and screening for specific nu-
`cleic acids using nucleic acid amplification techniques
`has been limited by the necessity of amplifying a single
`target sequence at a time. In instances where any of
`multiple possible nucleic acid sequences may be present
`(e.g., infectious disease diagnosis), performing multiple
`separate assays by this procedure is cumbersome and
`time—consuming. U.S. Pat. Nos. 4,683,195; 4,683,202 and
`4,800,159 describe the PCR. Although these inventors
`state that multiple sequences may be detected, no proce-
`dure for amplifying multiple target sequences simulta-
`neously is disclosed. When multiple target sequences
`are amplified, it is by sequentially amplifying single
`targets in separate PCRs. In fact, when multiple pairs of
`primers directed to different target sequences are added
`to a single PCR, the reaction produces unacceptably
`high levels of nonspecific amplification and back-
`ground. An improvement on the PCR which reportedly
`allows simultaneous amplification of multiple target
`sequences is described in published European Patent
`Application No. 0 364 255. This is referred to as multi-
`plex DNA amplification. In this method, multiple pairs
`of primers are added to the nucleic acid containing the
`target sequences. Each primer pair hybridizes to a dif-
`ferent selected target sequence, which is subsequently
`amplified in a temperature-cycling reaction similar to
`PCR.
`
`Certain nucleic acid amplification procedures have
`employed addition of defined sequences to the ends of
`nueleic acid fragments prior to amplification. U.S. Pat.
`No. 5,104,792 describes a modification of PCR which
`
`10
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`25
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`allows amplification of nucleic acid fragments for
`which the sequence is not known. The primers for the
`amplification reaction contain random degenerate se-
`quences at their 3’ ends and a defined sequence at their
`5’ ends. Extension of the primers produces fragments
`containing unknown sequences which are flanked by
`the defined sequence. These fragments may then be
`amplified in a conventional PCR using primers which
`hybridize to the known flanking sequence. Another
`method for PCR amplification of unknown DNA
`which flanks a known sequence is described by D. H.
`Jones and S. C. Winistorfer (1992. Nuc. Acids. Res. 20,
`595—600, “panhandle PCR”). In panhandle PCR, a sin-
`gle—stranded oligonucleotide complementary to a se-
`quence in the known DNA is ligated to the 3’ ends of a
`double stranded fragment. Upon denaturation and in-
`trastrand reannealing,
`the complementary sequences
`hybridize and the recessed 3’ end is extended with poly-
`merase, producing the unknown sequence flanked by
`the known sequence. The known sequence can then be
`used to prepare primers for amplification of the un-
`known sequence. Similar methods for generation of a
`hairpin structure and single primer amplification are
`described in published European Patent Application
`No. 0 379 369. WO 90/09457 describes a sequence-
`independent method for amplification of DNA sequen-
`ces which are entirely unknown. Universal oligonucleo-
`tide primer pairs are ligated to the target DNA by
`blunt-end ligation so that PCR. amplification may be
`primed using these known primers.
`Several methods are known which allow amplifica-
`tion of target sequences when only partial sequence
`information is known. A. R. Shuldiner, et al. (1990.
`Gene 91, 139—142) describe a modification of reverse
`transcription PCR in which a unique sequence is ap-
`pended to the 5’ end of the first strand during reverse
`transcription. First strand synthesis is primed by a hy-
`brid primer which is complementary to the RNA target
`at the 3’ end and contains the unique sequence at the 5’
`end. The cDNA is then amplified using a primer di-
`rected to the unique sequence and a primer directed to
`a target-specific sequence. This reportedly reduces am-
`plification of carryover contaminants. Published Euro-
`pean Patent Application No. 0 469 755 discloses a
`method for producing single stranded polynucleotides
`having two segments that are non-contiguous and com-
`plementary. A sequence complementary to an existing
`sequence in the polynucleotide is introduced by exten-
`sion of a primer which hybridizes to the polynucleotide
`at its 3’ end and has the complement of the existing
`sequence at its 5’ end. After extension of the primer the
`polynucleotide can be amplified using a single primer.
`V. Shyamala and G. F. L. Ames (1989. Gene 84, 1—8)
`teach a method for PCR amplification of DNA when
`the sequence of only one end is available (SSP-PCR).
`The unknown end is ligated to a genetic vector se-
`quence, and the fragment is amplified using a gene-
`specific primer and a generic vector primer. Similar
`methods are disclosed in Published European Patent
`Application No. 0 356 021. WO 90/01064 describes
`amplification of a sequence by synthesizing a comple-
`mentary strand primed with a sequence-specific primer
`directed to a known portion of the sequence. A homo-
`polymer is added to the 3’ end of the complement and
`the sequence is amplified using a homopolymer primer
`and a primer which is homologous to a region of the
`sequence-specific primer. Adaptation of PCR to foot-
`
`Ariosa Exhibit 1019, pg. 7
`|PR2013—00276
`
`Ariosa Exhibit 1019, pg. 7
`IPR2013-00276
`
`

`

`3
`printing is taught by P. R. Mueller and B. Wold (1989.
`Science 246, 780—786). For footprinting, a common oli-
`gonucleotide sequence is ligated to the unique end of
`each fragment of the footprint ladder. The fragments
`are amplified using a primer complementary to the
`common sequence and a primer complementary to the
`known sequence of the fixed end.
`The present methods provide a means for appending
`any adapter sequence or any pair of adapter sequences
`to any target prior to amplification by primer extension.
`The adapter sequences reduce the number of specific
`primers which are required for simultaneous amplifica-
`tion of two or more target sequences in a single primer
`extension amplification reaction (referred to herein as
`“multiplex amplification” or “multiplexing”). Conven-
`tional multiplexing involves putting into the reaction
`primers specific for amplification of each target se-
`quence, i.e., each target is amplified by a specific primer
`or pair of primers. Conventional multiplexing provides
`satisfactory results in certain circumstances, but has
`drawbacks in that multiple specific primers must be
`prepared for each multiplex reaction. Often, however,
`multiple sequences cannot be readily amplified and
`detected using conventional multiplexing due to genera-
`tion of high levels of nonspecific background amplifica-
`tion. The adapter mediated multiplexing of the inven—
`tion is an alternative to conventional multiplexing
`which gives improved results in certain cases. Of the
`foregoing publications, only EPO O 364 255 and Muel-
`ler and Wold address the problem of simultaneously
`amplifying multiple target sequences. Both teach simul-
`taneous amplification for PCR, which in part due to its
`temperature cycling provides significantly different
`reaction conditions as compared to isothermal amplifi-
`cations such as SDA. Although certain of the foregoing
`publications describe appending defined sequences to
`either end of a fragment prior to amplification, the addi-
`tion of the defined end and amplification are performed
`in separate reactions. Further, the present invention for
`the first time provides methods for simultaneously am-
`plifying multiple target sequences by SDA without the
`necessity of providing separate specific primers for each
`target. The inventive methods are particularly advanta-
`geous in that addition of defined adapter sequences to
`the ends of the target sequences and the amplification
`reaction occur in a single reaction mix and appear as a
`single step to the practitioner.
`SUMMARY OF THE INVENTION
`
`According to the present invention, a single primer or
`pair of primers can be used to coamplify multiple target
`nucleic acid sequences. Defined adapter sequences are
`appended to the ends of the target sequences within the
`context of the amplification reaction so that no addi-
`tional manipulations are involved to append the adapter
`sequences. That is, the target sequences with the ap-
`pended adapter sequences need not be isolated prior to
`amplification. In one embodiment for coamplifying two
`target sequences, a sequence corresponding to a termi-
`nal segment from one of the two strands of the first
`target sequence is appended to the 5’ end of one of the
`two strands of the second target sequence and a se-
`quence corresponding to a terminal segment from one
`of the two strands of the second target sequence is ap-
`pended to the 5’ end of one of the two strands of the first
`target sequence. Amplification of the two modified
`targets then requires only a single pair of amplification
`primers. One amplification primer of the pair hybridizes
`
`5
`
`10
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`15
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`20
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`
`5,422,252
`
`4
`to a sequence corresponding to the first target and the
`other amplification primer of the pair hybridizes to a
`sequence corresponding to the second target. Alterna-
`tively, a single pair of sequences from one target may be
`appended to the 5’ and 3' ends of each strand of any
`number of targets. In another embodiment, two arbi-
`trary “universal” adapter sequences may be appended
`to the ends of any number of targets. All such modified
`target sequences may then be amplified using a single
`pair of “universal” primers which hybridize to the ap—
`pended end-sequences.
`The methods of the invention are particularly advan-
`tageous for SDA, as the required number of amplifica-
`tion primer pairs containing restriction enzyme recogni-
`tion sites is reduced from one pair/target to a single
`pair, or, alternatively, to a single amplification primer.
`With fewer amplification primer pairs, the formation of
`primer dimers and nonspecific background amplifica-
`tion are reduced. The inventive methods also allow a
`reduction in the concentration of primers, as the adapter
`primer which contains the defined sequence is present
`at much lower concentration than the amplification
`primer it replaces.
`DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a diagram illustrating the method of the
`invention for appending adapter sequences to both ends
`of a target sequence.
`FIG. 2 is a diagram illustrating the method of the
`invention for coamplification of two target sequences.
`FIG. 3 is an autoradiograph showing the results of
`the experiment in Example 1.
`FIG. 4 is an autoradiograph showing the results of
`the experiment in Example 2.
`FIG. 5 is an autoradiograph showing the results of
`the experiment in Example 3.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The present invention provides methods for simulta-
`neous amplification of multiple target sequences by
`primer extension, particularly by SDA (multiplex
`SDA). The methods use a single pair of amplification
`primers or a single SDA amplification primer to coam-
`plify the multiple target sequences. This is accom-
`plished by appending a defined adapter sequence to the
`targets and amplifying by primer extension. The inven-
`tive methods are referred to herein as “adapter-
`mediated multiplexing.” This is in contrast to “conven-
`tional multiplexing” in which multiple pairs of target-
`specific primers are used to coamplify the multiple tar-
`gets without addition of. adapter sequences.
`The following terms are defined herein as follows:
`An amplification primer is a primer for amplification
`of a target sequence by primer extension. For SDA, the
`3’ end of the amplification primer (the target binding
`sequence) hybridizes at the 3’ end of the target sequence
`and comprises a recognition site for a restriction en-
`zyme near its 5’ end. The recognition site is for a restric-
`tion enzyme which will nick one strand of a DNA du-
`plex when the recognition site is hemimodified, as de—
`scribed by Walker, et al. (1992. PNAS 89, 392—396) and
`in US. Ser. No. 07/819,358, filed Jan. 9, 1992 (the dis-
`closure of which is hereby incorporated by reference).
`A hemimodified recognition site is a double stranded
`recognition site for a restriction enzyme in which one
`strand contains at
`least one derivatized nucleotide
`which prevents cutting of that strand by the restriction
`
`Ariosa Exhibit 1019, pg. 8
`|PR2013-00276
`
`Ariosa Exhibit 1019, pg. 8
`IPR2013-00276
`
`

`

`5,422,252
`
`5
`
`30
`
`25
`
`5
`enzyme. The other strand of the hemimodified recogni-
`tion site does not contain derivatized nucleotides and is
`nicked by the restriction enzyme. The preferred
`hemimodified recognition sites are hemiphosphorothi—
`oated recognition sites for the restriction enzymes Hin-
`CH, HindII, AvaI, NciI and Fnu4HI. For the majority
`of the SDA reaction, the amplification primer is respon-
`sible for exponential amplification of the target
`se—
`quence.
`An adapter primer has a sequence at its 3’ end (the 10
`target binding sequence) which hybridizes to the target
`sequence. At the 5’ end of the adapter primer is an
`adapter sequence. The adapter sequence may be a se-
`quence which is substantially identical to the 3’ end of
`one of the amplification primers or it may be any de- 15
`fined sequence for which amplification primers with
`complementary target binding sequences can be pre-
`pared.
`A bumper primer is a primer which anneals to a tar-
`get sequence upstream of either an adapter or amplifica— 20
`tion primer, such that extension of the bumper primer
`displaces the downstream primer and its extension prod-
`uct. Extension of bumper primers is one method for
`displacing the extension products of adapter and ampli-
`fication primers, but heating is also suitable.
`Identical sequences will hybridize to the same com-
`plementary nucleotide sequence. Substantially identical
`sequences are sufficiently similar in their nucleotide
`sequence that they also hybridize to the same nucleotide
`sequence.
`The terms target or target sequence refer to nucleic
`acid sequences to be amplified. These include the origi-
`nal nucleic acid sequence to be amplified and its com-
`plementary second strand (prior to addition of adapter
`sequences), either strand of an adapter-modified copy of 35
`the original sequence as described herein, and either
`strand of a copy of the original sequence which is an
`intermediate product of the reactions in which adapter
`sequences are appended to the original sequence.
`In the adapter-mediated multiplexing of the inven- 40
`tion, adapter sequences are appended to the ends of
`target sequences by means of adapter primers and a
`series of extension and strand displacement steps as
`described below. An adapter primer is an oligonudeo-
`tide comprised of (i) an adapter sequence at its 5’ end 45
`and (ii) a target binding sequence at its 3’ end. The 5’
`adapter sequence may be any sequence for which a
`suitable amplification primer can be prepared. The
`adapter sequence may be arbitrary or it may correspond
`to a segment of one of the target sequences. The adapter 50
`and target binding regions of an adapter primer may be
`contiguous, or they may be separated by a segment of
`unrelated sequence. Different adapter palmers may or
`may not have common 5' adapter sequences (as de-
`scribed below), but they will generally have different 3' 55
`target binding sequences to achieve hybridization to the
`various targets. A unique adapter primer will therefore
`usually be required for each target end to be modified
`by attachment of an adapter sequence. One or both ends
`of a target sequence may be modified by the attachment 60
`of adapter sequences. When both ends of a target se-
`quence are modified the two appended sequences may
`be identical or they may be different, depending on the
`choice of adapter primers.
`By way of example, the following detailed descrip- 65
`tion of the invention is directed to primer extension
`amplification by SDA. It will be apparent to one skilled
`in the art that these methods are readily applicable to
`
`6
`any method for DNA amplification which is based on
`extension of primers by polymerase. These include, for
`example, the 38R and PCR amplification methods dis-
`cussed above. To adapt the inventive methods to these
`amplification reactions, the SDA amplification primers
`would be replaced with amplification primers appropri—
`ate for the selected primer extension amplification reac—
`tion, as is known in the art. The adapter primers would
`be essentially unchanged regardless of the primer exten-
`sion amplification method selected.
`FIG. 1 illustrates a preferred method for modifying
`both ends of a target sequence by attachment of defined
`adapter sequences. A first adapter primer (A1) is hybrid-
`ized to a target DNA at the 3’ end of the target DNA
`sequence. The target binding sequence of A1 is at the 3'
`end and the selected adapter sequence is at the 5’ end. A
`first bumper primer (B1) as described by Walker, et al.
`(1992. Nuc. Acids Res. 20, 1691-1696) is hybridized to
`the target DNA upstream of the first adapter primer.
`The first adapter primer and first bumper primer are
`extended with polymerase. Extension of the upstream
`bumper primer displaces the extension product of the
`first adapter primer
`(Al-ext), which includes the
`adapter sequence at its 5' end but is otherwise comple-
`mentary to the original target DNA sequence. The
`target binding sequence (3' end) of a second adapter
`primer (A2) is then hybridized to the first adapter exten-
`sion product (Al-ext) at a position corresponding to the
`3’ end of the sequence complementary to the original
`target sequence. The 5’ end of A2 comprises a second
`adapter sequence, which may be the same as the A1
`adapter sequence or different from it. Polymerization
`and displacement by extension of a second bumper
`primer (B2) as before produces a single stranded copy of
`the original target sequence with adapter sequences
`appended to the 5’ and 3’ ends. Specifically, the 5’ end
`of this modified target derives directly from the second
`adapter primer, while the 3’ end of the modified target
`(which comes indirectly from the first adapter) is com-
`prised of a sequence complementary to the first adapter
`sequence.
`For brevity, FIG. 1 depicts target generation from
`only one of the two complementary target strands nor-
`mally present in a sample. Generation of modified tar-
`gets from the second strand is analogous to generation
`of modified targets from the first strand as shown in
`FIG. 1, except that the order of binding and extension
`of the first and second adapter primers is reversed. That
`is, A2 is bound and extended first and A1 binds to the
`extension product of A2 and is extended. Binding and
`extension of the first and second bumper primers is
`similarly reversed. The net result of the corresponding
`extension and displacement reactions for the second
`strand is a modified target fragment which has a se—
`quence identical to the first adapter sequence at the 5’
`end and a sequence complementary to the second
`adapter sequence at the 3’ end. All strands of the target
`sequences, terminally modified with adapter sequences,
`can then be amplified exponentially by amplification
`primers whose target binding sequences (at the 3’ end of
`the primer) are substantially identical to the first and
`second adapter sequences at the 5’ ends of the adapter
`primers used to generate the modified targets.
`The process illustrated in FIG. 1 can be used to mod—
`ify multiple target sequences simultaneously within a
`single reaction mixture provided the mixture contains
`an appropriate adapter primer for each target end to be
`modified. The various adapter primers may be designed
`
`Ariosa Exhibit 1019, pg. 9
`|PR2013-00276
`
`Ariosa Exhibit 1019, pg. 9
`IPR2013-00276
`
`

`

`5,422,252
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`7
`so that all modified target sequences generated contain
`the same pair of appended sequences. A11 terminally
`modified target strands may then be amplified by the
`same pair of amplification primers. Alternatively, if all
`adapter primers contain the same 5’ adapter sequence
`only a single amplification primer is needed to amplify
`all modified target strands.
`FIG. 2 illustrates an alternative embodiment of the
`
`present invention in which two target sequences are
`co-amplified using a single pair of amplification primers.
`Again, modification of only one strand of each target
`sequence is illustrated for clarity. In this embodiment,
`one end of each target strand is modified by appending
`to it a sequence substantially identical to a terminal
`segment of the other target. The other end of each
`target strand remains unmodified and retains its original
`complementarity to one member of the amplification
`primer pair. As detailed below, the resulting modified
`targets can then both be amplified by a single pair of
`amplification primers, one member of the pair being
`complementary to one of the two original target se—
`quences and the other member of the pair beingcomple-
`mentary to the other of the two original target sequen—
`ces. For the first target (A), an A-specific amplification
`primer (SA) is hybridized to the 3’ end of the target
`sequence and extended with polymerase. The nicking
`enzyme recognition site of the amplification primer is
`depicted in FIG. 2 as a raised portion of the primer. The
`resulting extension product is displaced by extension of
`a bumper primer (BAl) which hybridizes to the target
`upstream from 8,4. The displaced 8,; extension product
`(SA-ext) is hybridized to an adapter primer (AA) which
`binds to SA-ext at the 3’ end of the complement of the
`original target sequence. The 5’ end of AA comprises the
`adapter sequence (solid portion), which is substantially
`identical to the target binding sequence at the 3’ end of
`SB, an amplification primer which specifically binds to
`the second target (B). Extension of AA and displacement
`of the AA extension product (AA-ext) produces a single
`stranded copy of the A target sequence with a nicking
`enzyme recognition site and the A target sequence at its
`3' end and the S3 target binding sequence at its 5’ end.
`The second target (B) is treated similarly, first bind-
`ing and extending a B-specific amplification primer
`(83), then hybridizing an adapter primer (A3) to the
`extension product (SB-ext). SB hybridizes to B at a 3’
`terminal segment of B which is complementary to both
`the target binding sequence of SB and the adapter se-
`quence of AA. The 3’ end of adapter primer AB hybrid-
`izes at the 3’ end of the complement of the original
`target and the 5’ end of AB (open portion) is substan-
`tially identical to the target binding sequence of SA.
`Extension and displacement of the AB extension prod-
`uct (AB-ext) produces a copy of the second target se-
`quence with a nicking enzyme recognition site (raised
`portion) and the B target sequence at its 3’ end and the
`5,; target binding sequence at its 5’ end. The two adapt-
`er-modified copies of the target sequences are amplifia-
`ble by SDA using only the SA and SB amplification
`primers already present in the reaction. To begin SDA,
`AA-ext and AB—ext hybridize to their respective amplifi-
`cation primers, which are extended to produce the com-
`plement of the modified strand (i.e., extension of SA on
`the A modified strand and extension of 83 on the B
`modified strand),
`including the complement of the
`adapter sequence at the 3’ end. After nicking and dis-
`placement,
`the amplification primer of the opposite
`target can then bind to the 3’ end of this extension prod-
`
`10
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`15
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`20
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`35
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`8
`uct (i.e., Sp to the A-derived strand and SA to the B-
`derived strand) and is extended to produce a fragment
`with a nicking enzyme recognition site at each end. This
`fragment
`is amplified by conventional SDA as de-
`scribed by Walker, et al., supra.
`The double stranded reaction products which are
`produced after displacement of AA-ext and AB-ext may
`also participate in a reaction loop which generates addi-
`tional copies of AA-ext and AB-ext. Nicking the restric-
`tion enzyme recognition site of the bottom strand, ex-
`tending with polymerase and displacing the bottom
`strand produces targets which are similar to SA-ext and
`SB—ext but with half of a restriction enzyme recognition
`site at the 5’ end. The adapter primers can bind to these
`fragments as shown in FIG. 2 and can be extended and
`displaced to produce additional copies of AA-ext and
`AB-ext (also with half of a restriction enzyme recogni-
`tion site at the 5’ end) which enter the SDA reaction
`cycle as described above.
`FIG. 2 depicts the generation of modified targets
`from only one of the two complementary strands nor-
`mally present for each target sequence. Processes simi-
`lar to those shown also originate from the second strand
`of each target. In the case of the second strand, how-
`ever, the order of binding and extension of the primers
`is reversed. The adapter primers first bind directly to
`the target second strand and are extended on that tem—
`plate. After its subsequent displacement, the resulting
`adapter extension product hybridizes to the amplifica-
`tion primer, which is in turn extended and displaced to
`give a product containing the original second strand
`target sequence with a, recognition site for a nicking
`restriction enzyme at its 5’ end and a sequence comple-
`mentary to the adapter sequence at its 3’ end. This modi-
`fied fragment enters conventional SDA amplification
`by binding and extension of the amplification primer
`specific for the opposite target (i.e., SB binds to t