[19]
`United States Patent
`[11] Patent Number:
`6,027,923
`[45] Date of Patent: Feb. 22, 2000
`Wallace
`
`
`
`USOO6027923A
`
`[54] LINKED LINEAR AMPLIFICATION 0F
`NUCLEIC ACIDS
`
`[75]
`
`Inventor: Robert Bruce Wallace, Greenbrae,
`Calif.
`
`[73] Assignee: Bio-Rad Laboratories, Inc., Hercules,
`Calif.
`
`[21] Appl. No.: 08/826,532
`
`[22]
`
`Filed:
`
`Apr. 2, 1997
`
`Related U.S. Application Data
`
`[63] Continuation of application No. 08/475,605, Jun. 7, 1995,
`abandoned, which is a continuation—in—part of application
`No. 08/095,442, Jul. 23, 1993, abandoned.
`
`Int. Cl.7 ...................................................... C12P 19/34
`[51]
`[52] U.S. Cl.
`..................
`435/91.2; 435/6
`[58] Field of Search
`....... 435/6, 91.2
`
`
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`.................................. 435/6
`7/1989 Vary et al.
`4,851,331
`6/1995 Wallace et al.
`..
`435/91.2
`5,426,039
`
`1/1996 Cocuzza et al.
`.
`....... 435/6
`5,484,701
`6/1996 Newton .................................. 435/91.2
`5,525,494
`FOREIGN PATENT DOCUMENTS
`
`416817
`
`.
`8/1990 European Pat. Off.
`OTHER PUBLICATIONS
`
`Kleppe et al., J. Mol. Biol, 56:341—361 (1971).
`Saiki et al., Science, 230:1350—1354 (1985).
`Ph. Cuniasse et al., J. Mol. Biol, 213:303—314 (1990).
`R. Eritja et al., Nucleosides & Nucleotides, 6(4):803—814
`(1987).
`s. Randall et al.,J. Biol. Chem., 262(14):6864—6870 (1987).
`F. Seela et al., Nucleic Acids Research, 15(7):3113—3129
`(1987).
`Rychlik et al., Nucleic Acids Res., 18:6409—6412 (1990).
`Wu et al., DNA Cell Biol., 10:233—238 (1991).
`
`Wittwer et al., Nucleic Acids Res., 17:4353—4357 (1989).
`Wittwer et al., Biotechniques, 10:76—83 (1991).
`Petruska et al., PNAS USA, 85:6252—6256 (1988).
`Newton et al., Nucleic Acids Res., 17:2503—2516 (1898).
`Ugozzli et al., Methods, 2:42—48 (1991).
`Reed et al., Nucleic Acids Res., 13:7207—7221 (1985).
`Church et al., PNAS USA, 81:1991—1995 (1984).
`Panet
`et
`al.,
`Journal
`of Biological Chemistry,
`249:5213—5221 (1974).
`DB. Olsen et al., “Investigation of the .
`Biochemistry 29, 1990, pp. 9546—9551.
`A. Kornberg, DNA Replication, 1980, pp. 90—93.
`F. Eckstein et al., “Phosphorothioates in Molecular Biol-
`ogy”, TIBS 14, 1989, pp. 97—100.
`. Sequences”,
`.
`R.Y. Walder et al., “Use of PCR Primers .
`Nucleic Acids Research, 2], 1993, pp. 4339—4343.
`S]. Odelberg et al., “A Method for Accurate Amplification
`.
`.
`. Sequences”, PCR Methods and Applications 3, 1993, pp.
`7—12.
`
`. EcoR ”,
`
`.
`
`Newton et al., Nucleic Acids Res. 21(5):1115—1162, 1993.
`The Stratagene 1988 Catalog, p. 39.
`
`Primary Examiner—Scott W. Houtteman
`Attorney, Agent, or Firm—Rothwell, Figg, Ernst, & Kurz,
`p.c.
`
`[57]
`
`ABSTRACT
`
`The extensive synthesis (“amplification”) of a nucleic acid
`sequence of interest is attained through a linked series of
`multi-cycle primer extension reactions (LLA). The primers
`used in each of the primer extension reactions of the process
`contain non-replicable elements that halt nucleic acid syn-
`thesis and thereby prevent the synthesized molecules from
`serving as templates in subsequent cycles. Synthesized
`molecules accumulate during primer extension in a math-
`ematically linear fashion,
`thereby rendering the process
`relatively insensitive to contaminating nucleic acids. Mul-
`tiple primer sets are employed, thereby ensuring the accu-
`mulation of a large number of copies of the nucleic acid
`sequence of interest. The invention also provides for the
`detection of an amplified nucleic acid sequence of interest,
`as well as reagent kits for carrying out the reaction.
`
`45 Claims, 13 Drawing Sheets
`
`’l/I/I/I/A—
`—. 'l
`
`0 PRODUCT OF A PCR REACTION IS COMPLETELY DOUBLE STRANDED
`
`'///////4Z—
`—3I
`
`PRODUCTS OF AN LLA REACTION CAN BE ANNEALED TO
`‘
`FORM A DUPLEX WITH SINGLE STRANDED ENDS
`
`THIS PRODUCT IS SHORTER THAN THE CORRESPONDING
`‘
`PCR FRAGMENT
`
`I Non-replicable
`
`element
`
`Ariosa Exhibit 1021, pg. 1
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 1
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22,2000
`
`Sheet 1 0f 13
`
`6,027,923
`
`FIG . 1
`
`____x____
`
`(a)
`
`(b)
`
`(c)
`
`____x ................................................ (d)
`
`Ariosa Exhibit 1021, pg. 2
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 2
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 2 0f 13
`
`6,027,923
`
`FIG.2
`
`1O
`.........................................___f
`
`(e)
`
`......................................... /20
`
`Ariosa Exhibit 1021, pg. 3
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 3
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 3 0f 13
`
`6,027,923
`
`FIG.3
`
`_—_—x————
`
`----O----
`——v——x—o———CIIIIIIII.0.00I....IIDIIIOIIOUIIIOIOIIOOOI
`
`IOIIOOCCOCIOOOCCCII...ICIIIOOOIOOIOII—~——O———-
`
`————X——-—--—IIIIOIOOIOIOI..IUIUUOOICOIUOOOIOIIDIOID...
`
`(f)
`
`~———x-———-ID.0.IIOIIIOI.OOIOOOICOCCUCUCOOOOOOOO
`
`..._.__X————
`O.IIIUIOCUIOOIOOOOII.IIIIIt'll....0.......—-———0———-
`
`.0...0......OO.COO...OCC....IIOOOOCOCOCOIO————O————
`__._._x————
`
`————O.————
`
`Ariosa Exhibit 1021, pg. 4
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 4
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 4 0f 13
`
`6,027,923
`
`FIG.4
`
`l —
`
`-——x————.I.....I.I.CCCOOCCCIIIOOIIOCCCCOIIICIOOIOI
`
`I.IOUIOOCIOO.‘I..........I..I..II...’————O~-——
`
`——~—x————.I.II.IOIOO.CQOCICIOOOOIIOOOOUIOICIO......
`
`0..IIOIIOO0.0il.II.0.0...OOOOOIOOOOOO————O——_-
`
`I. I..00...I.U..‘COCDICIIIOUIUC.IIOII.————o————
`
`————x————II.IIOO.IIOOOOIIIOOCCIOICDIIOIIOIICIIOIIII
`
`—-v—-—-X---—..0..OO...O.CI...’......III...O...IOO
`
`-——-~X————O0.0I.IOOOOIOCOOOOOCIICIOOOIIOCIIOIOO
`0....IOOO0.0...COOO..0...IIOCICC.‘I...’...————o————
`
`.IOIIII.II.O...0.0..00......0000IDIOOII...————O————
`
`———-x-———.I.O.IOIIOOIUIOOIOOOOOOOOOIOOI000...-
`
`IIIOIDOC...III...IIO...0......OOOOICOOI...~———O————
`
`Ariosa Exhibit 1021, pg. 5
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 5
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 5 0f 13
`
`6,027,923
`
`FIG.5
`
`...........................................
`
`(a)
`
`\20
`
`I10
`
`(b)
`
`--—b—-—
`
`..........................................
`
`(C)
`
`~_-— 1:: ooooooooooooooooooooooooo u _________ b---
`
`Ariosa Exhibit 1021, pg. 6
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 6
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 6 0f 13
`
`6,027,923
`
`FIG.6
`
`OOOOIOOIIOOOOOOII.0......OOIOOIOOIIIIIO—-——O————
`--_a___
`
`_-_a-__....................................
`
`(d)
`
`—-—b—-—
`
`--—b---
`_—-—-—X————I.I.OOOOIIOUIIO.....I...OOOIOIOOOCOOOOC
`
`OOIUCCICIOIOII0.0CO...CC.00....C.......————0—————
`———-a——-—IIt.IIIO...0....IIUOIOICOOCOCOCOOOIO
`
`................................__-b__-//
`___a_-_....................................
`
`30
`
`(e)
`
`___a___.............................../\4o
`II.IIII.00...It.IUD...OOOOOOOOOOOCO———b—-—
`
`COO...OOOOOIOOOCOCOOCOOOOIOOOOOIOIl———b——-
`————x——-—I.00...0.0.0.0.0....OIOOOOCOIIOOIOOOIOI
`
`Ariosa Exhibit 1021, pg. 7
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 7
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 7 0f 13
`
`6,027,923
`
`REACTION 1
`
`REACTION 2
`
`QQBLESLRAEPEBM TEMPLATE
`
`BOEELELSLEAEQEEIflTéMi/WE
`
`J P1
`PRIMER EXCESS
`
`P2 \_
`PRIMER EXCESS
`
`m CYCLES 0F DENATURATION,
`ANNEALING AND PRIMER
`EXTENSION
`
`n CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`EXTENSION
`
`
`
`n COPIES
`
`REACTION 3
`
`REACTION 4
`
`Eh— ————— ->
`
`TEMPLATE I
`,
`_
`TEMPLATE II
`_
`:1:;/P1PZ\:¥::.:
`PRIMER EXCESS
`PRIMER EXCESS “m
`
`
`
`
`
`0 CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`p CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`EXTENSION
`
`EXTENSION
`
` IIIIIII
`
`IIIIIII
`
`0 COPIES
`
`p COPIES
`
`Ariosa Exhibit 1021, pg. 8
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 8
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 8 0f 13
`
`6,027,923
`
`FIG. 8A
`
`FIG. 8B
`
`REACTION 1A
`
`REACTION 2A
`
`DOUBLE STRANDED DNA TEMPLATE
`
`DOUBLE STRANDED DNA TEMPLATE
`
`PRIMER EXCESS
`
`m CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`‘
`
`EXTENSION
`
`PRIMER EXCESS
`
`P2
`
`
`
`n CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`EXTENSION
`
`
`
`IIA
`
`n COPIES
`
`FIG. 8C
`
`FIG. 8D
`
`REACTION 1B
`
`REACTION ZB
`
`DOUBLE STRANDED DNA TEMPLATE
`
`DOUBLE STRANDED DNA TEMPLATE
`
`P4
`
`'
`
`PRIMER L:z';
`
`EXCESS
`
`m' CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`l EXTENSION
`
`n' CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`l EXTENSION
`
`IIB
`
`n' COPIES
`
`Ariosa Exhibit 1021, pg. 9
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 9
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 9 0f 13
`
`6,027,923
`
`REACTION 3A
`
`REACTION 4A
`
`‘
`
`__.._. m
`TEMPLATE IIA
`
`m— ————— -»
`TEMPLATE 1A
`
`P1
`PRIMER EXCESS
`
`P21
`PRIMER EXCESS
`
`o CYCLES 0F DENATURATION,
`ANNEALING AND PRIMER
`
`p CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`EXTENSION
`
`EXTENSION
`
`_ _ _ _ -
`
`
`m— ————— —-—
`
`o COPIES
`
`p COPIES
`
`REACTION 3B
`
`REACTION 43
`
`
`
`m— ————— —-—
`
`TEMPLATE IIA
`
`TEMPLATE 1A
`
`PRIMER EXCESS
`
`P4—\
`PRIMER EXCESS 3
`
`
`
`o‘ CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`p‘ CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`
`EXTENSION
`
`EXTENSION
`
`éf—E E i NIB
`
`
`
`0' COPIES
`
`p' COPIES
`
`Ariosa Exhibit 1021, pg. 10
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 10
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 10 0f 13
`
`6,027,923
`
`REACTION 5
`
`REACTION 6
`
`
`TEMPLATE IVA
`
`fP3
`PR|MER EXCESS
`
`m— __ __ __ __ -.
`TEMPLATE IIIA
`
`P4‘\ :
`PRIMER EXCESS -
`
`
`
`o' CYCLES OF DENATURATION,
`ANNEALING AND PRIMER
`EXTENSION
`
`P' CYCLES 0F DENATURATION,
`ANNEALING AND PRIMER
`EXTENSION
`
`g E E E
`
`.___:_:m
`
`IIIB
`
`
`
`o' COPIES
`
`P' COPIES
`
`Ariosa Exhibit 1021, pg. 11
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 11
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22,2000
`
`Sheet 11 0f 13
`
`6,027,923
`
`FIG.11
`
`’Wl/l/I/A
`
`_W
`
`- PRODUCT OF A PCR REACTION IS COMPLETELY DOUBLE STRANDED
`
`'///////4 Z
`
`—3I
`
`0
`
`PRODUCTS OF AN LLA REACTION CAN BE ANNEALED TO
`
`FORM A DUPLEX WITH SINGLE STRANDED ENDS
`
`-
`
`THIS PRODUCT IS SHORTER THAN THE CORRESPONDING
`
`PCR FRAGMENT
`
`I Non-replicable
`
`element
`
`Ariosa Exhibit 1021, pg. 12
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 12
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 12 0f 13
`
`6,027,923
`
`
`
`l_..|«NNaB|L
`
`mm
`
`
`
`w<w<uwwu<UHo<<<<h<uwowkuwww<uuw<ww<uwwu<www<uw<wo<uuUHU<FuH<<uuwwhwaw<kuuu<u<uuw<wwpwhuuu<UHuu
`
`om#
`
`mmw
`
`0mm
`
`uwFUFu<<w<ww<whuUhu<wkuu<uoHO0H<uu<u<w<u<<<uHuu<<uw<Hu<uphwhwhu<<u<u<opuwpuwhhh<u<khuwph<hUH<uuII_|Imm3anVIIImmm-q8||..ln.|_
`
`
`
`<0<ww<<wkhwu<u<w<<u<Hku<<uH<wahhww<uowwhuuuww<whwuhwwhpo<<0H<wukwu<<wkwwqquwwwwhwhuuuwku<pru
`
`
`
`wdkhuuu<uuUHth<HuHowHH<FuuwHUFUHUHU<¢HU<uww<h<GPUprwwwHHuhu<w<<w<w<u<w<oohwp<uwwwku<<<w<h<<uu
`
`
`
`N_\.O_n_
`
`I_|lllllmmYQEIIIIIL
`
`muthUUHw<wHHHUkhww<0<uuu<wwkhuuu<FUkuwahuwHuw
`
`Ariosa Exhibit 1021, pg. 13
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 13
`IPR2013-00276
`
`

`

`US. Patent
`
`Feb. 22, 2000
`
`Sheet 13 0f 13
`
`6,027,923
`
`deE
`
`9040900004460400690000885UUUBUUGBQUUUUBBUBUmugBUEQflUGCUUdfiUUdUdUBHB
`
`VHHUEHmw
`
`
`
`UUOOHUUdUUdUOBUBUUBflUUDGUGU09.09.0490UU§U¢OBBBHBUUGfiUBfiBBUUUBBflUUfiUUUBB.‘m
`
`
`
`d0.4.0004009.300UgmuUUUBBBBBUUU<UBU040UgBUAmUdUOdGOngBgUUOBUUUUUUUBB
`
`
`
`
`
`
`
`UUOwn.UGBBUUdUflBUUUfiUUBUBUUfiUdewwfigufihwgngUBfiwwdwh04090004060408
`
`BUO.40doUUUUfiwfid09009940BfidfiwfiwggwwgvofidfidfivuU§U§GU¢HUUBUBBUUBUU
`
`NEG
`
`
`
`.muUUfigwggduggdUGGCUSUBUUUdUdGOOBBdBUBUdOdUBUBBHOBU.HUUU
`
`Ariosa Exhibit 1021, pg. 14
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 14
`IPR2013-00276
`
`

`

`1
`LINKED LINEAR AMPLIFICATION OF
`NUCLEIC ACIDS
`
`CROSS REFERENCE TO RELATED
`APPLICATION
`
`This is a continuation of application Ser. No. 08/475,605,
`filed Jun. 7, 1995, now abandoned, which was a
`continuation-in-part of Ser. No. 08/095,442, filed Jul. 23,
`1993, now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`1. Technical Field
`
`The present invention relates to the in vitro replication of
`nucleic acids. More specifically, the invention relates to a
`process for replicating a nucleic acid sequence of interest,
`with large quantities of the desired sequence ultimately
`resulting from the linkage of primer extension reactions
`wherein the sequence of interest accumulates in a math-
`ematically linear fashion.
`2. Brief Description of the Background Art
`The extensive replication of nucleic acids, today known
`as (and referred to herein as) nucleic acid “amplification,”
`finds wide utility, both practical and theoretical, in a variety
`of contexts. H. G. Khorana and his co-workers first proposed
`the use of an in vitro DNA amplification process to increase
`available amounts of double-stranded DNA (partial
`sequences of the gene for the major yeast alanine t-RNA)
`that had been created by the enzymatic ligation of synthetic
`DNA’s. See K. Kleppe et al.; J. Mol. Biol. 56:341—361
`(1971). Later,
`in vitro amplification was applied to the
`amplification of genomic DNA (Saiki et al., Science
`230:1350—1354 (1985)) as the technique now known as the
`polymerase chain reaction or “PCR.” Through the wide
`availability of synthetic oligonucleotide primers, thermo-
`stable DNA polymerases and automated temperature cycling
`apparatus, PCR became a widely-utilized tool of the
`molecular biologist.
`The PCR process is referred to in the literature as an
`“exponential amplification” process.
`In each round or
`“cycle” of primer extension, a primer binding site for the
`other primer is synthesized. Thus, each of the synthetic DNA
`molecules produced in any of the previous cycles is avail-
`able to serve as a template for primer-dependent replication.
`This aspect of the process, coupled with the presence of a
`sufficiently large number of primer molecules, results in
`synthetic DNA accumulating in a mathematically exponen-
`tial manner as the reaction proceeds.
`Although PCR has proven to be a valuable technique for
`the molecular biologist, and has been used extensively in the
`fields of human genetic research, diagnostics and forensic
`science, and even in the detection of antibodies, disadvan-
`tages nevertheless have been recognized. The PCR process
`can be difficult to quantify accurately, mainly because the
`amplification products increase exponentially with each
`round of amplification. The products of PCR, namely,
`double-stranded DNA molecules, are difficult to analyze or
`sequence per se. Strand separation typically must be carried
`out prior to sequencing or other downstream processes that
`requires single stranded nucleic acids, such as hybridization
`to a probe capable of detecting the sequence of interest.
`The PCR process also has proven to be quite susceptible
`to contamination generated through the transfer of previ-
`ously amplified DNA sequences into a new reaction. This
`problem appears to be caused by the facts that (1) very large
`amounts of DNA are generated in any given reaction cycle
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6,027,923
`
`2
`
`and (2) the process uses all product DNA strands as tem-
`plates in subsequent cycles. Even minute quantities of
`contaminating DNA can be exponentially amplified and lead
`to erroneous results. See Kwok and Higuchi, Nature
`339:237—238 (1989). Various methods to reduce such con-
`tamination have been reported in the literature (e.g. chemical
`decontamination, physical treatment, enzyme treatment and
`utilizing closed systems), as these contamination problems
`are widely recognized. See, John B. Findlay, “Development
`of PCR for in vitro Diagnostics,” presented at “Genetic
`Recognition,” Nov. 20, 1992, San Diego, Calif.
`There has remained a need for new nucleic acid (DNA)
`amplification methods that provide large amounts of DNA,
`and that selectively amplify only a specific sequence of
`interest, but which avoid the problems now associated with
`the “PCR” reaction. Specifically, there has remained a need
`for nucleic acid amplification methods that ultimately pro-
`duce large amounts of a nucleic acid molecule of interest, or
`large amounts of a molecule containing a nucleic acid
`sequence of interest, but are relatively insensitive to the
`presence of contaminating nucleic acids. There has also
`remained a need for nucleic acid amplification methods that
`generate single-stranded products.
`
`SUMMARY OF THE INVENTION
`
`The foregoing and other needs are met by the present
`invention, which in one aspect provides a process for
`amplifying a specific nucleic acid sequence of interest
`within complementary nucleic acid strands contained in a
`sample, the process including the steps of:
`(a) contacting the strands with a primer that contains a
`non-replicable element, under conditions such that first
`generation primer extension products are synthesized
`using said strands as templates, and wherein the primer
`for a strand is selected such that a first generation
`primer extension product synthesized thereon, when
`separated from the strand, can serve as a template for
`synthesis of a second generation primer extension
`product of the primer for the complement of the strand;
`(b) separating the first generation primer extension prod-
`ucts from their templates to produce single-stranded
`molecules; and
`(c) treating the first generation primer extension products
`with the primers of step (a) under conditions such that
`second generation primer extension products are syn-
`thesized using the first generation primer extension
`products as templates;
`wherein the second generation primer extension products
`contain at least a portion of the nucleic acid sequence
`of interest and cannot serve as templates for the syn-
`thesis of extension products of the primers which were
`extended to synthesize their templates.
`In other aspects of the invention, the products of step (c)
`are separated to produce single-stranded molecules, and the
`entire process is repeated at least once. Step (c) preferably
`is repeated many times, with the process being carried out in
`an automated fashion under the control of a programmable
`thermal cycling apparatus.
`Following the accumulation of second generation primer
`extension products, each of which is incapable of serving as
`a template for the primer extended to prepare its first
`generation template, a new set of primers that contain
`non-replicable elements can be employed. The new set of
`primers advantageously bind to the second generation syn-
`thetic products, bounding the sequence of interest to be
`amplified. The linear replication process is again carried out
`
`Ariosa Exhibit 1021, pg. 15
`|PR2013—00276
`
`Ariosa Exhibit 1021, pg. 15
`IPR2013-00276
`
`

`

`6,027,923
`
`3
`through a number of cycles. Such “linking together” of
`multi-cycle primer extension reactions ultimately results in
`thousand-fold or million-fold amplification of the original
`nucleic acid sequence of interest. Thus, the present process
`is deemed “linked linear amplification” or “LLA.”
`In another aspect of the invention, multiple (nested) sets
`of primers containing non-replicable elements can be pro-
`vided in a single amplification reaction mixture. The sets are
`selected so as to be capable of binding to their respective
`templates under decreasingly stringent conditions. Thus, all
`the components necessary to carry out several linked linear
`amplifications can be provided in a single reaction mixture.
`In yet another aspect of the invention, allele-specific
`nucleic acid replication is carried out according to the
`present invention with the use of primers directed to specific
`polymorphic sites on the template that are known to be
`indicative of a genetic disease or disorder, such as sickle cell
`disease. The allele-specific primers, containing non-
`replicable elements, are designed so that they prime nucleic
`acid synthesis of only those templates containing the desired
`allele.
`
`The synthetic nucleic acid molecules resulting from the
`present process can be used in the diagnosis of genetic
`disorders or diseases, as reagents in further techniques such
`as gene cloning, for forensic identification, etc.
`The process described herein also can be carried out using
`a single nucleic acid strand as a starting material. Such a
`process comprises:
`(a) contacting the strand with a first primer containing a
`non-replicable element, under conditions such that a
`first generation primer extension product is synthesized
`using the strand as a template;
`(b) separating the first generation primer extension prod-
`uct from its template to produce single stranded mol-
`ecules; and
`(c) contacting the first generation primer extension prod-
`uct with a second primer containing a non-replicable
`element under conditions such that a second generation
`primer extension product is synthesized using the first
`generation primer extension product as a template;
`wherein the primers are selected so that
`the second
`generation primer extension product cannot serve as a
`template for extension of the first primer. Steps (a)—(c)
`can be repeated many times, resulting in extensive
`nucleic acid synthesis, following which the reaction is
`linked to a subsequent reaction using a new set of
`primers.
`The processes described herein also can be carried out
`using primers containing cleavable elements. This process
`comprises:
`(a) contacting the strands of a nucleic acid template with
`a first primer containing a cleavable element, under
`conditions such that a first generation primer extension
`product is synthesized using the strand as a template;
`(b) separating the first generation primer extension prod-
`uct from its template to produce single stranded mol-
`ecules;
`(c) treating the single stranded molecules such that the
`first generation primer extension product is cleaved at
`the position of the cleavable element;
`(d) contacting the first generation primer extension prod-
`uct with a second primer under conditions such that a
`second generation primer extension product is synthe-
`sized using the first generation primer extension prod-
`uct as a template;
`the second
`wherein the primers are selected so that
`generation primer extension product cannot serve as a
`
`4
`template for extension of the first primer, when the first
`primer extension product has been cleaved at the cleav-
`able element. Steps (a)—(d) can be repeated many times,
`resulting in extensive nucleic acid synthesis, following
`which the reaction may be linked to a subsequent
`reaction using a new set of primers. Additionally, the
`second primer may optionally contain a cleavable ele-
`ment such that the linked linear amplification reaction
`can be carried out on either a double-stranded or a
`
`single-stranded starting template.
`The present invention also relates to a reagent kit for use
`in amplifying a particular nucleic acid sequence. Such kit
`includes, for example, a DNA polymerase, two or more
`primers for each sequence to be amplified wherein each of
`said primers comprises a non-replicable element or incor-
`porates a cleavable element, and, optionally, a control
`nucleic acid sequence capable of being replicated by the
`primers and DNA polymerase. The kit also may contain a
`nucleic acid probe capable of indicating the presence or
`absence of an amplification product of the particular
`sequence. Where the kit contains primers incorporating a
`cleavable element, it may also contain reagents for cleaving
`the primer at the cleavable element.
`
`10
`
`15
`
`20
`
`25
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIGS. 1—4 comprise a schematic representation of a
`nucleic acid amplification process of the present invention.
`FIGS. 5—6 comprise a schematic representation of a
`nucleic acid amplification process that has been linked to the
`process represented by FIGS. 1—4.
`FIG. 7 is a more detailed schematic representation of a
`nucleic acid amplification process carried out with two
`primers according to the present invention.
`FIGS. 8—10 present a detailed schematic representation of
`a linked linear nucleic acid amplification process carried out
`with four primers according to the present invention.
`FIG. 11 is a schematic representation of nucleic acid
`molecules prepared by the PCR process and by a process
`according to the present invention.
`FIG. 12 is a schematic representation of the sequence of
`the human B-globin gene (GenBank locus HUMHBB SEQ
`ID NO: 1) and of several primers described herein.
`FIG. 13 is the sequence of Human Growth Hormone
`Sequence SEQ ID NO: 15 amplified in Example 7. The
`relative positions of the oligonucleotides used in the experi-
`ment are underlined. Oligonucleotide GH1 hybridizes to the
`complement of the Human Growth Hormone sequence
`shown.
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`
`The nucleic acid replication process of the present inven-
`tion is capable of producing large quantities of a specific
`nucleic acid sequence of interest. The process in its preferred
`form comprises a linked series of multi-cycle primer exten-
`sion reactions. In each of the multi-cycle primer extension
`reactions, primer-dependent nucleic acid replication is car-
`ried out
`through a number of cycles, with the primer
`extension products accumulating in a numerically linear
`fashion from cycle to cycle. A unique primer, or set of
`primers,
`is provided for each nucleic acid strand in the
`starting sample that contains the sequence to be amplified.
`The linear accumulation of primer extension products from
`cycle to cycle is assured through the use of primers that
`contain non-replicable elements—elements that halt
`the
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Ariosa Exhibit 1021, pg. 16
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 16
`IPR2013-00276
`
`

`

`6,027,923
`
`5
`primer extension reaction, preventing the nucleic acid poly-
`merase from replicating the entire sequence of the primer.
`Alternatively, the linear accumulation of the desired primer
`extension products is assured through the use of primers that
`contain cleavable elements, where cleavage of the first
`primer extension product results in a second primer exten-
`sion product lacking the functional binding site for the first
`primer. Through the selection of appropriate primers con-
`taining such non-replicable or cleavable elements, and
`appropriate primer annealing conditions, it is ensured that
`the primer extension products which accumulate in greatest
`abundance (referred to herein as “second generation primer
`extension products”) cannot serve as templates themselves
`in subsequent cycles of primer extension using the same
`primers. Thus, unlike nucleic acid amplification processes
`(such as “PCR”) which utilize the primer extension products
`from each cycle as templates for subsequent cycles, “expo-
`nential amplification” does not occur from cycle to cycle.
`The process of the present invention utilizes and takes
`advantage of a number of important properties of oligo-
`nucleotide hybridization and the primer extension reaction.
`The invention takes advantage of the facts that:
`DNA polymerase is able to copy a template DNA many
`times by sequential cycles of denaturation and primer-
`dependent elongation.
`Primer-dependent elongation can occur, under appropri-
`ate conditions, even if the primer is not completely
`complementary to the template.
`Primer extension can utilize a template produced in a
`previous round of primer extension.
`Primer extension is inhibited by abasic sites or by non-
`nucleotide residues when such are present in the tem-
`plate nucleic acid. Alternatively, primer extension may
`be inhibited by physically removing a portion of the
`generation primer extension product by cleaving this
`molecule at the cleavable sites incorporated into the
`primers used in the primer extension reaction.
`Primer length and composition affect, in known ways, the
`conditions (e.g., temperature) at which a primer will
`“prime” polymerase-induced extension on a template.
`Primer extension reactions can be performed in rapid
`cycles with the aid of thermal cycling apparatus.
`The present process advantageously utilizes a series of
`linear amplification reactions, which can be carried out
`(linked) either in series or in parallel (i.e., simultaneously) to
`generate a very large number of copies of a nucleic acid
`sequence of interest. The nucleic acid sequence of interest
`may encompass essentially the entire length of the template
`strand(s), or it may comprise only a very minor portion of it.
`The template strand(s) containing the sequence of interest
`may be present in a substantially homogeneous sample or as
`part (even an extremely minor part) of a mixture of nucleic
`acids.
`
`In accordance with the present invention, a primer that
`contains a non-replicable or cleavable element is provided
`for each strand containing a sequence to be amplified. In the
`case of a double-stranded template, the primer(s) are added
`either prior to or following denaturation of the template. The
`primers are permitted to anneal to their respective starting
`templates, and are extended in the presence of a polymerase
`enzyme, under conditions appropriate for the function of the
`enzyme, to form first generation primer extension products.
`The process is repeated by denaturing the resulting duplexed
`nucleic acid, permitting the primers to anneal to the strands
`and again carrying out the primer extension reaction. Primer
`extension upon the first generation primer extension prod-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`ucts yields second generation primer extension products
`which, due to the presence of non-replicable elements,
`cannot serve as templates for those same primers in subse-
`quent cycles. Alternatively, where the primer contains a
`cleavable element,
`the first generation primer extension
`products are cleaved at the position of the cleavable element,
`so that when this first generation primer extension product is
`annealed to the appropriate primer and extended, the second
`generation primer extension product formed will
`lack a
`binding site for the primer used to generate the first primer
`extension product.
`FIGS. 1—5 present a schematic representation of one
`series of primer extension reactions (i.e., one linear ampli-
`fication reaction) carried out on a DNA template according
`to the process of the present
`invention. The process is
`illustrated starting in step (a) with a double-stranded DNA
`
`molecule having defined termini. The strands of the starting
`DNA are denoted by solid (
`) lines throughout FIGS.
`1—4.
`
`The starting duplex is denatured, preferably by heating in
`a buffer solution containing the same, and the resulting
`single strands are contacted with a pair of primers (step (b)).
`Each primer preferably is provided in substantial molar
`excess of the starting template strand and contains within its
`sequence a non-replicable or alternatively, a cleavable
`element, here denoted by (x) or (0) within the primer
`sequence. Under appropriate conditions, the primers anneal
`to their respective templates and are elongated (step (c))
`according to the primer extension reaction in the presence of
`a DNA polymerase and the four deoxyribonucleotides. Syn-
`thesized DNA is denoted by dotted (""") lines in FIGS.
`1—4, and the DNA synthesized using the starting duplex
`DNA as a template is denoted “first generation” DNA. The
`resulting templates again are denatured, and the primers are
`annealed (step (d)).
`As seen in step (e) of FIG. 2, primer elongation using first
`generation DNA as a template results in the preparation of
`second generation DNA and does not progress past the
`non-replicable element incorporated into the first generation
`synthetic DNA. Thus, DNA molecules denoted by reference
`numerals 10 and 20 are synthesized. These second genera-
`tion molecules do not participate further in the primer
`extension reaction because, as seen in the Figures, molecule
`10 has not incorporated an effective binding site for the
`primer containing non-replicable or a cleavable element (x),
`and molecule 20 has not incorporated an effective binding
`site for the primer containing the non-replicable or cleavable
`element (0). Thus, as seen in steps (f) and (g), second
`generation molecules accumulate in a mathematically linear
`fashion in subsequent rounds of primer extension. ***
`Following a desired number of cycles, the synthetic DNA
`is utilized as a starting material for (i.e., linked to) a second
`series of primer extension reactions using a second set of
`primers. As seen in FIGS. 5—6, primers containing non-
`replicable elements designated (a) and (b) are selected so as
`to be able to utilize molecules 10 and 20, the products of the
`reaction of FIGS. 1—4, as templates for further DNA syn-
`thesis. This series of primer extension reactions similarly
`results in the accumulation of synthetic DNA molecules,
`designated by reference numerals 30 and 40 in step (e) of
`FIG. 6, which cannot serve as templates for the primers
`utilized in those reactions. Following a desired number of
`cycles, these synthetic molecules can be linked to further
`replication using appropriate primers again containing non-
`replicable or cleavable elements. Where the primers contain
`cleavable elements, the first primer extension reaction is
`followed by a reaction in which the first primer extension
`
`Ariosa Exhibit 1021, pg. 17
`|PR2013-00276
`
`Ariosa Exhibit 1021, pg. 17
`IPR2013-00276
`
`

`

`6,027,923
`
`7
`product is cleaved at the position of the cleavable element,
`prior to the second primer extension reaction.
`It should thus be apparent that the synthetic nucleic acid
`products of any one series of cycles can themselves serve as
`templates for further amplification only if a new primer or
`set of primers is provided. Thus, if a first linear amplification
`is performe