lllllllllllllllllllllllllllllllllllllllllllllllllllll||H||l|lH|||l|||||||
`USO05270l 84A
`5,270,184
`[11] Patent Number:
`United States Patent
`Walker et al.
`[45] Date of Patent:
`Dec. 14, 1993
`
`
`[191
`
`[54] NUCLEIC ACID TARGET GENERA-non
`
`wo9o/14439 11/1990 pcr lnt‘l Appl.
`
`..................... 435/6
`
`[75]
`
`Inventors: George T. Walker, Chapel Hill;
`Michael C. Little, Raleigh; James G.
`Nfleau, Durham’ an of NC.
`
`[73] Assignee: Becton, Dickinson and Company,
`Franklin Lakes. N-I
`[21] APPL N0-1 794,399
`[22] Filed:
`No“ 19, 1991
`
`Int. c1.s ............................................ .. C12P 19/34
`[51]
`[52] U.S. CL ................................. 435/91.2; 435/91.21
`Of Search ......................... 435/6.91;
`435/501» 94
`
`[55]
`
`References Cited
`US‘ PATENT DOCUMENTS
`4,683,195
`7/1987 Mullis et al.
`............................ 435/6
`5,106,727 4/1992 Hartley ................................... 435/6
`FOREIGN PATENT DOCUMENTS
`0200362 12/1986 European Pat’ O0-_ .
`0395398 10/1990 European Pat. Off.
`.
`0497272
`8/1992 European Pat. Off.
`.
`
`OTHER PUBLICATIONS
`
`Maniatis et al., Molecular Cloning a Lab Manual, Cold
`Spring Harbor Lab., 1992, pp. 109, M4.
`John C. Guatelli ct al., Proc. Natl. Acad. Sci. USA (1990)
`Deborah Y. Kwoh et al., ABL (Oct. 1990) p. 14.
`Primary Examiner——Stephanie W. Zitomer
`Attorney, Agent, or Firm—Donna R. Fugit
`
`ABSTRACT
`[571
`The invention is a method for generating nucleic acid
`scquencgs ends
`comprisgs;
`(a) hybridizing a primer to a nucleic acid sequence,
`(b) hybridizing a primer to the nucleic acid sequence
`in (a) located 5’ to the primer in (a), and
`(c) polymerizing both primers so that the primer in
`(3) is displaced from the nucleic acid sequence,
`The invention provides a method for generating target
`nucleic acid sequences for subsequent amplification.
`The method 15 applicable to both DNA and RNA.
`
`7 Claims, 4 Drawing Sheets
`
`5' S1
`0.‘,T
`1
`
`
`
`
`
`5' T2
`
`
`9
`
`POLYMERIZE USING S2
`dGTP, dCTP, TTP AND
`dATP(ocS)
`
`1
`
`5
`
`
`
`POLYMERIZE AND
`
`DISPLACE STRAND
`
`HYBRIDIZE SDA PRIMERS T0 DISPLACED STRANDS
`
`Ariosa Exhibit 1018, pg. 1
`|PR2013—OO276
`
`Ariosa Exhibit 1018, pg. 1
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Dec. 14,1993
`
`Sheet 1 of 4
`
`5,270,184
`
`F l G _ 1
`
`DENATURE TARGET
`BIND PRIMERS
`T
`2
`.
`
`
`
`
`—\T5’
`S2
`2
`51
`5'E‘..\_ TI
`
`
`_
`
`_
`
`PRIMER EXTENSION AND_
`DISPLACEMENT av Exo
`
`I<LENow
`
`
`
`O O I
`
`S
`\‘
`
`S2-EXT $2\
`S1—EXT
`
`0 I O
`
`B1
`
`
`
`5
`e,\ 1
`
`. '51
`
`3,
`
`BIND OPPOSITE PRIMERS
`TO S1—EXT AND S2—EXT
`
`S2-EXT 52\
`S1-EXT
`
`B2
`S2
`PRIMER EXTENSION AND
`DISPLACEMENT av Exo'
`KLENOW
`
`BINDING or OPPOSITE
`RRINERS
`
`EXTENSION av Exo”
`KLENOW
`
`51
`
`32 \‘
`
`\ 1
`
`2
`
`52
`
`¢sDA
`
`T0 FIG-2
`
`Ariosa Exhib_it 1018, pg. 2
`|PR2013—OO276
`
`Ariosa Exhibit 1018, pg. 2
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Dec. 14, 1993
`
`Sheet 2 of 4
`
`5,270,184
`
`FIG-2
`
`T2
`5’
`5’ S1
`
`
`
`s
`
`5‘ POLYMERIZE USING 32
`
`dGTP, dCTP, TTP AND
`dATP(°<S)
`
`
`5!
`
` T
`
`A
`
`
`
`$ mcx wnn HINC n
`
`
`
`j
`
`
`POLYMERIZE AND
`DISPLACE STRAND
`
`
`
`N
`4
`
`Un-f'§___-f
`
`
`
`HYBRIDIZE SDA PRIMERS TO DISPLACED STRANDS
`
`Ariosa Exhibit 1018, pg. 3
`|PR2013—OO276
`
`Ariosa Exhibit 1018, pg. 3
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Dec. 14, 1993
`
`Sheet 3 of 4
`
`5,270,184
`
`FIG-3
`
`5'
`— -G—T—T—G—A—C-
`
`PRIMER
`
`TARGT POLYMERIZE USING
`
`dCTP, dGTP, TTP AND
`dATP(oc$)
`
`5
`
`$ NICK WITH HINC u
`
`— —G-T —T-G—A-C-
`— -C-AsAsC—T—G-
`
`— -G—T-T1G-A—C-
`
`- -C—AsAsC-T-G-
`
`®
`
`Gm
`
`POLYMERIZE AND
`DISPLACE STRAND
`\c
`
`— -G—T—T-GsA-C-
`— -C-AsAsC—T—G—
`
`—. —G—T—T1GsA—C-
`
`— -C-AsAsC—T—G—
`
`‘ NICK wnn HINC n
`
`®
`
`51,4
`
`~c\
`
`POLYMERIZE AND
`
`DISPLACE STRAND
`
`
`
`— —G—T-T—GsA—C—
`-—- —C-AsAsC—T—G-
`
`Ariosa Exhibit 1018, pg. 4
`|PR2013—OO276
`
`Ariosa Exhibit 1018, pg. 4
`IPR2013-00276
`
`

`
`U.S. Patent
`
`Dec. 14, 1993
`
`Sheet 4 of 4
`
`5,270,184
`
`FIG-4
`
`TARGET RESTRICTION FRAGMENT
`
`5.
`
`P1
`
`T
`
`,2
`
`T
`
`‘
`
`T
`
`RESTRICT/DENATURE
`HYBRIDIZE AMPLIFICATION
`PROBES
`
`5'
`
`
`
`POLYMERIZE AND
`DISPLACE STRAND
`
`HYBRIDIZE AMPLIFICATTON PROBES TO DISPLACED STRANDS
`
`Ariosa Exhib_it 1018, pg. 5
`|PR2013—OO276
`
`
`
`P2
`
`POLYMERIZE
`WITH dNTP(°<S)
`
`Ariosa Exhibit 1018, pg. 5
`IPR2013-00276
`
`

`
`1
`
`5,270,184
`
`NUCLEIC ACID TARGET GENERATION
`
`FIELD OF THE INVENTION
`
`This invention relates to a method for preparing a
`target nucleic acid prior to amplification.
`
`BACKGROUND OF THE INVENTION
`
`Nucleic acids may be either in the form of deoxyribo-
`nucleic acids (DNA) or in the form of ribonucleic acids
`(RNA). DNA and RNA are high molecular weight
`polymers
`formed from many nucleotide building
`blocks. Each nucleotide is composed of a base (a purine
`or a pyrimidine), a sugar (either ribose or deoxyribose)
`and a molecule of phosphoric acid. DNA is composed
`of the sugar deoxyribose and the bases adenine (A),
`guanine (G), cytosine (C) and thymine (T).
`The nucleotides are assembled into a linear chain to
`form the genetic code. Each sequence of three nucleo-
`tides can be “read” as the code for one amino acid
`through the process of translation (DNA must first be
`converted into RNA through the process of transcrip-
`tion). By varying the combination of bases in each three
`base sequence, different amino acids are coded for. By
`linking various three base sequences together, a se-
`quence of amino acids can be made which forms prote-
`ins and polypeptides. The entire coding unit for one
`protein is referred to as a gene. There can be one or
`more copies of a gene in an organism. Some genes are
`present in hundreds or thousands of copies, others are
`present only as a single copy.
`Regardless of the number of copies, genes are linked
`together in an organism to form higher structural units
`referred to as chromosomes in higher organisms. In
`some lower organisms, genes may occur in extra chro-
`mosomal units referred to as plasmids. Genes need not
`be linked directly to each other in an end—to-end fash-
`ion. Certain non coding regions (i.e., introns: sequences
`of bases that do not translate into amino acids) may
`occur between genes or within a gene. The arrangement
`of nucleotides in an organism determines its genetic
`makeup which may be referred to as its genome (hence,
`DNA isolated from an organism is referred to as geno-
`mic DNA).
`DNA in most organisms is arranged in the form of a
`duplex wherein two strands of DNA are paired to-
`gether in the familiar double helix. In this model, hydro-
`gen bonds are formed between A and T and between C
`and G on the paired strands. Thus, on one strand, the
`sequence ATCG (5’—-3') will have on its complemen-
`tary strand the sequence TAGC (3’—>5’). Both strands,
`however, contain the same genetic code only in a com-
`plementary base-paired manner. One could read, there-
`fore, either strand of DNA in order to determine the
`genetic sequence coded for.
`For a further description of the organization, struc-
`ture and function of nucleic acids, see Watson, Molecu-
`lar Biology of the Gene, W. J. Benjamin, Inc. (3rd edit.
`1976), especially Chapters 6-14.
`Understanding and determining the genetic sequence
`of nucleic acids present in a sample is important for
`many reasons. First, a number of diseases are genetic in
`the sense that the nucleotide sequence for a “normal"
`gene is in some manner changed. Such a change could
`arise by the substitution of one base for another. Given
`that three bases code for a single amino acid, a change
`in one base (referred to as a point mutation) could result
`in a change in the amino acid which,
`in turn, could
`
`5
`
`l0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`2
`result in a defective protein being made in a cell. Sickle
`cell anemia is a classic example of such a genetic defect
`caused by a change in a single base in a single gene.
`Other examples of diseases caused by single gene de-
`fects include Factor IX and Factor VIII deficiency,
`adenosine deaminase deficiency, purine nucleotide
`phosphorylase deficiency, omithine transcarbamylase
`deficiency, argininsuccinate synthetase deficiency, beta-
`thalassemia, a1 antitrypsin deficiency, glucocerebrosi-
`dase deficiency, phenylalanine hydroxylase deficiency
`and hypoxanthine-guanine phosphoribosyltransferase
`deficiency. Still other diseases, such as cancers, are
`believed to be caused by the activation, increase in copy
`number and/or removal of suppression of genes known
`to be present in the genome (referred to as oncogenes).
`Examples of oncogenes believed to be relevant to cer-
`tain cancers include N myc for neuroblastomas, retino-
`blastomas and small cell
`lung cancers and c-abl for
`chronic myelogenous leukemia. For a further descrip-
`tion of the relevance of oncogenes to the diagnosis of
`cancers and for a listing of specific oncogenes, see
`Weinberg, Sci. Amer., Nov. 1983, Slamon et al., Sci-
`ence, 224:256 (1984), U.S Pat. No. 4,699,877 and
`4,918,162.
`Second, in addition to changes in the sequence of
`nucleic acids, there are genetic changes that occur on a
`structural level. Such changes include insertions, dele-
`tions and translocations along a chromosome and in-
`clude increased or decreased numbers of chromosomes.
`In the former instance, such changes can result from
`events referred to as crossing over where strands of
`DNA from one chromosome exchange various lengths
`of DNA with another chromosome. Thus, for example,
`in a “normal” individual,
`the gene for protein “X”
`might reside on chromosome 1; after a crossing over
`event, that gene could now have been translocated to
`chromosome 4 (with or without an equal exchange of
`DNA from chromosome 4 to chromosome 1) and the
`individual may not produce X.
`In the instance of increased or decreased chromo-
`some number (referred to as aneuploidy), instead of a
`“normal” individual having the correct number of cop-
`ies of each chromosome (e.g., two of each in humans
`[other than the X and Y chromosomes]), a different
`number occurs. In humans, for example, Down’s syn-
`drome is the result of having three copies of chromo-
`some 2l instead of the normal two copies. Other aneu-
`ploid conditions result from trisomies involving chro-
`mosomes l3 and 18.
`
`Third, infectious diseases can be caused by parasites,
`microorganisms and viruses all of which have their own
`nucleic acids. The presence of these organisms in a
`sample of biological material often is determined by a
`number of traditional methods (e.g., culture). Because
`each organism has its own genome, however, if there
`are genes or sequences of nucleic acids that are specific
`to a single species (to several related species, to a genus
`or to a higher level of relationship), the genome will
`provide a “fingerprint” for that organism (or species,
`etc.). Examples of viruses to which this invention is
`applicable include HIV, HPV, EBV, HSV, Hepatitis B
`and C and CMV. Examples of microorganisms to which
`this invention is applicable include bacteria and more
`particularly include H.
`influenzae, mycoplasma,
`le-
`gionella, mycobacteria, chlamydia, candida, gonocci,
`shigella and salmonella.
`
`Ariosa Exhibit 1018, pg. 6
`|PR2013—OO276
`
`Ariosa Exhibit 1018, pg. 6
`IPR2013-00276
`
`

`
`5,270,184
`
`3
`In each example set forth above, by identifying one or
`more sequences that are specific for a diseases or organ-
`ism, one can isolate nucleic acids from a sample and
`determine if that sequence is present. A number of
`methods have been developed in an attempt to do this.
`While it is critical that one or more sequences specific
`for a disease or organism be identified, it is not impor-
`tant to the practice of this invention what the target
`sequences are or how they are identified. The most
`straightforward means to detect the presence of a target
`sequence in a sample of nucleic acids is to synthesize a
`probe sequence complementary to the target nucleic
`acid (instrumentation, such as the Applied BioSystems
`380B,
`is presently used to synthesize nucleic acid se-
`quences for this purpose). The synthesized probe se-
`quence then can be applied to a sample containing nu-
`cleic acids and, if the target sequence is present, the
`probe will bind to it to form a reaction product. In the
`absence of a target sequence and barring non specific
`binding, no reaction product will be formed. If the
`synthesized probe is tagged with a detectable label, the
`reaction product can be detected by measuring the
`amount of label present Southern blotting is one exam-
`ple where this method is used.
`A difficulty with this approach, however, is that it is
`not readily applicable to those instances where the num-
`ber of copies of the target sequence present in a sample
`is low (i.e., less than 107). In such instances, it is difficult
`to distinguish signal from noise (i.e., true binding be-
`tween probe and target sequences from non specific
`binding between probe and non target sequences). One
`way around this problem is to increase the signal Ac-
`cordingly, a number of methods have been described to
`amplify the target sequences present in a sample.
`One of the best known amplification methods is the
`polymerase chain reaction (referred to as PCR) which is
`described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202
`and 4,800,159. Briefly, in PCR, two primer sequences
`are prepared which are complementary to regions on
`opposite complementary strands of the target sequence.
`An excess of deoxynucleoside triphosphates are added
`to a reaction mixture along with a DNA polymerase
`(e.g., Taq polymerase). If the target sequence is present
`in a sample, the primers will bind to the target and the
`polymerase will cause the primers to be extended along
`the target sequence by adding on nucleotides. By raising
`and lowering the temperature of the reaction mixture,
`the extended primers will dissociate from the target to
`form reaction products, excess primers will bind to the
`target and to the reaction products and the process is
`repeated.
`Another method for amplification is described in
`EPA No. 320,308, published Jun. 14, 1989, which is the
`ligase chain reaction (referred to as LCR). In LCR, two
`complementary probe pairs are prepared, and in the
`presence of the target sequence, each pair will bind to
`opposite complementary strands of the target such that
`they abut. In the presence of a ligase, the two probe
`pairs will link to form a single unit. By temperature
`cycling, as in PCR, bound ligated units dissociate from
`the target and then serve as “target sequences" for liga-
`tion of excess probe pairs. U.S. Pat. No. 4,883,750 de-
`scribes a method similar to LCR for binding probe pairs
`to a target sequence but does not describe an amplifica-
`tion step.
`A still further amplification method is described in
`PCT Appl. No. PCT/US87/00880, published Oct. 22,
`1987, and is referred to as the Qbeta Replicase method.‘
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`4
`In this method, a replicative sequence of RNA which
`has a region complementary to that of a target is added
`to a sample in the presence of an RNA polymerase. The
`polymerase will copy the replicative sequence which
`can then be detected.
`
`Still other amplification methods are described in GB
`Appl. No. 2 202 328, published Sep. 21, 1988, and in
`PCT Appl. No. PCT/US89/01025, published Oct. 5,
`1989. In the former application, “modified” primers are
`used in a PCR like, template and enzyme dependent
`synthesis. The primers may be modified by labelling
`with a capture moiety (e.g., biotin) and/or a detector
`moiety (e.g., enzyme). In the latter application, an ex-
`cess of labelled probes are added to a sample. In the
`presence of the target sequence, the probe binds and is
`cleaved catalytically. After cleavage,
`the target se-
`quence is released intact to be bound by excess probe.
`Cleavage of the labelled probe signals the presence of
`the target sequence. And finally U.S. patent application
`Ser. No. 07/648,257 filed Jan. 31, 1991 discloses and
`claims a method for amplifying nucleic acid (referred to
`as strand displacement amplification) which comprises
`target generation prior to amplification in which restric-
`tion enzymes are employed.
`Each of the above referenced amplification methods
`benefit from access to the desired nucleic acid sequence
`to be amplified. In addition, the need to generate am-
`plifiable target fragments with defined 5’- and 3‘-ends
`(i.e., ending at specific nucleotide positions) is a contin-
`ually sought after goal.
`SUMMARY OF THE INVENTION
`
`The invention is a method for generating nucleic acid
`sequences which comprises;
`(a) hybridizing a primer to a nucleic acid sequence,
`(b) hybridizing a primer to the nucleic acid sequence
`in (a) located 5’ to the primer in (a), and
`(c) polymerizing both primers so that the primer in
`(a) is displaced from the nucleic acid sequence.
`The invention provides a method for generating tar-
`get nucleic acid sequences with defined 5’- and 3’-ends
`for subsequent amplification and cloning. The method is
`applicable to both DNA and RNA.
`In addition, the method can employ a heat step after
`(c), above, and repeat over and over.
`When the target fragments comprise double stranded
`nucleic acids, the method further comprises denaturing
`the nucleic acid fragments to form single stranded tar-
`get sequences only once. Where the nucleic acids com-
`prise RNA, it is preferable to use reverse transcriptase
`to convert RNA to DNA.
`
`Subsequent strand displacement amplification (SDA)
`is enhanced because a larger number of modified nick-
`ing sites are generated from each original target strand.
`The previously described SDA method involved target
`restriction prior to binding primers and produced only
`two double stranded fragments with a modified nicking
`site at just one end. The restriction step was necessary
`to provide target fragments with defined 5’-and 3’-ends.
`Subsequent binding of the SDA primers to the 3’-ends
`of these target fragments and subsequent extension by
`an exonuclease deficient form of klenow (exo—k1enow)
`provided nicking enzyme recognition sites from which
`SDA initiated. The current target generation method
`produces four double stranded fragments with a total of
`six modified nicking sites (see FIG. 1). In addition,
`target sequences can now be chosen without regard for
`convenient flanking restriction sites. Finally, SDA can
`
`Ariosa Exhibit 1018, pg. 7
`|PR2013—OO276
`
`Ariosa Exhibit 1018, pg. 7
`IPR2013-00276
`
`

`
`5
`now be applied to either double or single stranded tar-
`get DNA target samples. Previously, targets had to be
`double stranded for cleavage by the restriction en-
`zyme(s). As used in this document, “nicking” refers to
`preferential cleavage of one of two strands present in a
`double-stranded recognition site.
`The invention further relates to methods for the sepa-
`ration and/or detection of amplified products generated
`by the invention. Methods for separation of amplified
`products comprise magnetic separation, membrane cap-
`ture and capture on solid supports. In each method, a
`capture moiety may be bound to a magnetic bead, mem-
`brane or solid support. The beads, membrane or solid
`support then can be assayed for the presence or absence
`of amplified products. An example of a capture moiety
`includes a nucleic sequence complementary to the am-
`plified products produced and an antibody directed
`against a receptor incorporated into the primer or am-
`plified product. The separation system may or may not
`be coupled to a detection system.
`The invention further relates to methods of generat-
`ing amplified products which can function as probes or
`templates for sequence analysis. In this format,
`the
`above described method and steps are used to generate
`amplified products. The amplified products can then be
`treated to remove the nicking enzyme recognition se-
`quence from the amplified product, for example by
`using a restriction enzyme. In this manner, the recogni-
`tion sequence is removed and the remaining amplified
`product comprises a probe which can he used in other
`systems.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1 Target generation scheme for strand displace-
`ment amplification. This figure depicts the initial steps
`which transform the original
`target sequence into
`“steady state" amplification as depicted in FIG. 2. A
`target DNA sample is heat denatured. Four primers
`(B1, B2, S1 and S2), present in excess, bind the target
`strands at positions flanking the sequence to be ampli-
`lied. Primers S1 and S2 have Hincll recognition sequen-
`ces at their 5'-ends. The four primers are simultaneously
`extended by exo‘ klenow using dGTP, dCTP, TTP
`and dATP(aS). Extension of B1 displaces the extension
`product from primer S1 (S1-ext). Likewise, extension of
`B2 displaces the S2 extension product, S2-ext. B2 and S2
`bind to displaced S1-ext. B1 and S1 bind to displaced
`S2-ext. Extension and displacement reactions on tem-
`plates S1-ext and S2-ext produce two fragments with a
`hemiphosphorothioate HincII site at each end and two
`longer fragments with a hemiohosphorothioate Hincll
`site at just one end. Hincll nicking and exo‘ klenow
`extension/displacement reactions initiate at these four
`fragments, quickly reaching the “steady state” set of
`reactions depicted in FIG. 2. Sense and antisense DNA
`strands are differentiated by thin and thick lines. Hincll
`recognition sequences are depicted by raised portions
`on the lines.
`FIG. 2 The “steady-state” series of strand displace-
`ment amplification (SDA) reactions. The following
`reaction steps continuously cycle during the course of
`amplification. Present in excess are two SDA primers
`(S1 and S2). The 3’-end of S1 binds to the 3'-end of a
`displaced strand serving as target (T1), forming a duplex
`with 5'-overhangs. Likewise, S2 binds to the displaced
`strand, T2. The 5’-overhangs of S1 and 52 contain a
`recognition sequence (5’GTTGAC3') for the restriction
`enzyme Hincll. Exo‘ klenow extends the 3’-ends of the
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`5,270,184
`
`6
`duplexes using dGTP, CCTP, TTP and dATP(aS),
`which produces hemiphosphorothioate recognition
`sites on S1T1 and S2T2. Hincll nicks the unprotected
`primer strands of the hemiphosphorothioate recogni-
`tion sites, leaving intact the modified complementary
`strands. Exo‘ klenow extends the 3'-end at the nick on
`S1T1 and displaces the downstream strand that is func-
`tionally equivalent to T2. Likewise, extension at the
`nick on S2T2 results in displacement of a downstream
`strand functionally equivalent to T1. Nicking and poly-
`merization/displacement steps cycle continuously on
`S1T1 and S2T2 because extension at a nick regenerates a
`nickable HincII site. Target amplification is exponential
`because strands displaced from S1T1 serve as target for
`S2 while strands displaced from S2T2 serve as target for
`S1. Sense and antisense DNA strands are differentiated
`by thin and thick lines. I-Iincll recognition sequences
`are depicted by raised portions on the lines. The partial
`HincII recognition sequences 5’GAC and its comple-
`ment 5’GTC are present at the 5‘- and 3’-ends of dis-
`placed strands, respectively, as represented by small
`boxes.
`
`FIG. 3 Schematic showing steps of strand displace-
`ment amplification (SDA) using only one SDA primer
`which produces linear amplification.
`FIG. 4 Schematic showing steps of strand displace-
`ment amplification (SDA) using two SDA primers and
`a restriction digestion step for target generation (com-
`pared to the present invention target generation) prior
`to SDA. Use of two SDA primers (as opposed to one
`SDA primer) produces exponential amplification.
`DETAILED DESCRIPTION
`
`To practice this invention, a sample may be isolated
`from any material suspected of containing the target
`nucleic acid sequence. For animals, preferably, mam-
`mals, and more preferably humans, the sources of such
`materials may comprise blood, bone marrow, lymph,
`hard tissues (e.g.,
`liver, spleen, kidney,
`lung, ovary,
`etc.), sputum, feces and urine. Other sources of material
`may be derived from plants, soil and other materials
`suspected of containing biological organisms.
`The isolation of nucleic acids from these materials
`can be done any number of ways. Such methods include
`the use of detergent lysates, sonication, vortexing with
`glass beads and a French press. In some instances, it
`may be advantageous to purify the nucleic acids isolated
`(e.g., where endogenous nucleases are present). In those
`instances, purification of the nucleic acids may be ac-
`complished by phenol extraction, chromatography, ion
`exchange, gel electrophoresis or density dependent
`centrifugation.
`Once the nucleic acids are isolated, it will be assumed
`for purposes of illustration herein only that the genomic
`nucleic acid is DNA and is double stranded.
`To begin target generation according to this inven-
`tion for subsequent SDA, the nucleic acid to be ampli-
`fied (e.g., target) is heat denatured in the presence of an
`excess of 4 primers (FIG. 1). Two primers (S1 and S2)
`are typical SDA primers, with target binding regions at
`their 3’-ends and nicking enzyme recognition sequences
`located 5‘-
`to the target complementary sequences.
`(Two SDA primers (S1 and S2) are necessary for expo-
`nential amplification, only one SDA primer (S1 or 52) is
`necessary for linear amplification. The other two prim-
`ers (B1 and B2) consist simply of target binding regions.
`(Only one upstream primer, B1 or B2, is absolutely nec-
`essary). S1 and S2 bind to opposite strands of the target
`
`Ariosa Exhibit 1018, pg. 8
`|PR2013—OO276
`
`Ariosa Exhibit 1018, pg. 8
`IPR2013-00276
`
`

`
`5,270,184
`
`8
`
`7
`at positions flanking the sequence to be amplified. B1
`and B2 bind upstream of S1 and S2, respectively. Exo-
`klenow simultaneously extends all 4 primers using 3
`deoxynucleoside triphosphates and one modified deox-
`ynucleoside triphosphates. Extension of S1 and S2 forms
`two products, S1-ext and S2-ext. Extension of B1 and B2
`results in displacement of S1-ext and S2-ext from the
`original target templates. Displaced and single-stranded
`S1-ext serves as target for S2 and B2. Likewise, displaced
`S2 ext is target for S1 and B1. All four primers are ex-
`tended on templates S1-ext and S2-ext. Combined exten-
`sion and displacement steps produce 2 double-stranded
`fragments with modified nicking enzyme recognition
`sites located at each end and two longer double-
`stranded fragments with modified recognition sites at
`only one end. Although the method has been described
`in steps,
`the method actually occurs simultaneously.
`Amplification can now proceed by the strand displace-
`ment amplification (SDA) procedure described below
`and described also in U.S. patent application Ser. No.
`07/648,257, incorporated herein by reference.
`The invention eliminates the conventional restriction
`step prior to binding primers. Thus workflow and costs
`are reduced in addition to simplifying target choice of
`sequences by the elimination of the need to locate target
`sequences flanked by convenient restriction sites. Also,
`the invention demonstrates increased sensitivity, pro-
`vides more freedom in choosing target nucleic acid
`sequences for amplification, and shortens time involved
`in amplification.
`This method for target generation can work with
`other amplification procedures such as polymerase
`chain reaction (PCR), PCR Technology, H. A. Erlich,
`Ed. (Stockton Press, New York, N.Y., 1989), transcrip-
`tion-based amplification system (TAS), Proc. Natl.
`Acad. Sci. USA 86:ll73 (1989), ligation amplification
`reaction (LAR), Genomic: 4:560 (1989),
`ligase based
`amplification system (LAS), Gene 89:11? (1990), and Q.
`B. replicase, Lomell et al., Clin. Chem. 35:1826 (1989),
`and 3SR, J. Guatelli et al., Proc. Natl. Acad. Sci. USA
`87:l874 (1990).
`If the target generation method of this invention is
`employed with amplification methods other than SDA,
`then several modifications can be made. There is no
`need for primers to contain any recognition sequences if
`subsequent amplification is by polymerase chain reac-
`tion. Likewise, if amplification is by Q beta replicase,
`then the recognition sequence should contain an RNA
`polymerase promotor for transcription of an RNA se-
`quence recognized by Q beta replicase (F. R. Kramer et
`al., Nature 3:39:40] (1989). If 3SR is used to amplify the
`DNA then primers S1 and/or S2 would contain RNA
`polymerase promoter sites. If amplification is to pro-
`ceed using a phi29 replication system (see Genentech
`W0 90/ 10064) then S1 and S2 would contain origin of
`replication sequences.
`In addition, the method can be practiced by essen-
`tially repeating FIG. 1 over and over. Heat is generally
`applied to denature the double stranded fragments.
`Such a method would eliminate the need for nicking
`enzymes and eliminate the need for modified nucleo-
`sides. Thus the methods essentially becomes an amplifi-
`cation method.
`Following target generation according to the scheme
`in FIG. 1, the target nucleic acid sequence can now be
`amplified by SDA using HincII as the amplification
`nicking enzyme. The four target fragments at the end of
`FIG. I automatically enter a “steady state" series of
`
`amplification cycles where Hincll nicks the hemiphos-
`phorothioate recognition site, and exo‘ klenow extends
`the 3’-end at the nick while displacing the downstream
`strand that serves as target for the opposite SDA primer
`(FIG. 2). These steps continuously repeat over the
`course of amplification. For example, 107-fold amplifi-
`cation theoretically derives from ~23 repetitions or
`cycles of the steps in FIG. 2 (223: 107). Each displaced
`strand in FIG. 2 contains the sequence 5'GAC at the
`5'-end and the complement 5'GTC at the 3'-end. 5'GAC
`is the portion of the HincII recognition sequence lo-
`cated 3' to the nick site.
`The transition between FIGS. 1 and 2 may become
`clearer with a detailed account for the last double
`stranded fragment at the bottom of FIG. 1. If Hincll
`nicks the hemiphosphorothioate site on the left end of
`the fragment containing the S1 primer sequence, exo-
`- klenow initiates replication at the nick and displaces a
`downstream strand with 5’GAC at the 5’-end and a full
`complement of the primer S2 at the 3'-end. S2 binds this
`displaced strand, and exo‘ klenow extends S2 forming
`the double-stranded fragment depicted as the product
`of the step labelled "polymerize using dGTP, dCTP,
`TTP and dATP (a.S)” on the right side of FIG. 2. Simi-
`lar reactions occur with the entire set of fragments
`depicted at the bottom of FIG. 1.
`The first primer (e.g., see S1 and S2 of FIG. 1) used in
`this method generally has a length of 20-100 nucleo-
`tides. Primers of approximately 25-40 nucleotides are
`preferred. This sequence should be substantially homol-
`ogous to a sequence on the target such that under high
`stringency conditions binding will occur. The primer
`also must contain a sequence (toward the 5' end) that
`will be recognized by an endonucleases, if subsequent
`SDA occurs, and polymerases (DNA or RNA), Q beta
`replicases, enzymes that recognize origins of replication
`(such as phi29) and the like, depending on subsequent
`amplification procedures (referred to as “recognition
`sequences“). The recognition sequences generally, al-
`though not necessarily, are non palindromic when using
`SDA. Although two primers are preferred, only one is
`necessary and as many as one chooses can be employed
`(more amplifiable targets are generated with more prim-
`ers).
`The second primer (e.q., see B1 and B2 of FIG. 1) is
`designed in the manner as just described, however, the
`second primer does not have to contain a recognition
`sequence, although it may, and preferably it does con-
`tain a recognition sequence. Preferably the second set of
`primers are from about 8 to about 100 nucleotide bases
`long, more preferably the second set of primers are
`about 25 to about 4-0 nucleotide bases long, with recog-
`nition sites. If the primers do not contain recognition
`sites they are preferably about l0 to about 25 nucleotide
`bases long.
`The second primer, which should bind upstream (5')
`of the first primer, is extended by a DNA polymerase
`with strand displacement ability and lacking 5’-3’ exo-
`nuclease activity. As the second primer is extended, the
`extended product of the first primer is displaced. Al-
`though two outside or “second" primers are preferred
`for use, only one is necessary (e.g., B1) and as many as
`one chooses may be employed (the more primers used
`in the target generation scheme (FIG. 1) the greater the
`number of amplifiable target fragments containing nick-
`able recognition sites. This results in a greater amplifica-
`tion factor.)
`
`IO
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`Ariosa Exhibit 1018, pg. 9
`|PR2013—OO276
`
`Ariosa Exhibit 1018, pg. 9
`IPR2013-00276
`
`

`
`5,270,184
`
`10
`enzyme could be used in an amplification system not
`requiring modified deoxynucleoside triphosphates. As
`an additional example, the restriction enzyme EcoRI
`has been shown to preferentially cleave one strand in
`noncanonical recognition sites or when its canonical
`recognition site is
`flanked by an oligopurine tract
`(Thielking et al. (1990) Biochemistry, 29, 4682; Lesser et
`al. (1990) Science 250, 776; Venditti & Wells (1991) J.
`Biol. Chem. 266, 16786). As another example, the re-
`striction enzyme Dpnl (available from New England
`Biolab