© 1992 Oxford University Press Nucleic Acids Research, Vol. 20, No. 7 1691-1696

Strand displacement amplification—an isothermal, in vitroDNA amplification technique

G.Terrance Walker*, Melinda S.Fraiser, James L.Schram, Michael C.Little, James G.Nadeauand Douglas P.MalinowskiDepartment of Molecular Biology, Becton Dickinson Research Center, PO Box 12016,Research Triangle Park, NC 27709, USA

Received December 4, 1991; Revised and Accepted March 10, 1992

ABSTRACT

Strand Displacement Amplification (SDA) Is anisothermal, in vitro nucleic acid amplification techniquebased upon the ability of Hindi to nick the unmodifiedstrand of a hemiphosphorothioate form of itsrecognition site, and the ability of exonuclease deficientklenow (exo~ klenow) to extend the 3'-end at the nickand displace the downstream DNA strand. Exponentialamplification results from coupling sense and antisensereactions in which strands displaced from a sensereaction serve as target for an antisense reaction andvice versa. In the original design (G. T. Walker, M. C.Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl.Acad. Scl 89, 392 - 396), the target DNA sample is firstcleaved with a restriction enzyme(s) in order togenerate a double-stranded target fragment withdefined 5'- and 3'-ends that can then undergo SDA.Although effective, target generation by restrictionenzyme cleavage presents a number of practicallimitations. We report a new target generation schemethat eliminates the requirement for restriction enzymecleavage of the target sample prior to amplification. Themethod exploits the strand displacement activity ofexo" klenow to generate target DNA copies withdefined 5'- and 3'-ends. The new target generationprocess occurs at a single temperature (after initial heatdenaturation of the double-stranded DNA). The targetcopies generated by this process are then amplifieddirectly by SDA. The new protocol improves overallamplification efficiency. Amplification efficiency is alsoenhanced by improved reaction conditions that reducenonspecific binding of SDA primers. Greater than107-fold amplification of a genomic sequence fromMycobacterium tuberculosis is achieved in 2 hours at37° C even in the presence of as much as 10 ng ofhuman DNA per 50 yL reaction. The new targetgeneration scheme can also be applied to techniquesseparate from SDA as a means of convenientlyproducing double-stranded fragments with 5'- and3'-sequences modified as desired.

INTRODUCTION

Strand Displacement Amplification (SDA) is an isothermal, invitro method of amplifying DNA. The original SDA designprovides powerful amplification compatible with diagnosticnucleic acid assays (1). However, it requires restriction enzymecleavage of the DNA sample prior to amplification in order togenerate an amplifiable target fragment with defined 5'- and3'-ends. The target restriction step not only complicates theexperimental protocol, but it also limits the choice of target DNAsequences because the sequence must be flanked by convenientenzyme restriction sites. In addition, the target DNA typicallymust be double-stranded for restriction enzyme cleavage. Wehave simplified the SDA protocol by replacing the targetrestriction step with a novel method of generating amplifiablefragments. SDA reactions can now be performed in three simplesteps: 1) the target DNA sample is heat denatured in the presenceof primers and other reagents, 2) Hindi and exo~ klenow areadded, and 3) the sample is incubated at 37°C.

As in the original report on SDA (1), we applied this improvedversion to genomic DNA samples from Mycobacteriumtuberculosis (M. tb), the causative agent of tuberculosis. Enhancedamplification was achieved by virtue of the new target generationscheme and improved reaction conditions which lowerbackground reactions that attentuate target amplification.

The new target generation scheme is a novel means ofproducing a double-stranded fragment with 5'- and 3'-endsmodified as desired. We propose a few general applications forthe method.

MATERIALS AND METHODSMaterialsThe large fragment of E. coli DNA polymerase I (klenow) waspurchased from Boehringer Mannheim (sequencing grade).Exonuclease deficient large fragment of E. coli DNA polymeraseI (exo~ klenow) (2) was purchased from United StatesBiochemical. HincII was purchased from New England Biolabsat a concentration of 75 units//xL. Ultra pure human placentalDNA (Sigma) was phenol/chloroform extracted and ethanolprecipitated to remove Na2EDTA. Deoxynucleoside

* To whom correspondence should be addressed

at National U

niversity of Singapore on Novem

ber 10, 2014http://nar.oxfordjournals.org/

Dow

nloaded from

1692 Nucleic Acids Research, Vol. 20, No. 7

Table I. Amplification of M. tb genomic DNA

InitialM. tbgenomecopies(molecules)

50000050000500050050zero+

100100100

AmplifiedProduct(molecules)

> l x l O 1 2

> l x l 0 1 2

7 x 1 0 "2 x 1 0 "2 x l 0 1 0

4x10"4 x 1 0 "7xlO1 0

2x l0 1 0

AmplificationFactor*

>2xlO 5

>2xlu*lx lO 7

4xlO7

4X107

4X108

7 x l 0 7

2 x l 0 7

HumanDNA Oig)

0.50.50.50.50.50.50.11

10

denature tirgetbind prlmeri

•Amplification factors were calculated considering there are — 10 copies of thetarget IS6110 sequence per M. tb genome (1).+Signal due to accidental contamination with the equivalent of ~\ M. tb genomecopy.

5'-triphosphates (dGTP, dCTP, TTP) and 2'-deoxyadenosine5'-O-(l-thiotriphosphate) (dATPS) were purchased fromPharmacia. Oligodeoxynucleotides were synthesized on anApplied Biosystems, Inc. 380B instrument and purified bydenaturing polyacrylamide gel electrophoresis. M. tb cultureswere generously provided by Salmon Siddiqi, Becton DickinsonDiagnostic Instrument Systems, Sparks MD. Daryl Shank (BectonDickinson Research Center) kindly prepared genomic DNA fromM. tb as previously described (1). Aerosol Resistant Tips (ART)(Continental Laboratory Products) were used to reducecontamination of SDA reactions with previously amplifiedproducts.

SDA ReactionsGenomic M. tb DNA was serially diluted in 50 mM KjPO.,, pH7.4 containing 0.01 or 0.1 ng/fiL human placental DNA. SDAwas performed in 50 /iL reactions containing varying amountsof M. tb DNA, 0.1-10 fig human placental DNA as indicated,150 units HincII, 5 units exo~ klenow, 1 mM each dGTP, dC-TP, TTP and dATPS, 50 mM KiPO4, pH 7.4, 6 mMMgAcetate2, 3% (v/v) 1-methyl 2-pyrrolidinone (NMP)(Sigma), 3% glycerol (from stock enzyme solutions), 500 nMSDA primers S, and S2 and 50 nM primers B, and B2. Priorto addition of Hindi and exo" klenow, reaction samples wereheated 4 minutes at 95 °C to denature target DNA followed by1 minute at 37°C to anneal primers. Upon addition of HincIIand exo" klenow, amplification proceeded 2 hours at 37°C andwas then terminated by heating 2 minutes at 95 °C.

Amplified products were detected using a oligodeoxynucleotidedetector probe (Figure 3 and Table I) or SDA primers (Figure 4)which were 5'-32P-labelled using T4 polynucleotide kinase in a50 nL reaction containing 6.9 mM Tricine (pH 7.6), 50 mMTRIS-HC1 (pH 8), 10 mM MgCl2 5 mM DTT, 2.3 /iM 7-32P-ATP (3000 Ci/mmol, 10 mCi/mL), 50 units kinase (New EnglandBiolabs) and either 1 /iM oligodeoxynucleotide detector probeor 10 /iM SDA primer. 32P-labelling was carried out for 1 hourat 37 °C and terminated by heating 2 minutes at 95 °C. ForFigure 3, aliquots (2 /tL) of the 32P-detector probe kinase mixwere added to 10 /iL aliquots from SDA reactions, and themixture was heated 2 minutes at 95 °C and then 2 minutes at37°C. The 5'-32P-detector probe was extended to a diagnosticlength upon addition of 2 /xL of 1 unit//*L klenow and incubation

primer extension anddisplacement by exo"klenow using dGTP,dCTP, TTP and dATPaS

**•*»

bind opposite primersto Sj-ext and S2-ext

N; S^eit

displacement by exo"klenowbinding of oppositeprimersexteniion by exo"klenow

Figure 1. Target generation scheme for SDA. This figure depicts the initial stepsin an SDA reaction which transform the original target sequence into theamplification cycle depicted in Figure 2. A target DNA sample is heat denatured.Four primers (B,, B2, S, and S2), present in excess, bind the target strands atpositions flanking the sequence to be amplified. Primers Si and Sj have HincIIrecognition sequences ( GTTGAC) located 5' to the target complementarysequences. The four primers are simultaneously extended by exo" klenow usingdGTP, dCTP, TTP and dATPS. Extension of B, displaces the S, primerextension product, S|-ext. Likewise, extension of B; displaces S^-ext. B2 andS2 bind to displaced S,-ext. B, and S, bind to displaced S^-ext. Extension anddisplacement reactions on templates S,-ext and S -ext produce two fragmentswith a hemiphosphorothioate HincII at each end and two longer fragments witha hemiphosphorothioate HincII site at just one end. HincII nicking and exo"klenow extension/displacement reactions initiate at these four fragments,automatically entering the SDA reaction cycle depicted in Figure 2. Sense andantisense DNA strands are differentiated by thin and thick lines. HincII recognitionsequences are depicted by ( —HH— ).

at 37°C for 10 minutes. For Figure 4, 5'-32P-labelled SDAprimers were used directly in amplification reactions. Sampleswere mixed with an equal volume of 50% (w/v) urea, 20 mMNa2EDTA, 0.5xTBE (3), 0.05% bromophenol blue/xylenecylanol and analyzed by denaturing gel electrophoresis (10%acrylamide:bis (18:1), 50% (w/v) urea, 0.5xTBE). X-ray film(Kodak X-OMAT AR) was exposed for 30 minutes at room

at National U

niversity of Singapore on Novem

ber 10, 2014http://nar.oxfordjournals.org/

Dow

nloaded from

Nucleic Acids Research, Vol. 20, No. 7 1693

5' s,

5' , . . S2 5'. polymerize usingI dGTP, dCTP. TTP

and d T f t S )

hybridize SDA primeri to displaced strands

Figure 2. The SDA reaction cycle. These reaction steps continuously cycle duringthe course of amplification. Present in excess are two SDA primers (S, and S2).The 3'-end of S, binds to the 3'-end of the displaced target strand T,, forminga duplex with 5'-overhangs. Likewise, S2 binds T2. The 5'-overhangs of S, andS contain the Hinc II recognition sequence (5 GTTGAC). Exo~ klenow extendsthe 3'-ends of the duplexes using dGTP, dCTP, TTP and dATPS, which produceshemiphosphorothioate recognition sites on S, -T, and S2T2. HincII nicks theunmodified primer strands of the hemiphosphorothioate recognition sites, leavingintact the modified complementary strands. Exo~ klenow extends the 3'-end atthe nick on S| T, and displaces the downstream strand that is equivalent to T2.Likewise, extension at the nick on S2T2 results in displacement of T,. Nickingand polymerization/displacement steps cycle continuously on S| -T, and S2T2because extension at a nick regenerates a nickable HincII recognition site. Targetamplification is exponential because strands displaced from S( -T, serve as targetfor S2 while strands displaced from SyT2 serve as target for S|. Sense andantisense DNA strands are differentiated by thin and thick lines. Intact and ruckedHincII recognition sequences are depicted by _ ^ H _ and - • • - ,respectively. The partial HincII recognition sequence 3 GAC and its complement3 GTC are present at the 5'- and 3'-ends of displaced strands as represented by•_ and —•.

temperature using intensifying screens. 32P-labelled gelelectrophoresis bands were excised and quantified by liquidscintillation counting using appropriate gel electrophoresisbackground controls. The number of amplified product molecules(Table I) was calculated based upon the specific activity of the5'-32P-detector probe which was measured from aliquotspurified by gel electrophoresis. Amplification factors werecalculated considering there are ~ 10 copies of the IS6110 targetsequence per M. tb genome (1).

RESULTS

A limitation of the original SDA design is the requirement forrestriction enzyme cleavage of the target DNA sample prior toamplification (1). The restriction step is necessary to providetarget fragments with defined 5'- and 3'-ends. Subsequent bindingof the SDA primers to the 3'-ends of these target fragments andextension by exo~ klenow create hemiphosphorothioate HincIIrecognition sites from which amplification initiates.

We have developed a new method of target generation whicheliminates the need for restriction enzyme cleavage of the targetprior to SDA (Figure 1). The target DNA sample is heatdenatured in the presence of an excess of 4 primers. Two primers(S| and SJ are typical SDA primers, with target binding regionsat their 3'-ends and HincII recognition sequences (5GTTGAC)located 5' to the target complementary sequences. The other twoprimers (B, and B2) consist simply of target binding regions. S,and S2 bind to opposite strands of the target at positions flanking

the sequence to be amplified. B, and B2 bind upstream of S| andS2, respectively. Exo~ klenow (present in excess over thenumber of target sequences) simultaneously extends all 4 primersusing dGTP, dCTP, TTP and dATPaS. Extension of S, and S2forms two products, S]-ext and S2-ext. Extension of B| and B2results in displacement of S|-ext and S2-ext from the originaltarget templates. Displaced and single-stranded S|-ext thenserves as target for primers S2 and B2. Likewise, displacedS2-ext is target for S| and B,. All four primers are extended ontemplates S r ext and S2-ext. Combined extension anddisplacement steps produce 2 double-stranded fragments withhemiphosphorothioate HincII recognition sites at each end andtwo longer double-stranded fragments with ahemiphosophorothioate HincII site at only one end. In thepresence of HincII (which can be added at the top of Figure 1along with exo~ klenow), the four target fragments at thebottom of Figure 1 automatically enter the strand displacementamplification cycle (Figure 2), where HincII nicks thehemiphosphorothioate recognition site, and exo~ klenowextends the 3'-end at the nick while displacing the downstreamstrand that in turn serves as target for the opposite SDA primer.These steps, repeated continuously over the course of the reaction,produce exponential growth in the number of target sequences.107-Fold amplfication (Table I) theoretically derives from ~23repetitions or cycles of the steps in Figure 2 (223= 107). Eachdisplaced strand in Figure 2 contains the sequence 5'GAC at the5'-end and the complement 5GTC at the 3'-end. 5GAC is theportion of the HincII recognition sequence located 3' to the nicksite.

The transition between Figures 1 and 2 may become clearerwith a detailed account for the last double-stranded fragment atthe bottom of Figure 1. If HincII nicks the hemiphosphorothioatesite on the left end of the fragment containing the S) primersequence, exo~ klenow initiates replication at the nick anddisplaces the downstream strand containing 5GAC at the 5'-endand the full complement of the primer S2 sequence at the 3'-end.S2 binds to this displaced strand, and exo~ klenow extends S2forming the double-stranded fragment depicted as the productof the step labelled 'polymerize using dGTP, dCTP, TTP anddATP(aS)' on the right side of Figure 2. Similar reactions occurwith the entire set of fragments depicted at the bottom of Figure 1.

Duplexes containing two HincII sites in Figure 1 may alsoundergo more complex reactions in which the two restriction sitesare nicked simultaneously. Following dissociation of HincII fromthe nicked sites, strand extension/displacement may proceed fromone or both sites. Although we have not yet determined therelative importance of these different pathways, all such processesultimately lead to amplifiable species found in Figure 2.

Amplification of M. tb DNAA set of samples containing varying amounts of target genomicM. tb DNA and the indicated amounts of human DNA wassubjected to SDA. The two SDA primers (S| and S2) containtarget binding regions at their 3'-ends that are complementaryto opposite strands of the IS6110 sequence of M. tb (4) atnucleotide positions 972-984 and 1011-1023 (S, = 5'dTTG-AATAGTCGGTTACTTG7TG/iajGCGTACTCGACC, S? =5'dTTGAAGTAACCGACTATTG77U/iCACTGAGATCCCC-T). (HincII recognition sites are italicized). The two outsideprimers (B| and B2) bind to opposite strands at nucleotidepositions 954-966 and 1032-1044 [B, = 5'dTGGACCCGC-CAAC.-B2 = 5'dCGCTGAACCGGAT|. After the SDA

at National U

niversity of Singapore on Novem

ber 10, 2014http://nar.oxfordjournals.org/

Dow

nloaded from

1694 Nucleic Acids Research, Vol. 20, No. 7

35-mer

P-detectorprobe

(15-mer)

DNA

M tbgenome

copieshuman

cio'n

01 10

io3

n0.1 10

zeron0.1 10

M.W.ladder

1

79-mers

32P-SDAprimers

(37-mers)—- 40-mer

- 33-mer

21-mers

Figure 3. Denaturing gel electrophoresis analysis of SDA reactions using a5'- P-labelled detector probe. SDA products were detected by extension of a5'-32P-labelled probe (15-mer) to a length of 35 or 56 nucleotides. Aliquots (10fiL) from each 50 nL SDA reaction were analyzed. The number of M. tb genomecopies present in each 50 pL reaction is shown above each lane. Each samplecontained 0.5 /ig of human DNA. Longer autoradiography exposures reveal targetspecific bands in the zero target lane. Band intensities are nearly constant forsamples containing >5000 intial targets due to the limited amount of 32P-labelleddetector probe. Unextended ^P-labelled detector probe appears as multiple bandsdue to degradation by the 3'-5' cxonuclease activity of klenow used in the detectionstep. DNA polymerase extension reactions of the 5'-32P-probe were not allowedto proceed below 37"C in order to minimize background detection reactions.

— 20-mer

Figure 4. Denaturing gel electrophoresis analysis of SDA reactions using 5'-32P-labelled SDA primers. SDA reactions were performed using 5'-32P-labelled SDAprimers (S, and S2) in the presence of 0.1 or 10 ^g of human DNA. Aliquots(10 nL) from each 50 #iL SDA reaction were analyzed. The number of M. tbgenome copies present in each 50 yL reaction is shown above each lane. Unreacted5'-32P-labelled SDA primers are 37-mers. Target specific S'-^P-labelledamplification products are a set of 21- and 79-mers. Background reaction productsappear as a high molecular weight smear and more distinct bands over 55- to110-mers. The 21-mer bands also derive in part from background reactions (seetext). Reactions were not allowed to proceed below 37°C in order to minimizebackground reactions.

reaction is terminated, all species shown in Figure 2 are presentand detectable. In the present study, amplified target fragmentswere detected by DNA polymerase extension of a 5'-32P-labelled probe (5'-32P-dCGTTATCCACCATAC) (IS6110nucleotide positions 992 — 1006) that is complementary to aninternal segment of the target sequence. The 5'-32P-labelleddetector probe is extended to a length of either 35 or 56nucleotides when hybridized to one or the other of the followingtwo strands produced during SDA: 5'dG/lCACTGAGATCCC-CTATCCGTATGGTGGATAACGTCTTTCAGGTCGAGT-ACGCCGrC and 5'dTTGAATAGTCGGTTACTTG77G/lCA-CTGAGATCCCCTATCCGTATGGTGGATAACGTCTTTC-AGGTCGAGTACGCCG7TC. (Partial and complete Hindirecognition sequences are italicized). The first strand is one ofthe strands displaced during SDA and corresponds to IS6110nucleotide positions 972-1023 with 5GAC and 5GTCappended to the 5'- and 3'-ends. The second longer strand is thesame strand before nicking by HincII and as such has a 5'-endidentical to primer S|.

Results from amplification of genomic M. tb DNA are shownin Figure 3 and Table I. The amount of amplified product isdependent upon the number of initial targets. (However, levelsof amplified product are nearly constant for > 5000 initial M.tb genome copies due to the limited amount of the 32P-detectorprobe used.) Longer autoradiography exposures reveal targetspecific 32P-bands in the zero target lane of Figure 3; we

estimate each sample is contaminated with the equivalent of — 1genome copy of M. tb. Although this contamination problemhinders accurate determination of sensitivity, SDA enables 32P-detection of as few as 10—50 initial target DNA molecules (1 —5genome copies containing - 10 copies of the target IS6110sequence) (Table I and unpublished observations).

Background AmplificationAs previously discussed (1), background amplification reactionscompete with target specific amplification and are more prevalentat higher non-target (human) DNA concentrations. The sensitivityof SDA therefore depends upon the amount of non-target DNApresent (Table I). Background reactions presumably begin withextension of primers spuriously bound to non-target sequences.In processes similar to those depicted in Figure 1, thesebackground extension products are then displaced andsubsequently hybridized to a second SDA primer, which is inturn extended and displaced. In background reactions, these initialpriming events will generally involve imperfectly pairedsequences and therefore should occur far less efficiently than thecarefully designed priming/extension reactions leading to bona-fide target generation. However, once a non-target sequence isattached to two primer-derived nickable HincII sites (see bottomof Figure 1), it will undergo amplification as effectively as thegenuine target sequence.

at National U

niversity of Singapore on Novem

ber 10, 2014http://nar.oxfordjournals.org/

Dow

nloaded from

Nucleic Acids Research, Vol. 20, No. 7 1695

To examine the background reactions, SDA was performedusing 5'-32P-labelled primers (S, and S2) at various target levelsand in the presence of either 0.1 or 10 /tg of human DNA(Figure 4). Excess unreacted 5'-32P-labelled SDA primers are37-mers. Target specific SDA products appear as two32P-21-mers and a set of four 32P-79-mers. The 21-mers are thenucleotide sequences located 5' to the Hindi nicking sites onthe SDA primers (the short strands indicated in Figure 2 abovethe step labelled 'polymerize and displace strand')- The set offour 79-mers are the unnicked amplification strands shown inFigure 2 above the step labelled 'nick with Hindi'. In Figure 4,background amplification products appear only in lanes forreactions containing high (10 ng) human DNA, where they runas a high molecular weight smear and more distinct bands rangingin size from 55-110 nucleotides. The ~43-mer bands, whichare present in all lanes in Figure 4, appear in the absence ofHindi and as such are not SDA background reaction products(e.g., 'primer-dimers'). (The 43-mer probably represents exo~klenow extension of a minor hairpin conformation of primerSi.) The 21-mer bands in Figure 4 also arise from backgroundreactions as well as from target amplification because they derivefrom the target-independent sequences at the 5'-ends of the SDAprimers; notice that 21-mers are apparent in the zero target samplewith 10 /xg of human DNA although no target specific 79-mersare discernible.

The apparent lack of background products in the low (0.1 /ig)human DNA lanes (Figure 4) supports the hypothesis thatbackground reactions arise from spurious amplification of humanDNA sequences. Background amplification predominates inreaction mixtures containing high human DNA and low numbersof initial target sequences (< 104 genome copies). Under theseconditions, target amplification proceeds efficiently for only ~2hours before high levels of background product accumulate anddramatically reduce target amplification by competing for limitingcomponents like Hindi. The more target copies initially present,the more target-specific amplification that can occur beforeessential components are depleted. Consequently, in reactionscontaining low initial target numbers, the final level of amplifiedtarget depends on the initial copy number (1). At very high initialtarget levels (rMO6 M. tb genome copies), however,background reactions are attenuated by target amplification,which predominates under these conditions, and the final levelof amplified target is independent of the number of targets initiallypresent (data not shown).

DISCUSSION

We have streamlined the original SDA experimental protocol byeliminating the need to cleave a target DNA sample by arestriction enzyme(s) prior to amplification (1). The new targetgeneration scheme uses 4 primers in a concerted series of DNApolymerase extension and displacement reactions. Although theoverall reaction mechanism of SDA appears complex as depictedin Figures 1 and 2, individual reaction steps are invisible to theexperimenter. In fact, the experimental protocol consists simplyof 1) heat denaturing a target DNA sample in the presence ofprimers and other reagents 2) addition of Hindi and exo~klenow, and 3) incubation at 37°C.

This new target generation scheme offers a number ofadvantages over the previous method of generating targetfragments by restriction enzyme cleavage (1). Amplification isenhanced because a larger number of hemiphosphorothioate

Hindi sites is generated from each original double-strandedtarget. The previous method of target generation produces twodouble-stranded fragments each with a hemiphosphorothioateHindi site at one end (1), whereas the current method producesfour double-stranded fragments with a total of sixhemiphosphorothioate Hindi sites (Figure 1). In addition, targetsequences can now be chosen without regard for convenientflanking restriction sites. Furthermore, SDA can now be appliedto either double- or single-stranded target DNA target samples.Previously, targets generally had to be double-stranded for targetgeneration by restriction enzyme cleavage. SDA is nowcompatible with sample preparation methods which render targetgenomic DNA single-stranded prior to amplification.

Primers B, and B2 in Figure 1 can also be typical SDAprimers containing target binding regions at their 3'-ends andHindi recognition sites located toward their 5'-ends. The resultis an enhancement of amplification from additional nicking andextension/displacement reactions at these outsidehemiphosphorothioate Hindi sites (data not shown). Displacedstrands from these outside sites serve as target for both oppositeprimers.

Performance of SDA with just one SDA primer (S| or S2)generates single-stranded displaced strands in a linearamplification mode. In a related sense, SDA could be performedwith an excess of one SDA primer over the other (e.g.,[S|]> >[S2]) which should produce exponential amplificationof a target with a predominance of one amplified strand overthe other in a manner analogous to 'asymmetric' PCR. In eithercase, the resultant single-stranded displaced strands could serveas either detector probes (e.g., Southern blots) or templates forDNA sequence analysis.

The ability of SDA to amplify low target numbers at 37°Cis quite remarkable when compared with PCR, which achievesexquisite sensitivity only when non-specific priming reactions arediminished by stringent (high temperature) reaction conditions(5). Although the absence of temperature cycling in SDA reducesthe number of recurrent opportunities for errant hybridizationand primer extension, considerable non-specific priming surelyoccurs at the 37°C reaction temperature. However, initiation ofSDA for any sequence (target or background) must take placevia multiple steps similar to those shown in Figure 1. Theefficiency of this concerted series of events will be greatlydiminished if the primer extension and strand displacement stepsmust wait for the adventitious, nonspecific hybridization ofprimers, as will generally be the case for background reactions.Once non-target amplicons are generated through this inefficientprocess, however, they will be amplified as readily as bona-fidetarget sequences. Consequently, significant accumulation ofbackground product can result if non-target DNA is present inlarge excess over specific target sequences. To reduce thepotential for these background reactions, it is important tomaintain the highest level of stringency compatible with enzymestability. Accordingly, SDA reactions are not allowed to proceedat temperatures less than 37 °C, and we increased stringency byadding organic co-solvents to the reaction. With thesemodifications to our original reaction conditions (1), we can nowamplify specific target sequences > 107-fold even in thepresence of as much as 10 /xg of human DNA per 50 /tL reaction.

SDA and PCR are affected by common experimentalparameters (e.g., target length, target dG-dC content, primersequences, and organic solvents such as 1-methyl 2-pyrrolidinone,glycerol and formamide). Because it operates under conditions

at National U

niversity of Singapore on Novem

ber 10, 2014http://nar.oxfordjournals.org/

Dow

nloaded from

1696 Nucleic Acids Research, Vol. 20, No. 7

of low stringency (37 °C) through a strand displacementmechanism, SDA is especially sensitive to experimentalparameters. For example, SDA amplification factors decrease— 10-fold for each 50 nucleotide increase in target length, overa range of 50 to 200 nucleotides (unpublished observations); thismay be related to the non-processive nature of the polymerase.In addition, the best choice for an organic solvent and its optimalconcentration appear to depend on the particular sequence to beamplified (unpublished observations); a literature survey suggeststhis is also true for PCR. As with PCR, care must be taken tochoose primer sequences that do not support 'primer-dimer'amplification reactions, which can arise in SDA reactions by thehybridiztion of primer sequences 3' to their Hindi recognitionsites.

The scheme in Figure 1 represents a general and convenientmethod of producing double-stranded fragments with defined 5'-and 3'-ends as illustrated by the following potential applications:1) The Hindi recognition sequences in Si and S2 could besubstituted by any restriction sites in order to produce fragmentsfor ligation into a cloning vector. (The scheme in Figure 1 canbe performed with dATP instead of dATPS.) 2) The Hindirecognition sequences in S) and S2 could be substituted by RNApolymerase promoters for production of transcription templates.3) The technique could be applied to synthesis of double-strandedcDNA using a 5'-3' exonuclease deficient reverse transcriptasepossessing strand displacement ability (does not require a reversetranscriptase with RNase H activity).

Various isodiermal DNA amplification techniques requireformation of a double-stranded DNA fragment containingterminal sequences specific for the given amplification technique.For example, the self-sustained sequence replication (3SR) systemproduces a double-stranded DNA fragment with a bacteriophageRNA polymerase promoter at one or both ends (6). Such afragment can be generated from target DNA through 2 cyclesof PCR using primers containing promoter sequences at their5'-ends. Alternatively, one can first cleave target DNA with arestriction enzyme(s), creating a fragment with defined 5'- and3'-ends to which 3SR primers can bind after denaturation in amanner analogous to the original target generation scheme ofSDA (1). (It should be noted that 3SR can directly amplify anRNA target sequence without these target generation steps.)Alternatively, the initial steps for 3SR amplification of targetDNA could proceed according to the target generation schemein Figure 1 by simply substituting the Hindi sites in S) and S2with promoter sequences. This target generation scheme couldalso be applied to amplification by Q-beta technology involvingformation of 'target-dependent replicatable RNAs' (7) or toamplification techniques using terminal origins of replication forthe bacteriophage Phi 29 (8,9).

The scheme in Figure 1 illustrates the possibility of performingPCR reactions with multiple primers and a DNA polymeraselacking 5'-3' exonuclease activity and possessing stranddisplacement ability. The procedure would simply consist ofrepeated cycles of the steps in Figure 1 using 'nested' primersets and an intervening heat denaturation step between cycles.Although a possible increase in background amplification mustbe considered, the use of multiple primer sets should enhancePCR especially during early cycles. For example, two nestedprimer sets (analogous to Bu B2, S, and S2) should produce6-fold amplification for the first cycle, although the amplificationfactor converges toward a value of 2-fold at later cycles as theshorter product prevails. In addition to exo~ klenow, the

thermophilic large fragment of DNA polymerase I from Bacillusstearothermophilus (Bio-Rad) strand displaces and lacks 5'-3'exonuclease activity.

REFERENCES1. Walker, G. T., Little, M. C , Nadeau, J. G. and Shank, D. D. (1992) Proc.

Nail. Acad. Sci. USA 89, 392-396.2. Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman,

J. M., Joyce, C. M. and Steitz, T. A. (1988) Science 240, 199-201.3. Maruatis, T., Fritsch, E. F. and Sambrook, J. (1982) Molecular Cloning:

A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,NY).

4. Thierry, D., Cave, M. D., Eisenach, K. D., Crawford, J. T., Bates, J. H.,Gicquel, B. and Guesdon, J. L. (1990) Nucleic Acids Res. 18, 188.

5. Mullis, K. B. (1991) PCR 1, 1-4.6. Guatelli, J. C , WhitfieW, K. M., Kwoh, D. Y., Barringer, K. J., Richman,

D. D. and Gingeras, T. R. (1990) Proc. Nail. Acad. Sci. USA 87,1874-1878.

7. Kramer, F. R. and Ljzardi, P. M. (1989) Nature 339, 401-402.8. Miller, H. I. (1990) PCT International Patent Publication Number WO

90/10064.9. Kessler, C. and Ruger, R. (1991) PCT International Patent Publication

Number WO 91/03573.

at National U

niversity of Singapore on Novem

ber 10, 2014http://nar.oxfordjournals.org/

Dow

nloaded from