Quantitative Reverse Transcription Strand Displacement Amplification: Quantitation of Nucleic Acids Using an Isothermal Amplification Technique

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

259, 226 –234 (1998)

AB982641

Quantitative Reverse Transcription Strand Displacement Amplification: Quantitation of Nucleic Acids Using an Isothermal Amplification Technique Colleen M. Nycz,1 Cheryl H. Dean, Perry D. Haaland, Catherine A. Spargo, and G. Terrance Walker Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, North Carolina 27709

Received November 14, 1997

Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500 –500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting. © 1998 Academic Press

Viral load testing for HIV has become an important tool for monitoring patient therapy allowing physicians to determine the effectiveness of their clinical regimen for each patient (1– 4). Physicians are able to make more timely decisions concerning when to change therapy by tracking increasing levels of virus due to the emergence of drug-resistant 1

To whom correspondence should be addressed at Becton Dickinson Research Center, P.O. Box 12016, Research Triangle Park, NC, 27709. Fax: 919-990-2274. E-mail: [email protected]. 226

strains. Viral load methods need to be accurate, simple, and fast so that the clinical laboratory can provide timely results for a large number of clinical specimens. Strand displacement amplification (SDA)2 is an isothermal nucleic acid amplification technique that provides greater than 1010-fold amplification in 15 min (5). It is based on the ability of a restriction enzyme to nick a hemithiolated recognition sequence and a DNA polymerase to initiate synthesis at that nick and displace the downstream strand during polymerization. Figure 1 depicts the basic outline of the reaction. Two sequential steps are involved-target generation followed by exponential amplification. Target generation produces an amplifiable target with defined ends flanked by nickable restriction sites. This defined target is then exponentially amplified by repeated nicking, strand displacement, and primer hybridization. Figure 1 outlines the general procedure for amplification of an RNA target sequence (RT-SDA) using a reverse transcriptase to perform the first step of target generation (cDNA synthesis). The present report describes quantitative RT-SDA (QRT-SDA) using a control sequence for competitive amplification. In addition, we included a single-strand binding protein to improve amplification efficiency. The amplification protocol takes 30 min and occurs in a single tube without temperature cycling. SDA is ideally suited for viral quantitation because of its simple workflow, fast time to result, and freedom from sophisticated equipment. For the preliminary demonstration of QRT-SDA, we chose to coamplify a portion of the HIV gag-1 gene sequence. 2

Abbreviations used: SDA, strand displacement amplification; RT, reverse transcription; QRT, quantitative reverse transcription; AMV, avian myeloblastosis virus. 0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

STRAND DISPLACEMENT AMPLIFICATION

227

MATERIALS AND METHODS

Generation of RNA Transcripts

FIG. 1. Schematic of the RT-SDA reaction. RT-SDA is performed using an excess of four oligodeoxynucleotide primers (S1, S2, B1, and B2). S1 and S2 contain target binding regions at their 39 ends and recognition sites (59-CTCGGG) for BsoBI located 59 to the target binding regions (BsoBI recognition sites are designated by the raised boxes). S1 and S2 bind to opposite strands of the target sequence, flanking the region to be amplified. B1 and B2 are simply target binding sequences (containing no BsoBI recognition sites) that hybridize the target sequence at positions 59 to that of S1 and S2. (A) RT-SDA initiates with a target generation step during which the primers S2 and B2 anneal to the denatured target RNA sequence. AMV reverse transcriptase, extends both primers in the presence of dATP, dGTP, dUTP, and dCTPaS and displaces the downstream extension product of primer S2 (S2-ext). This newly generated cDNA strand (S2-ext) then binds primers S1 and B1. The DNA polymerase exo2 Bst extends S1 and B1, resulting in displacement of the S1 extension product (S1-ext). Extension of an S2 primer on S1-ext produces a doublestranded target fragment with nickable BsoBI sites on each end. BsoBI nicks the original primer (nonthiolated) strand of the hemiphosphorothioate recognition site on either end of the target fragment (right side shown) and exo2 Bst extends the 39 end at the nick and displaces the downstream strand. An S1 primer binds to this displaced strand and is extended, forming an intermediate in the SDA cycle shown in B as indicated by the dashed arrow. (B) The SDA cycle is where the majority of the amplification occurs. During each round of the cycle, the 39 end of S1 binds to the 39 end of the displaced target strand T2, forming a duplex with 59 overhangs. Likewise, S2 binds to T1, the complement of T2. Exo2 Bst extends the recessed 39 ends of the duplexes, producing hemiphosphorothioate recognition sites that are nicked by BsoBI. These nicking and extension/displace-

A portion of the HIV GAG-1 gene was cloned into a pGEM11Zf(1) vector (Promega, Madison, WI). The cloned GAG sequence was identical to HIV MN and corresponds to positions 1222–1839, as described by Gurgo et al. (6, 7). Control plasmids for quantitation were generated from this parent plasmid using random site-directed mutagenesis. A 27-bp region internal to the SDA primers (positions 1309 –1335) was randomly mutagenized using site-directed mutagenesis essentially as described by Kunkel (8) and cloned into Escherichia coli DH5a. Mutant plasmids were identified by an NcoI site inserted into the plasmids (positions 1304 –1309) as a part of the mutagenesis. The wildtype HIV and mutant control plasmids were linearized using SalI digestion and purified by phenol chloroform extraction/ethanol precipitation. The wild-type and mutant control plasmids were then used to generate RNA transcripts using T7 polymerase. The RNA was digested with DNase I to remove any contaminating DNA and purified by phenol chloroform extraction/ ethanol precipitation followed by Centricon 100 filtration. Concentrations were determined by spectrophotometry and RNA was stored at 108 copies/ml in 10 ng/ml human placental DNA (Sigma, St. Louis, MO) in separate aliquots at 270°C. Dilutions were made fresh from these stock aliquots for each experiment. The appropriate mutant control transcript was selected based on comparable amplification efficiency to the wild-type HIV sequence. SDA Oligodeoxynucleotides The positions of the primers and detector oligodeoxynucleotides are illustrated on the map in Fig. 3. Primer B1 corresponds to positions 1223–1241 of the GAG sequence (6, 7), and its sequence is 59-dTACATCAGGCCATATCACC. Primer B2 corresponds to positions 1424 –1408, and its sequence is 59-dGCAGCTTCCTCATTGAT. Primer S1 is 59-dACCGCATCGAATGCATGTCTCGGGTGGTAAAAGTAGTAGAAG and its binding region corresponds to positions 1260 –1276. The italicized bases are the BsoBI recog-

ment steps cycle continuously [short upturned arrows] because extension at a nick regenerates a nickable BsoBI recognition site. The strand displaced from the S1 z T2 duplex is identical to T1. Likewise, the displaced strand from the S2 z T1 duplex is identical to T2. Consequently, target amplification is exponential because each displaced T2 binds a new S1 primer while each displaced T1 binds a new S2 [long upturned arrows]. Sense and antisense strands are differentiated by thin and thick lines. Intact and nicked BsoBI recognition sequences are depicted and ■ ■ . The partial BsoBI recognition sequence 59-TCGGG by and its complement 59-CCCGA are present at the 59 and 39 ends of displaced strands as represented by ■ and ■.

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NYCZ ET AL.

nition site and the underlined bases comprise the target binding region. The binding region of primer S2 corresponds to positions 1368–1348, and the entire sequence is 59-dCGATTCCGCTCCAGACTTCTCGGGGTGTTTAGCATGGTGTT. The detector probe for the HIV sequence (DHIV) corresponds to positions 1313–1339, and its sequence is 59-dCAGCATTATCAGAAGGAGCCACCCCAC. The detector probe for the control plasmid sequence (Dcontrol) corresponds to positions 1334 –1307 and its sequence is 59-dTTTTCCACTATATACCGTCGTATCGCCA. The detector probes were 59-32P labeled at a concentration of 5 mM in a 10-ml volume containing 50 mM Tris–HCl, pH 8.0, 10 mM MgCl2, 20 units T4 polynucleotide kinase, and 0.06 mCi [g-32P]ATP (3000 Ci/mmol, 10 mCi/ml, DuPont New England Nuclear) for 45 min at 37°C and terminated by heating for 3 min in a boiling water bath. The detector probes were used without further purification. All SDA oligodeoxynucleotides were synthesized on an Applied Biosystems Inc. 380B Synthesizer, purified by HPLC, and stored in distilled H2O. SDA Reagents SDA reagents were purchased from Sigma, Gibco BRL (Gaithersburg, MD), or as described below. The SDA enzymes, BsoBI and exo2 Bst DNA polymerase, large fragment, were obtained from New England Biolabs (Beverly, MA). The deoxynucleoside 59-triphosphates (dGTP, dATP, dUTP), 29-deoxycytosine 59-O-(1thiotriphosphate) (dCTPaS), and the T4 gene 32 single-strand binding protein (T4gp32) (9, 10) were purchased from Pharmacia (Piscataway, NJ). Avian myeloblastosis virus (AMV) reverse transcriptase was purchased from Gibco BRL.

mixed in a 45-ml volume. The samples were heated to 70°C for 30 s for RNA target denaturation and subsequently equilibrated at 45°C for 2 min. BsoBI restriction enzyme, exo2Bst polymerase, AMV reverse transcriptase, and T4gp32 were added for a final volume of 50 ml. The reaction was incubated at 45°C for 15 min for reverse transcription and then incubated at 53°C for an additional 15 or 25 min as indicated. Amplification was terminated by freezing samples in a dry ice/ethanol bath. Analysis of SDA products was performed by denaturing gel (8%) electrophoresis. Five microliters of the completed SDA reaction was mixed with 5 ml of a denaturing solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue and 0.05% xylene cyanol (Amersham, Arlington Heights, IL) and heated in a boiling water bath for 3 min immediately before gel loading. Autoradiography was performed at 270°C using Fuji RX or Kodak XAR-5 film and DuPont intensifying screens. A Molecular Dynamics PhosphorImager Model 425 was used for quantitation of SDA products. The total number of SDA products (molecules) in the aliquot analyzed by gel electrophoresis was calculated from [Bpdt /(Bpdt 1 Bdet )]D, where Bpdt is the PhosphorImager counts from the combination of electrophoresis bands representing nicked and unnicked SDA products, Bdet is the PhosphorImager band counts corresponding to the unreacted detector probe, and D is the total number of original detector probe molecules in the aliquot analyzed by gel electrophoresis. SDA factors correspond to the number of product molecules divided by the original number of targets (T0) for a given volume of the SDA reaction. Statistical Analysis

SDA SDA reaction conditions were as follows: 35 mM potassium phosphate, pH 7.6, 0.1 mg/ml acetylated bovine serum albumin, 1 mM primer S1, 0.75 mM primer S2, 0.05 mM each primer B1 and B2, 0.4 mM of each detector probe, 1.4 mM dCTPaS, 0.5 mM dUTP, 0.2 mM each dATP and dGTP, 7.5 mM MgOAc2, 500 ng human placental DNA, 5% (v/v) DMSO, 8% (v/v) glycerol, 320 units BsoBI restriction enzyme, 20 units exo2Bst DNA polymerase (from Bacillus stearothermophilus) (11–13), 4.8 mg T4gp32 singlestrand binding protein, 2.5 units AMV reverse transcriptase, and the HIV and control targets as indicated. Although each SDA reaction contained detector probes for both the HIV and the control target sequence, parallel SDA reactions were run in which only one of the two detector probes was 32 P-labeled. SDA was performed as follows. All reagents, except for T4gp32 and the enzymes, were

The statistical analysis leading to quantification of unknowns is based on an extension of the models and analysis that Raeymaekers (24) proposed for PCR. Walker et al. (14) suggested the following model for SDA: T ~t ! 5 T 0 z e lT t

[1]

S ~t ! 5 S 0 z e lS t ,

[2]

where T (t ) and S (t ) are the amounts of amplified target and control sequence produced after t time units of SDA, T (0) 5 T 0 and S (0) 5 S 0 are the initial target and control sequence concentrations and lT and ls are the amplification rates for the target and control sequence. The ratio of the amplified products is then related to the initial amounts as follows:

STRAND DISPLACEMENT AMPLIFICATION

log~T~t!/S~t!! 5 log~T0 ! 2 log~S0 ! 1 log~elT t/elS t ! 5 log~T0 ! 2 log~S0 ! 1 t z ~lT 2 lS !.

[3] [4]

229

Then, if r 5 1, the initial target concentration can be written as ˆ j 0 5 exp~2bˆ j /m ˆ !. T

[8]

Note that e l Tt is the amplification factor for the target, or in other words, the ratio of the final to initial amounts of target at time t. The ratio of the amplification factors for the target and control is

If r Þ 1, then we have determined empirically that a good estimate is

r 5 ~e lT t /e lS t ! .

ˆ j 0 5 exp~2bˆ j /m ˆ !/rˆ . T

[5]

We have typically found that the amplification factors for the target and the control are not equal so that r Þ 1 cannot be ignored. Substituting r into Eq. [3] we get log~T ~t !/S ~t !! 5 log~T 0 ! 2 log~S 0 ! 1 log~r ! log~T ~t !/~S ~t ! z r !! 5 log~T 0 ! 2 log~S 0 ! .

[6] [7]

In the quantitation that follows differences in amplification rates between the two reactions can thus be compensated for via estimation of r. Note that another interpretation of Eq. [6] and [7] is that the graph of log(T (t )/S (t )) versus log (S0) should be linear with slope equal to 21 but with intercept that depends on the relative amplification rates. We have found that in practice the slope is often not equal to 21, but that accurate quantitation can still be carried out. In the Appendix, we outline a mathematical model for SDA that extends the above equations to this case. Given the relationship, Eq. [7], there are many ways to carry out the actual quantitation. We proceed as follows: 1. Regress log(T (t )/S (t )) versus log(S0) for a set of standards that have known initial concentrations of control and a common initial amount of target. Test for curvature in the regression line and drop points at the highest and lowest concentrations of the controls until the regression is linear. This step determines the approximate range of quantitation for the assay. A corresponding analysis of log(T (t )/S (t )) versus log(T0) should yield similar results when all samples have a common initial amount of control and varying initial concentrations of target. 2. Calculate the estimated amplification ratio, rˆ, by taking the average of the calculated amplification ratios over the range in which the regression is linear. Calculate the estimated slope, m ˆ , and intercept, bˆ, from the linear regression of log (T (t )/S (t ) z r ) against log(S0). 3. For each unknown sample, j 5 1, 2, . . . k, assume that the slope m j 5 m and estimate b j , as ˆ z log~S 0 ! bˆ j 5 log~T j ~t !/S j ~t !! 2 m

Note that it is not necessary to perform a complete quantitation type curve for each unknown in order to quantify the initial target concentrations as long as the slope and amplification ratio are not affected by the initial concentration of target. That is, m j 5 m, for all j. We have typically found this to be true (data not shown) as long as the initial target concentration falls within the dynamic range of quantitation as defined above. RESULTS

SDA is a DNA amplification technique that consists of a target generation step that generates a target fragment with defined 59 and 39 ends followed by an exponential amplification phase that replicates the target sequence through a series of primer extension, nicking, and strand displacement steps (5, 14, 16 –18). SDA can be adapted to RNA targets by including a reverse transcriptase to perform the target generation step (Fig. 1). Detection of SDA products can be accomplished by the addition of a sequence specific detector probe during the SDA reaction. The target-specific detector is simultaneously extended during SDA in a manner such that the level of extended probe mirrors the concentration of SDA product (Fig. 2) (19 –23). We have developed a QRT-SDA protocol for the coamplification and detection of an HIV gag sequence and control sequence using the same SDA primers. For quantitation it is most desirable to include an internal control sequence which is easily distinguishable from the target of interest but contains many of the same features. In this study, the control sequence was identical to the HIV sequence except for an internal 27-bp region which had been randomly mutagenized (Fig. 3). This allows us to amplify the control sequence and HIV target sequence using the same SDA primers but distinguish the two products with different detector probes. Detector probes for the HIV and control sequence were designed to confer specificity. As shown in Fig. 3, the detector probes for each sequence do not bind to the same product strand. The corresponding HIV detector probe products are 57 nucleotides (structures II, III, and V, Fig. 2) and 76 nucleotides (structure VI, Fig. 2)

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NYCZ ET AL.

FIG. 2. Schematic of SDA with concurrent detection. The left side of the figure depicts the exponential SDA cycle (Fig. 1) in the presence of a 59 32P-labeled detector probe (*—) which binds downstream of one of the SDA primers (upper left-hand corner of figure). This detector probe/target complex is duplicated on the right side of the figure (structure I) along with subsequent reaction products (structures II–VI). The DNA polymerase extends both the detector probe (structure II) and the SDA primer (IV) which displaces the detector probe extension product into solution (structure III). The complementary SDA primer binds to this product (structure V) and is extended by the DNA polymerase to the end of the detector probe (structure VI). Relatively short (structures II, III, and V) and long (structure VI) extension products from the detector probe indicate target-specific amplification.

in length, while the respective control sequence products are 81 and 100 nucleotides in length. Since we are coamplifying the HIV target and the control sequence with simultaneous extension of corresponding 32P-labeled detector probes, there are a number of protocol variations possible. For all the experiments in the present report, each SDA reaction contained both the HIV detector probe and the control sequence detector probe. Additionally parallel reactions were run for each level of HIV target

FIG. 3. Alignment of HIV pGAG and control sequences including SDA primers and detector probes. Number positions reflect alignment along the HIV gag-1 sequence as described by Gurgo et al. (6, 7). The boxed sequence represents the randomly mutagenized region. The NcoI site is designated at the beginning of the mutagenized region. DHIV and Dcontrol are the detector probes for the HIV and control sequences, respectively. S1, S2, B1, and B2 are the SDA primers.

FIG. 4. Autorad depicting the quantitation curve from the quantitative RT-SDA reaction. Control sequence RNA levels tested were 102 to 106 copies/reaction. Target HIV RNA was held constant at 104 copies/reaction. The reaction conditions were 15 min at 45°C followed by 15 min at 53°C.

and control sequence evaluated but only one of the two detector probes in each SDA reaction was 32Plabeled. SDA products (32P-detector probe extension products) and unreacted 32P-detector probes were separated by denaturing gel electrophoresis (Fig. 4) and quantitated for statistical analysis as described under Materials and Methods. It is possible, in theory, to run a single SDA reaction with both detector probes 32P-labeled and then differentiate the extension probe products by gel electrophoresis based on molecular weight differences. However, we found the present approach of running parallel SDA reactions to be more accurate for quantitation because of minor background gel electrophoresis bands. Raeymaekers (24, 25) describes a quantitative PCR to be a relationship between the amplification of a target sequence and a known amount of control sequence (internal standard). The ideal relationship is one where the target and control sequences amplify at the same rate or efficiency using the same primer pair. Raeymaekers describes the ability to quantitate in the post-exponential, linear phase of a PCR assuming both amplification reactions move into the non-exponential phase concurrently since both targets depend on the same set of reagents. Because amplification reactions are limited in the amount of total product, amplification of both sequences tends to simultaneously enter the post-exponential phase as soon as the level of one reaches the total allowable level of product dependent on the limiting concentration of a common reagent. If the amplification efficiency is the same for both sequences, then the ratio of their products will remain constant throughout both the exponential and linear phases, and the amount of unknown target can be

STRAND DISPLACEMENT AMPLIFICATION

determined from the ratio of the two products and the known initial level of the control sequence. For a series of reactions containing the same unknown level of target and various known amounts of control target, a regression curve plotting the log of the ratio of the products (target/control) versus the log of the initial input copy number of control is used to determine the amount of the unknown target. Ideally the plot will have a slope of 21 and pass through 0. The predicted amount of the unknown target is equal to that point on the input control axis where the curve passes through 0. Coamplification of the target and control sequences should be correlated for accurate quantitation. Correlation is ensured by the principles described by Raeymaekers, which include coamplification using a common primer pair, similar amplification efficiencies for both sequences, and simultaneous entry into the linear amplification phase. There are some instances where coamplification is no longer correlated. Any reaction condition which affects the amplification of one sequence and not the other would eliminate correlation. These factors could be a simple as temperature for example, where a slight change in temperature differentially affects the annealing of one of the two detector probes. Additionally, if one SDA product shows a greater tendency to self-anneal (T1 annealing to T2 in Fig. 1) toward the end of SDA thereby competing with primer hybridization, then amplification of the target and control sequence will no longer correlate. Regardless of the source, noncorrelation produces a deviation from linearity for a Raeymaekers’ analysis and threatens accurate quantitation over a broad range of initial target levels. Other conditions restricting the range of linear regression in a Raeymaekers’ analysis include large differences in the amplification efficiencies of the target and control sequence or signs of depletion of reagents at high initial levels of target or control sequence. As long as the coamplified sequences remain, correlated quantitation can be performed across a wide dynamic range. We established that our model system was not amplifying both sequences at the same rate. However, both reactions moved into the linear phase at the same time indicating that both reactions were being affected equally by a limiting reagent. We believe this to be a limiting amount of restriction enzyme because final SDA product levels increase with increasing amounts of restriction enzyme in a linear fashion (stoichiometric not kinetic limiting) (data not shown). Quantitation using sequences that do not amplify at the same efficiency can be achieved provided that the difference in amplification efficiency is known. The ratio of amplification efficiencies (r) can be used to correct the ratio of products for the target and control sequence in the Raeymaekers’ analysis. Evaluation of the linear phase of amplification indicated that

231

FIG. 5. The linear regression of log(T(t)/(S(t)*r)) (total target produced/total control produced times the amplification ratio) against log(SO) (initial control target level) for the QRT-SDA protocol involving 15 min at 45°C followed by either 15 min at 53°C (30 min RT-SDA, ■) or 25 min at 53°C (40 min RT-SDA, F). The results for 102 and 106 control copies were not included in the regression fits as indicated by the open squares and circles, whereas the points used in the regression fit are shown with closed symbols. The amplification ratio, r, was calculated using only those points included in the linear regression fit.

RT-SDA follows the principles as described by Raeymaekers (as shown below). The current RT-SDA protocol included 15 min at 45°C followed by 15 min at 53°C (30-min RT-SDA). While RT-SDA can instead be performed with a single 30-min incubation at 53°C, we included the 45°C incubation for better reproducibility of reverse transcription due to the limiting thermal stability of AMV reverse transcriptase. A mock quantitative RT-SDA experiment was performed following the conditions outlined above. We performed a Raeymaekers’ quantitative experiment that consisted of 102–106 copies of the control sequence in the presence of 104 HIV RNA copies. We estimated an amplification ratio (r) of 2.05 from the quantitation curve, while a plot of the Raeymaekers’ linear regression analysis (ln(T(t)/S(t) z r) versus log S0) yielded a slope of 20.936 (Fig. 5). SDA, being an isothermal reaction, is monitored over time, not by cycle number as with PCR. We therefore evaluated the effects of SDA incubation time on the Raeymaekers’ analysis. We extended the 53°C incubation step from 15 to 25 min (40-min RT-SDA) and found that we were able to generate a straight line but the slope was 20.782 (Fig. 5). Additionally, r 5 2.26 for the longer incubation time. The dynamic range, as calcu-

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NYCZ ET AL.

FIG. 6. Estimation of unknown HIV RNA target levels for QRTSDA protocols involving a 15-min incubation at 45°C followed by a 53°C incubation for 15 or 25 min as indicated (30 or 40 min RT-SDA). The closed symbol represents an aberrant data point.

lated under Materials and Methods, for both RT-SDA incubation times was 500 –500,000 copies of HIV RNA. Although the slope of the curves differed, the dynamic range was not affected. In addition to the typical Raeymaekers’ analysis, we performed a reverse experiment with samples containing 104 copies of control sequence in the presence of 102–106 copies of HIV RNA (data not shown). Linear regression analysis produced slopes of 0.93 and 0.62 and amplification factors, r, of 0.24 and 0.96 for shorter and longer incubation times consistent with the curves in Fig. 5. In the same experiment, we amplified four samples containing unknown levels of HIV RNA in the presence of 104 copies of control sequence over the two RT-SDA incubation times (Fig. 6). Seven of the eight data points predicted the levels of initial HIV RNA within 15%. The individual coefficient of variation of these seven points was 30%, but this was driven higher by one sample at the lowest concentration that had quite high variation (CV 5 85%). Results indicated that quantitation of RNA could be achieved during the linear amplification phase of the reaction over a 10-min time frame for total SDA duration of 30 or 40 min.

scheme compared with the stepwise nature of PCR, the two techniques generally follow the quantitative principles that Raeymaekers originally described for PCR (24, 25). For the purpose of our work, we have converted these principles into a constant time function as described in the Appendix. We were able to quantitate over an extended period of SDA during the postexponential phase of the reaction. Shortly after the QRTSDA system entered the linear phase after 30 min of amplification, its characteristics modeled those as described by Raeymaekers generating a quantitative curve with a slope very close to 21. However, as SDA continued well into the linear phase (40 min of amplification), the system remained quantitative but its characteristics changed producing a quantitative curve with a slope deviating substantially from 21. We believe that the cause for this change is the limiting concentration of one of the detector probes. We determined that at the end of 40 min of amplification we were depleting the control detector probe at high input levels of control target. This is the most probable explanation for the observed results. SDA is easily adaptable to multiple detection formats and different clinical settings because it does not require sophisticated equipment. The system developed here involves real-time extension of a detector probe with a single endpoint analysis. The radioactive label and gel electrophoresis analysis used here could easily be substituted by a microtiter sandwich hybridization assay using a variety of colorimetric, fluorescent, or chemiluminescent labels (26). However, simultaneous SDA and extension of detector probe is ideally suited to a real-time fluorescence detection format. Homogeneous real time detection offers the opportunity to continually monitor a reaction in a closed tube, eliminating the false-positive problems associated with amplicon contamination due to accidental dispersal of amplicons in the laboratory during heterogeneous detection. Spears et al. (23) described real-time detection for an SDA reaction using a fluorescently labeled probe and fluorescence polarization measurements. The format should enable quantitative measurements of an SDA reaction based on the time at which a positive signal is generated (high target samples go positive earlier than low target samples). We are continuing our efforts in real time detection to provide an acceptable assay format for the clinical setting. APPENDIX

DISCUSSION

Quantification When Amplification Rates Are Not Constant in Time

SDA can be used for quantitation of nucleic acids. The current QRT-SDA technique has a dynamic range of approximately 500 –500,000 copies of HIV RNA. Although SDA employs a continuous amplification

One of the main assumptions in the above model for SDA, (1) and (2), is that the amplification rate, l, is assumed to be constant in time. This is not generally the case in actual SDA because there is generally a

233

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ramp up period, a period of pure exponential amplification, and a plateau period. In Raeymaekers’ paper, quantitation for PCR was to be performed during the exponential phase. In order to extend the results quantitation in either the exponential or plateau phase of SDA, we propose the following model: Let l(t) be the amplification rate as a function of time, t. Suppose that l(t) is continuous and differentiable. Then the change in product over a small period of time, Dt, would be T ~t 1 Dt ! < T ~t ! z e l~t! z Dt

[A1]

Following this approach we get for an arbitrary time t and large number of approximation intervals, n, T ~0! 5 T 0

[A3]

T ~2t /n ! < T 0 z e l ~t/n! z t/n z e l

Pe

~2t/n! z t/n

[A4]

n

T ~t ! < T 0 z

[A5]

i51

Pe n

l ~it/n! z t/n

E 1 log~e

5 lim T 0 e

O

E 5T e 0

l ~it /n ! z t/n

i51

n3`

o lT ~s !ds

E /e

t

o

ls ~s !ds

!

[A9]

The situation just mentioned above implies that for a range of values of T0 and S0

E log~e

t

E /e

t

o

ls ~s !ds

! 5 a z log~S0 ! 1 r,

[A10]

[A11]

ACKNOWLEDGMENTS

t

o

t

which is a general formula for a straight line with intercept and slope that may deviate from the ideal case (b 5 log(T 0 ) and m 5 21) depending on the particular circumstances of the coamplification.

n

l ~s !ds

[A6]

Now we can apply this relationship to the quantitation problem as follows: log~T ~t !/S ~t !!

E 5 log~T ! 2 log~S ! 1 log~e 0

log~T ~t !/S ~t !! 5 log T 0 2 log S 0

log~T ~t !/S ~t !! 5 b 1 m z log~S 0 !

i51

n3`

[A8]

where we interpret r as an overall ratio of amplification factors as in Eq. [7] and we interpret a as the relative ratio of amplification factors that depends on both T0 and S0. This leads us to the final model that we use for quantitation, namely,

so that in the limit

T ~t ! 5 lim T 0 z

lS ~s!ds

o

When l T (t) ; l s (t ) or, more generally, * to l T (s )ds 5 * to ls (s)ds, then the initial target level can be quantified based on the linear model, as discussed below. Note also l T (t ) 5 l T and l s (t ) 5 l s are constant, then Eq. [A8] reduces to Eq. [4]. We have found that in some cases the graph of log (T (t )/S (s )) versus log S0 is linear with slope not equal to 21 for values of T0 and S0, in a restricted range, and is nonlinear outside that range. In order to clarify this situation, rewrite Eq. [A8] as follows:

o lT ~s !ds

l ~it/n! z t/n

E

t

lT ~s!ds 2

o

[A2]

T ~t /n ! < T 0 z e l ~t/n! z t/n

0

E

t

5 log~T0 ! 2 log~S0 ! 1

t

o

lT ~s !ds

!

E 2 log~e

t

o

lS ~s !ds

!

[A7]

The authors thank Dr. Kent Lohman for supplying the gGEM clone used in this work. Also, we thank Julie Dickson for production and purification of the RNA transcripts.

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