Isothermal, in Vitro Amplification of Nucleic Acids by a Multienzyme
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Proc. Natl. Acad. Sci. USA Vol. 87, pp. 1874-1878, March 1990 Biochemistry Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication (reverse transcriptase/RNase H/T7 RNA polymerase) JOHN C. GUATELLI*t, KRISTINA M. WHITFIELDt, DEBORAH Y. KWOHt, KEVIN J. BARRINGERt, DOUGLAS D. RICHMAN*t, AND THOMAS R. GINGERASO§ *Departments of Medicine and Pathology, University of California San Diego School of Medicine, and tSan Diego Veterans Administration Medical Center, San Diego, CA 92161; and tThe Salk Institute Biotechnology/Industrial Associates, Inc., La Jolla, CA 92037 Communicated by Ronald M. Evans, December 18, 1989 (receivedfor review October 6, 1989) ABSTRACT A target nucleic acid sequence can be repli- RNA Purification. Human immunodeficiency virus type 1 cated (amplified) exponentially in vitro under isothermal con- (HIV-1) RNA was extracted in total RNA from HIV- ditions by using three enzymatic activities essential to retroviral 1-infected CEM cells by the guanidinium isothiocyanate/ replication: reverse transcriptase, RNase H, and a DNA- cesium chloride gradient procedure (6) and quantitated by dependent RNA polymerase. By mimickisg the retroviral comparative hybridization to standards of known concentra- strategy of RNA replication by means of cDNA intermediates, tion. this reaction accumulates cDNA and RNA copies ofthe original 3SR Reaction. One hundred-microliter 3SR amplification target. Product accumulation is exponential with respect to reactions contained the target RNA, 40 mM Tris-HCI at pH time, indicating that newly synthesized cDNAs and RNAs 8.1, 20 mM MgCl2, 25 mM NaCl, 2 mM spermidine hydro- function as templates for a continuous series of transcription chloride, 5 mM dithiothreitol, bovine serum albumin (80 and reverse transcription reactions. Ten million-fold amplifi- ,ug/ml), 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, cation occurs after a 1- to 2-hr incubation, with an initial rate 4 mM ATP, 4 mM CTP, 4 mM GTP, 4 mM UTP, 250 ng of of amplification of 10-fold every 2.5 min. This self-sustained oligonucleotide 88-211 (primer B, 5'-AATTTAATACGACT- sequence replication system is useful for the detection and CACTATAGGGATCTATTGTGCCCCGGCTGGTTTTGC- nucleotide sequence analysis of rare RNAs and DNAs. The GATTCTA-3'), and 250 ng ofoligonucleotide 88-347 (primer analogy to aspects of retroviral replication is discussed. A, 5'-AATTTAATACGACTCACTATAGGGATGTACTAT- TATGGTTTTAGCATTGTCTAGTGA-3'). [Each priming The transfer of genetic information from RNA to DNA and oligonucleotide contains the T7 RNA polymerase binding then back to RNA is a scheme characteristic ofretroviruses. sequence (italic) and the preferred transcriptional initiation Such a scheme provides a mechanism for the replication of site (boldface). The remaining sequence is complementary to RNA genomes (reviewed in refs. 1-3). In exploring variations the target HIV-1 sequence.] After heating at 650C for 1 min ofan in vitro transcription-based amplification system (TAS) and cooling at 370C for 2 min, 30 units of AMV reverse (4), it was discovered that it was possible to devise a transcriptase, 100 units of T7 RNA polymerase, and 4 units concerted, three-enzyme, in vitro reaction to carry out an ofE. coliRNase H were added to each reaction. All reactions isothermal replication oftarget nucleic acid sequences, anal- were incubated at 370C for 1 hr or the amount of time ogous to the strategy used in retroviral replication. This indicated and stopped by placing the reaction on ice. reaction is a self-sustained sequence replication (3SR) system Bead-Based Sandwich Hybridization. The 3SR amplifica- involving the collective activities of avian myeloblastosis tion product and 32P-labeled oligonucleotide 87-81 (5'- virus (AMV) reverse transcriptase, Escherichia coli RNase AATTAGGCCAGTAGTATCAACTCAACT-3') were dena- RNA tured in 30 ttl of 10 mM Tris, pH 8.1/1 mM EDTA at 650C for H, and T7 polymerase. The accumulation ofboth target 5 min in an Eppendorf tube. To this, 30 ul of a solution nucleic acid-specific RNA and cDNA has been observed, hybridization mixture [10x standard saline phosphate/EDTA quantitated, and characterized. Several aspects of this 3SR (6)10%o (wt/vol) dextran sulfate/0.2% SDS] was added. The amplification protocol provide features not observed in the solution was mixed and incubated at 420C for 2 hr. use ofeither the polymerase chain reaction or TAS protocols. Approximately 25 mg (wet weight) of Trisacryl beads These features and the parallels between the strategies em- containing oligonucleotide 86-273 (5'-AGTCTAGCAGAA- ployed in the 3SR reaction and during retroviral replication GAAGAGGTAGTAATTAGA-3') was prehybridized in 250 are noted and discussed. pA of hybridization solution (5 x standard saline phosphate/ EDTA/10% dextran sulfate/0.1% SDS) in a 2-ml microcol- MATERIALS AND METHODS umn (Isolab) for 30 min at 370C. The prehybridization solu- tion was removed, and 40 p.1 of fresh hybridization solution Materials. AMV reverse transcriptase was purchased from was added to the beads, together with the 60 p.1 of solution Life Sciences (Saint Petersburg, FL); T7 RNA polymerase from the solution hybridization step. The beads were then was from Stratagene; E. coliRNase H and RNase-free DNase incubated at 370C for 1 hr with occasional mixing and then I were from Bethesda Research Laboratories. Oligonucleo- washed six times with 1 ml of 2x standard saline citrate (6) tides were synthesized by phosphoramidate chemistry by at 370C. The radioactivity of the beads and the combined using an Applied Biosystems model 380A DNA synthesizer. washes was measured by Cerenkov counting for 1 min. The Trisacryl beads containing capture oligonucleotides, which amount of target detected was determined by calculating the were used for sandwich hybridizations, were prepared sim- percentage of total radioactivity captured on the beads and ilarly to those described previously (5). Abbreviations: 3SR, self-sustained sequence replication; TAS, tran- The publication costs of this article were defrayed in part by page charge scription-based amplification system; HIV-1, human immunodefi- payment. This article must therefore be hereby marked "advertisement" ciency virus type 1; AMV, avian myeloblastosis virus. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 1874 Downloaded by guest on October 2, 2021 Biochemistry: Guatefli et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1875 then multiplying this percentage by the femtomoles of 32P- Step 1 5' 3'RNA labeled oligonucleotides used for the hybridization. 3 primer 2 I3) RNA Nuclease Digestion. Samples of 3SR reactions were di- 5' t gested with RNase-free DNase I in 50 mM sodium acetate at 4RT DNA pH 6.5, 10 mM MgCl2, and 2 mM CaC12 in the presence of 3 5' | >'RNA ___ RNaseA, H DNA enzyme (25 pug/ml) at 370C for 1 hr. Digestions were termi- 4 RNA nated by the addition of EDTA to 12.5 mM and were - %rie DNA ~ DNA extracted once with an equal volume of phenol/chloroform 5 prior to separation in a 5% denaturing polyacrylamide gel. 3' |T DNA Direct 3SR Transcript Sequencing. One hundred-microliter 6 3m+ DNA 1100 DNA 3SR reactions were extracted once with an equal volume of T7RNA polymerase phenol/chloroform, adjusted to 0.3 M sodium acetate, and __3' 5' precipitated with the addition of 2.5 volumes of 100o etha- RNA (sense) RNA (anbsense) |3primer® 5' primer® nol. The reaction products were pelleted at 16,000 x g for 10 8 RNA ® ( DNA min, rinsed with 70%o (vol/vol) ethanol, and dried completely R- DNA RNA before being dissolved in 50 of sterile H20. Two percent A.l 9T ARNA - DNA of the transcript (derived from a 107-fold amplification from - DNA RNA 100 amol of target HIV) was sequenced (7) by using a primer RNase H RNase H complementary to the major antisense 3SR RNA product and 10 "'sa- RNA - DNA DNA Adoa oooRNA located within the targeted env region (5'-GACGTTCAATG- |'primer® Tprimer ) GAAC-3'). cDNA Cloning and Sequencing. The 3SR reactions were -DNA - DNA extracted once with phenol/chloroform, precipitated with 9 ADNA ethanol, and converted into first- and second-strand cDNAs -DNA by the single-tube method first described by D'Alessio and FIG. 1. Strategy of the 3SR scheme. The 3SR reaction depends Gerard (8). The second-strand synthesis reaction was termi- on a continuous cycle of reverse transcription and transcription nated by heating, and the cDNA ends were repaired by the reactions to replicate an RNA target by means of cDNA intermedi- direct addition of20 units ofthe large fragment ofE. coli DNA ates. Oligonucleotides A and B prime DNA synthesis and encode the polymerase I, followed by a 1.5-hr incubation at 22°C. promoter sequence for the T7 RNA polymerase (black boxes). Steps End-repaired cDNAs were extracted once with one volume 1-6 depict the synthesis of a double-stranded cDNA, which is a of phenol/chloroform and- then precipitated (in the presence transcription template for 17 RNA polymerase. Complete cDNA of 10 ,g of glycogen as a carrier) with 3 volumes of 100%o synthesis is dependent on the digestion of the RNA in the interme- ethanol. Precipitated cDNAs were phosphorylated with T4 diate RNA*DNA hybrid (step 4) by RNase H. Transcription- kinase into that had been competent cDNAs yield antisense RNA copies ofthe original target polynucleotide (6), ligated pUC19 (step 7, right). These transcripts are converted to cDNAs containing cut with Sma I and treated with phosphatase, and used to double-stranded promoters on both ends in an inverted repeat transform E. coli strain MC1061. 3SR clone-containing trans- orientation (steps 7-12). These cDNAs can yield either sense or formants were identified by colony hybridization by using a antisense RNAs, which can reenter the cycle. Thin lines, RNA; thick kinased oligonucleotide probe.