AU623602B2 - Transcription-based nucleic acid amplification/detection systems - Google Patents

Description

CORRE;,TED

ERSION*J P3 A5- d crcing~cs k~fP/QCtcd by 1 t ew WORLD INTELLECTUAL PROPERTY ORGAN jTION PCT ~~~Inte~aonal~eau 2C 6 __INTERNATIONAL APPLICATION 16LI D TH A COOPERATION TREATY (PCT) (51) International Patent Classification 4 1111 Internatinal, Piublication Nu~mber: WO 88/ 103151 C12Q 1/68, C12N 15/00 Al (43) International Publication Date: 29 )ecember 1988 (29.12.88) (21) International Application Number: PCT/US88/02108 (22) International Filing Date: (31) Priority Application Numbers: 17 June 1988 (17.06.88) 064,141 202,978 19 June 1987 (19.06.87) 6 June 1988 (06.06,88) us (32) Priority Dates: (33) Priority Country: (74) Agents: W ITT, Phillip, H. et al.; Fitch, Even, Tabin Flannery-, Room 900, 135 Soutlt LaSalle Street, Chicago, IL 60603 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), BR, CH (European patent), DE (European patent), DK, FI, FR (European patent), GB (Europe&n patent), HU, IT (European patent), JP, KR, LU (European patent), NL (Europea.- patent), NG, SE (European patent), SU.

Published With international search report.

Before the expiration of the time limit/or amending the claims and to be republished in the event of the receipi of amendments.

(71) Applicant: SISKA DIAGNOSTICS, INC. (UJS/US]; 10280 North Torrey Pines Road, Suite 270, La Jolla, CA 92037 (US).

(72) Inventors: GINGERAS, Thomas, Raymond 1528 Juniper Hill Drive, Encinitas, CA 92024 MERT- EN, Ulrich ;4422 Leon Street, San Diego, CA 92107 KWOH, Deborah, Yantis -2404 Jacaranda Avenue, Carlsbad, CA 92009 (US), (54) Title: TRANSCRIPTION-F(ASED NUCL.VIC ACID AMPLIFICATION/DETEFCTION SYSTEMS (57) Abstract The present invention is predicated on the novelty or' certain fILNA transcripts, their prod~uction, optional replication, and use, to achieve desired qrnplification and detection of corresponding (in sequentoe) target aucleic acid sequence. The transcripts correspond in sequence to a target nucleic acid sequence contained in an original sample amongst a mixture of nucleic acids, and therefore, the presence of the transcripts in amplified form provides for their detecdog, and hence by correspondence, the in vitro or x-vivo detection of tho presence of the target nucleic acid sequence. In said sample.

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0,1(m OBA 201 20th 1 00h 200th pARV HBA -w lw I fn "M OAtM Ol1go 86-31 0lig0 87-0~9 (Referred to In PCT Gazette N!o. 04/1989, Section 11) WO 88/10315 PCT/US88/02108 1 TRANSCRIPTION-BASED NUCLEIC ACID AMPLIFICATION/DETECTION

SYSTEMS.

This is a continuing application under 35 U.S.C.

120/121 of USSN 07/064141 filed 19 June 1987, the contents of which are hereby incorporated by reference.

Field of the Invention The present invention relates generally to advances in molecular biology and molecular genetics.

More particularly, the present invention relates to novel methods and kits containing requisite reagents and means for increasing'the in vitro or x-vivo copy number of, amplifying, at least one selected segment (sequence) of nucleic acid or its complezment or hybridizing homologous segment, in a sample comprising one or more nucleic acids, which may include RNA, DNA or bcth.

Among the applications in which the methods and kits of this invertion find utility are 1) in analyses of body fluids and tissues for the detection of specific nucleic acid sequences characteristic of a genetic or pathogenic disease or condition by in vitro or x-vivo nucleic acid probe hybridization assays and 2) in the selective cloning of single-copy or rare or low-expression genes.

Background of the Invention Much of the work in molecular biology, molecular genetics and applications thereof, such as for example, nucleic acid probe hybridization assays for blood-borne pathogens or defective genes, involves the detection or isolation of a particular nucleic acid sequence. A wn al /11n1 2 fundamental problem in such work is to detect or isolate and then quantitate a nucleic acid sequence of interest.

The problem has been a difficult one because biological materials, such as cell cultures, tissue specimens, and blood samples, typically are comprised of a complex mixture of RNA and DNA entities of which at most only a minuscule fraction has a sequence of interest.

Indeed, practical applications of nucleic acid probe hybridization assays have been limited because the sensitivity of the assays, when carried out with reagents suitable for routine use and over acceptably short time periods, is too low to detect sequences of interest at the low concentration at which they occur in real samples.

Two fundamentally different approaches have been taken to address the problem of detecting a nucleic acid sequence of interest ("target segment") present at a low level in a complex mixture of nucleic acids.

In the first approach, the amount of nucleic acid (including the target segment) in a sample is nut altered; instead, a signal-generating system is associated with the target segment and produces a detectable signal representative of the number of molecules of target segment. For example, a nucleic acid probe, with a sequence complementary to that of a subsegment of the target segment is linked to an enzyme such as alkaline phosphatase and is mixed with sample under hybridization conditions that will effect hybridization between the probe and the target segment (but not appreciably between probe and other nucleic acid sequences in the sample).

After removing enyme-linked probe that failed to hybridize, a chromo(enic substrate for alkaline phosphatase is added under suitable conditions and, in principle, a large number of detectable, colored molecules is rapidly produced for each probe molecule hybridized to target segnvnt.

SWO 88/10315 PCT/US88/02108 3 Numerous other systems for detecting nucl ic acid sequences without altering the amount of target nucleic acid in the sample are known to the art. Another example is the commonplace labeling of a nucleic acid probe with S radioactive atoms such as 3 2 P and then detecting probe hybridized to target via amplified signal initiated by decay of the radioactive nuclei. Still another example involves linking to probe for the target segment another nucleic acid that is capable of replication such %hat it can be readily detected by known techniques. Certain RNAs are known that are susceptible to (autocatalytically) replicase-induced replication by certain polymerase, such as bacteriophage RNA-dependent RNA polymerase, such as Q2 replicase and the replicase from brome mosaic virus (BMV).

In such a system, both an RNA and the RNA of complementary sequence ar- templates for replication by the RNA polymerase; consequently the amount of replicated RNA increases exponentially with time (as long as the number of RNA template molecules does not exceed the number of RNA polymerase molecules in a system). See Miele et l., J. Mol. Biol. 171, 281 (1983). A system in which probe for a target segment is linked to an RNA capable of being replicated by Qp replicase is described by Chu et al., Nucl. Acids Res. 14, 5591 (1986) and that by BMV replicase by Marsh et al., Positive Strand RNA Viruses, Alan R. Liss (publr.; New York) (1987; Proceedings of UCLA Symposium, 1986).

The first approach has two serious drawbacks.

First, in many instances, the copy number of target segment in a sample of practical size is so low that, even for reasonably rapid signal generating systems, the time required to generate detectable signal that is significantly above background is impracticably long.

Second, signal generation occurs at essentially the same rate from "background" signal generating molecules as from signal generating molecules associated with target. In any _i j WO 88/1315 PCT/US88/02108

I

4 assay for a target segment, a signal due to "backg,' und" is unavoidable; that is, invariably there is some signal due to probe that non-specifically adheres to filters or other solid supports or hybridizes to segments with sequences closely similar to that cf target segment. If the copy number of target is too low, the time constant ratio of signal from target plus background (i.e.

"signal") to signal from background "noise") will be too low to be detectable significantly above background.

These and other drawbacks have led the art to a second approach of addressing the problem of detecting a target segment present at a low level in a complex mixture of nucleic acids.

This second approach is fundamentally different and involves increasing the copy number of the target segment itself, preferably to an extent greater than that of other sequences in a sample, particularly those that might erroneously be detected as target segment because of similarities in sequence. Examples of this second approach include various culture techniques in which cells that harbor the target segment are caused to increase in number, sometimes more rapidly than numbers of other cells, or in which particular nucleic acids plasmids, RNAs) therein having disposed target segment are caused to increase in number.

Such culture techniques have the disadvantages of being cumbersome and problematic and time-consuming and manifest the inevitable: nucleic acids other than those which include target segment are simultaneously increased in copy number, thus potentially increasing "background," Another disadvantage is the resultant growth of potentially dangerous organisms as a necessary step to achieve anmplification.

A'1ct27 example of this second approach is amplification of a DNA target segment in a so-called "polymerase chain reaction" This technique is a WO 88/10315 PCT/US88/02108 1 L borrowed adaptation of known, naturally occurring processes occurring in the replicative process of, for example, single-stranded DNA genomes of certain virus entities, and in all events,, represents an application akin to cDNA preparation ala Hong, Bioscience Reports 1, 243 (1981); Cooke et al., J. Biol. Chem. 255, 6502 (1980); and Zoller et al., Methods in Enzymoloqy 100, 468-500 (1983). By this technique, a particular segment increases in copy number exponentially with a number of cycles, each of which entails bybridizing to a 3'-terminal subsegment of each of the target segment and its complement the segment of sequence complementary to that of target segment) a DNA primer, (2) extending each of the primers with a DNA polymerase, and rendering single stranded by thermal denaturation the duplexes resulting from step This technique is described in Saiki et Scienc 1350 (1985), and iullis et al., European Patent Application Publication Nos. 200362 and 201184. See also U.S. Patents 4683195 and 4683202. Reportedly, in applying the technique for cycles over about 3 hours, the copy number of a target segment can be increased by a factor of about 105.

Because only those segments to which a specific primer hybridizes with 4 sufficient stability to initiate chain extension by the pclymeras, to form the complement and which have a complement to 'qhich another specific primer hybridizes similarly to yield target segment upon chain extension, they increase exponentially in copy number while other non-target segments not erroneously hybridized with the employed primer increase, if at all, at most linearly in copy number as a function of number of cycles.

The polymerase chain reaction technique can greatly increase not only the copy number of target segment but also the ratio of thA amount of target segment to that of background-causing segmerts in a sample.

-i WO 88/10315 PCT/us88/02108

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6 Of course, it follows that this second approach can be applied to a sample in conjunction with use of the first approach applied with the amplified target segment to provide even a stronger detection signal.

It is an object of the present invention to solve the problems addressed by the prior art and to c:vercome the disadvantages enumerated in prior researchers endeavors to solve those problems. It is a further object of the present invention to provide a straightforward technique that :an be utilized in an acceptc'-ly short time, employing the convenience of known reagents and having the precision necessary to reach consistent scientific results; one that can be employed in a reproducible assay setting and that is adaptable for use in kits for laboratory/clinical analyses.

It is thus an object of the present invention to increase the detectability of certain nucleic acid sequences (target segments) by amplification of said target sequences in an in vitro or x-.rivo system devoid of the disadvantages enumerated thus far by prior art endeavors.

The present invention is concerned with a novel technique for carrying out the second approach to detecting a target segment present at a low level in a complex mixture of nucleic acids. It employs a novel RNA transcript production step in conjunction with, and derived from, a synthesized double-stranded cDNA copy of the target sequence as a complete cycle. Multiple cycles can be employed. By virtue of the transcription step being the dominant aspect of novelty, it is conveniently referred to herein as a transcription-based amplification system (TAS). The novel technique of the present TAS invention results in rapid increase in copy number of a selected target segment by making use of two properties of DNA-dependent RNA polymerase: appreciable initiation of transcription from only a small number of sequences 4 WO 88/10315 PrT/Ts8/2n1in0 7 specific for each polymerase, see, Brown et al., Nucl. Acids Res. 14, 3521 (1986); and rapid production of a large number of transcripts from each copy of a promoter (typically 102-104 per hour) recognized by an RNA polymerase. See Milligan et al., Nucleic Acids Res. 8783 (1987). The technique of the invention can also utilize the ability of RNAs with certain sequences to be rapidly (autocatalytically) replicated by RNA-dependent RNA replicases. See also Miele et al., supra. In addition, it provides a standardization technique making possible unambiguous measurement of the amount of target DNA present in a sample.

The present invention (unless induced (autocatalytic) replication is employed) yields a single-stranded RNA transcript, or an RNA-DNA duplex formed therefrom when measures are not undertaken to prevent its formation, that has a subsegment that has the sequence of the target segment or the sequence complementary to that of the target segment, and that is present in large excess relative to nucleic acid with a subsegment of complementary sequence. This excess initially of a single-stranded RNA transcript is advantageous in certain methods for detecting amplified product with a labeled nucleic acid probe because little segment of complementary sequence is present to compete with probe for hybridizing to the amplified product.

Also, the single-stranded RNA transcript product hereof is struck-off more or less continuously and provides for direct detection of ta-get segment without the necessity of cumbersome, arror-prone repeaied PCR cycles and strand separation. Such advantages are not provided by the PCR technique that yields double-stranded DNA (one strand of which comprises target segment and the other strand of which comprises complement of target segment) that need to be separated before detection and only after a large WO 88/10315 PCT/US88/02108 8 number of repeated cycles necessary to reach acceptable amplification levels.

The techniques of the present invention provide amplification of a selected target segment to an extent at least as great as the PCR technique over about the came period of time, but in a far more simplistic and reproducible manner and distinct from natural processes or other art.

Summary of the Invention We have discovered how bacteriophage DNAdependent RNA polymerase can be used 'o rapidly amplify increase the copy number of) a selected ta±iet nucleic acid sequence (target sequence or segment) present in a sample of nucleic acids. Further, we have discovered how bacteriophage DNA-dependent RNA polymerase can be used together with bacteriophage RNA-dependent RNA polymerase to accomplish the same result. Heretofore, it has not been appreciated that such bacteriophage RNA polymerase could be used for this purpose.

The invention entails methods based on these discoveries and kits for carrying out the methods.

These methods and kits are particularly usefully applied in connection with nucleic acid probe hybridization assays for a nucleic acid which includes a target segment amplified in accordance with the invention.

Thus, the invention also entails methods, and kits for carrying out the methods, for detecting the presence or absence of a segment in a sample of a nucleic acid, which comprises a particular segment, by means of a probe hybridized to that segment after amplification in accordance with the invention.

The present invention is predicated on the novelty of certain RNA transcripts, their production, optional replication, and use, to achieve desired amplification and "'WO 88/10315 PCT/US88/02108 9 detection of corresponding (in sequence) target nucleic acid sequence. The invention is practiced in an in vitro or x-vivo setting, and is employed conjunctively with the synthesis of a double-stranded cDNA copy of the targsequence in order to produce a double-stranded nucleic acid template used in turn for production of said RNA transcripts. This process of double-stranded cDNA synthesis and RNA transcription constitutes a single cycle of the present transcription-based amplification system (TAS). If desired, this cycle may be repeated in order to achieve even higher levels of amplification. By virtue of the method by which they are produced (and reproduced), the transcripts correspond (identically or complementarily) in sequence to a target nucleic acid sequence contained in an original sample amongst a mixture of nucleic acids, and therefore, the presence of the transcripts in amplified-form provides for their detection, and hence by correspondence, the in vitro or xvivo detection of the presence of the target nucleic acid sequence in said sample.

Thus, the present invention involves tne in vitro or x-vivo detection of at least one specific nucleic acid sequence (target sequence or segment) in a sample containing nucleic acid. The present invention reduces to a method comprising preparing a double-stranded nucleic acid containing a sequence corresponding to a target sequence operably linked to a promoter therefor, emplcing said double-stranded nucleic acid as a double-stranded nucleic acid template for the preparation of a plurality of RNA transcripts therefrom, each bearing an RNA sequence corresponding to said target sequence, and detecting the presence of said RNA sequence and by analogy the presence of target sequence.

The present invention is directed to all methods and means associated with the preparation and use of such RNA transcripts. Thus, the present invention is directed 4 10 to the optionally repetitive method of preparing said double-stranded nucleic acid template defined above comprising: providing a first nucleic acid primer, containing a promoter sequence operably linked to a sequence corresponding to a first segment of the target sequence, said first segment including the 3'-end of the target sequence, and a second nucleic acid primer, which is hybridi2able to a sequence complementary to that of a second segment of the target sequence, said second segment including the 5'-end of the target sequence; hybridizing under suitable conditions said first nucleic acid primer with target sequence in a sample containing nucleic acid; extending said hybridized first nucleic acid primer in a polymerase extension reaction complementarlly to the target sequence to form a corresponding, first duplex nucleic acid; separating the strand, of said first duplex; 15 hybridizing under suitable conditions to the resulting, separated promoter-sequence-containing strand said second nucleic acid

S

:primer; extending said hybridized second nucleic acid primer in a polymerase ,xtension reaction complementarily to said promoter-sequencecontaining strand to form a second dupeX nucleic acid; and prior to separating the strands of said second duplex, employing said second duplex as a template for the preparation of a plurality of RNA transcripts therefrom In a reaction catalyzed by an RNA polymerase that recognizes the promoter corresponding to the promoter 25 sequence of the first primer, each of said transcripts bearing an RNA sequence corresponding to said target sequence.

The present. invention is further directed to further, and alternative, methods and means of preparing said double-stranded nucleic acid template (supra), for example, an essentially single-pot reaction comprising providing a first nucleic acid primer containing a promoter sequence operably linked to a sequence complementary to a segment of a target sequence and a second nucleic acid primer having a sequence identical to a segment of a target sequence, said primers Corresponding to different regions of said target sequence but not, or not substantially, overlapping in their correspondence to the target and being selected such that an extension product of one when separated from IOA its complement can serve as a template for an extension product of the other, contacting a sample containing nucleic acid including target sequence with said primers under sequential hybridlzing and strand separation conditions so as to produce, in extension products of said primers.

4 4 4 *4 4* 4* 44** 4 4 .4.4 IS/1385u S* 'WO 88/10315 PCT/US88/02108 11 The present invention is further directed to methods and means of employing said double-stranded nucleic acid supra., as a template for the preparation of a plurality of RNA transcripts therefrom in a reaction catalyzed by a DNA-dependent RNA polymerase that recognizes the promoter thereof, and detecting and measuring, the presence of said RNA transcripts.

The present invention is further directed to methods and means to standardize the amount of target sequence detected (by correspondence to the amount of RNA transcript detected) by further correlation to the presence of known nucleic acid used as an internal standard. The copy number of the standard is predetermined, the standard shall experience the same conditions, hence fate, during the practice of this invention as does target sequence and it shall therefore serve as an evaluation means in determining relative amounts of transcripts prepared in parallel for it and for target sequence.

The present invention is further directed to the further replication of obtained RNA transcripts d-fined above via the presence therein of replicase recogrition sites. This is conveniently accomplished by providing said first nucleic acid primer defined above additionally bearing a replicase recognition site sequence, preferably between the promoter sequence and sequence corresponding to target sequence, and/or additionally containing a replicase recognition site on the second nucleic acid primer defined above. On subsequent transcription, the consequent transcripts bear the replicase recognition site(s), such that presence of a replicase (in the reaction locus or kit for example) (autocatalytically) induces replication of the transcripts producing additional copies to further failitate detection.

The present invention is further directed to kits comprising requisite reagents and associated means useful WO 88/10315 PCT/US88/02108 "i 12 in the in vitro or x-vivo detection of at least one specific nucleic acid sequence (target sequence) in a sample containing nucleic acid, employing the methods and means defined supra.

Detailed Description of the Invention 1. Brief Description of the Drawings Figures 1A, 1B and 1C illustrate a method according to the invention for amplifying a target segment of a nucleic acid (Nucleic Acid which is a DNA or RNA, wherein many copies of a first RNA (RNA I) with a segment with a sequence complementary to that of target segment are made, Figures 2A, 2B, 2C illustrrte the further amplification according to the invention of a segment of an RNA I, made as illustrated in Figures 1A, 1B and 1C, to make many copies of a second RNA (RNA II) with a segment with a sequence the same as that of a subsegment of the target segment, which was amplified to make the RNA I.

Figure 3 depicts autoradiograms showing various concentrations of HIV RNA amplified by TAS simultaneously with a fixed concentration of human f-globin nucleic acid.

Figure 4 displays a general strategy whereby RNA produced through a DNA-dependent RNA polymerase yields an RNA molecule that can serve as a template for an RNAdependent replicase.

2. General Methods and Definitions Reference is made to standard textbooks of molecular biology that contain definit!ons and nmethods and means for carrying out basic techniques of the present invention such as: DNA probe or primer preparation, including DNA synth-sis; hybridization methodology L WOQ88/10315 PCT/US88/02108 13 including variations in stringMency conditions. for, producing more or less hybv:idilzation certaihty deipending upon the degree of homology of the primer to a target DNA sequence; identification, isolation or preparation of Promot'irs, or more specifically promoters or sites recognized by bacteriophae DNA-dependent RNA polymerase and bacteriophage RNA-depen(lent RNA polymerase, or in the employment of eukaryo systems, viral DNA- and RNAdependent RNA polymerases, for example, adenovirus encoded RNA polymerase and brome mosaic virus RNA polyinerase; conditions conducive to the productlon of RNA transcripts, including so-called transcription enhancer sequenlces; the mechanism and methodology for (ind,\iced) replication; polymcarase chain reaction methods including the reagents used therein; and so forth. See, for example, Maniatis et al. Molecular Cloning: A Lab, rat.ory Manual, Cold Spring Harbor Laboratory, New York, (1982) e~nd the various references cited therein; U.S. Patent 4683195; U.S.

Patent 4683202; Hong, Bioscience Reports 1, 243 (1981); Cooke 9,,tal. J. Biol. Chem. 255 6502 (1980) and Zoller et al. Meh(ds in Enzymology 100, 468-500 (1983) Crea e" al., NucleicAqids Res. 8, 2331 (1980); Narang It Meth. Enzym. 68, 90 (1979) Beaucage et al. Tetrahedroi IL1tters 22,j 1859 (1981) Brown et Meth. Enzym. 68, 101 (1979) Caruthers et Meth. Enzym. 154, 287 (19,85) Hitzeman et al. J. Biol. Chem., 255, 2073 (1980); Lee et Science 239_, 1288 (1988);* Milligan et al., Ajjclf "c Acids Res. 15, 8783 (1987) Miller et al., Virolo~v 125, 236 (1983) Ahiquist et al., J. Mol. Biol.

1532, 23 (1981); Miller et al., Nature 313, 68 (1985); Ahiguist et al., J. Mol. Biol. 172, 369 (1984); Ah~tquist et al., Eln Mol, fiol. 3, 37 (1984) Ou et a1~ PN 9 5235 (1982); Chu et Nuc. Acids Res. 14, 5591. (1986); European Patent Application Publn. No. (EPA) 194809; MaI.sh et al., Positive Strand RNA Viruses, p. 327-336, Alan IR.

Liss (publ.; New York) (1987; ',roceeding~s of UCLA isolation of a particular nucleic acid sequence. A WO 88/10315 PCT/US88/02108 14 Symposium, 1986); Miller et al., J. Mol. Biol. 187, 537 (1986) Stoflet et al., Science 239, 491 (1988); and Murakawa et al., DNA 7, 287 (1988).

Al,l of the aforecited publications are by this reference hereby incorporated by reference herein.

By the term "promoter" is meant a nucleic acid sequence (naturally occurring or synthetically produced or a product of restriction digest) that is specifically recognized by an RNA polymerase that binds to a recognized sequence and initiates the process of transcription whereby an RNA transcript is produced. It may optionally contain nucleotide bases extending beyond the actual recognition site, thought to impart additional stability toward degradation processes. 3n principle, any promoter sequence may be employed for which there is a known and available pdlymerase that is capable of recognizing the initiation sequence. Typical, known and useful promoters are those that are recogn.zed by certain bacteriophage polymerase such as bacteriophage T3, T7 or SP6. See Siebenlist et al., Cell 20, 269 (1980). These are but examples of those polymerase which can be employed in the practice of the present invention in conjunction with their associated promoter sequences.

The "RNA transcript" hereof is the ribonucleic acid sequence produced after transcription initiation following RNA polymerase recognition of the promoter sequence (See supra). The production of such transcripts is more or less continuous, dependent in part on the amount of polymerase present.

By the term "primer" in the present context is meant a nucleic acid sequence (naturally occurring or synthetically produced or a product of restriction digest) that has sufficient homology with the target sequence such that under suitable hybridization condition, it is capable of hybridizing, that is binding to, the target sequence.

A typical primer is at least about 10 nucleotides in S WO 88/10315 PCT/US88/02108 length, and most preferably is of approximately 35 or more nucleotide bases in length, and in its most preferred embodiments, it shares identity or very high homology with the target sequence. See, for example, EPA 128042 (publd.

12 Dec 84).

The term "operably linked" in particular in connection with the linkage of a promoter sequence within a primer sequence, refers to the functionality of the ultimat~ "double-stranded nucleic acid template" of the present invention such that it, the template, is capable of producing corresponding RNA transcripts when the promoter is recognized by the suitable polymerase--see supra.

The primer extension reaction to produce a duplex is known per se. See references supra. Polymerase useful for this purpose include E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, reverse transcriptase, and so forth.

The techniques of forming a detection signal such as via radioactive labeling or chromogenic means uing a chromogenic susceptible enzyme are also well known and documented in the art. See discussion supra.

The use of a "replicase" for (autocatalytic) induction of replication of the RNA transcripts of the present invention are generally known in the art.

Suitable examples of such replicases that are useful in the present invention include the so-called QB virus replicase that recognizes certain nucleic acid sequence sites at both the and ends of the given RNA transcript and the so-called brome mosaic virus (BMV) as well as the alpha virus replicases which are thought to recognize nucleic acid sequence sites at the end of a given RNA transcript. These replicases serve to replicate, that is reproduce, the RNA transcripts and complements so as to multiply copies thereof. When such enzyme is present in the reaction locus during the process WO 88/10315 PCT/US88/02108 16 of transcription, it can be foreseen that the multiple transcripts that are produced during transcription can themselves undergo replication so as to exponentially increase the amount of RNA transcript product.

Internal standardization is defined as a process which is used to: a) insure that the TAS amplification process has not failed because of a procedural error, and b) measure the levels of target nucleic acid relative to a predetermined quantity of nucleic acid which is always associated with the sample of interest. Such internal standardization occurs by coamplifying a portion of the target sequence, as well as an endogenous sequence, in the same reaction. For example, by knowing the cell count present within the biological sample, a single-copy gene ki-globin) could be used as an internal standard and, since it is not expressed in the form of RNA, its initial copy number is equal to two times the total number of cells in the sample. By coamplifying portions of the A-globin and target sequences of interest, the ratios of the amplified signals can be compared to quantitate the levels of target sequence of interest. Since every cell has two copies (in diploid cells) of this internal standard, irrespective of whether the biological sample contains separate target sequences (e.i4, HIV), each sample is expected to produce a positive amplification signal resulting from the internal standard. See Groudine et al., Nucleic Acids Research 12, 1427 (1984) and McKnight, Cell 31, 355 (1982). See also British Patent Application Phbln. No. 2187283 A (publd. 3 Sept. 87).

3. Detailed Description of Preferred Aspects In one of its aspects, the invention is a method for amplifying a target nucleic acid segment of Formula I 3 -(first subsegment) -(second subsegment) t i -i SWO 88/10315 PCT/US88/02108 17 (third subsegment)t- 5

I

wherein (first subsegment)t is a nucleic acid segment of known sequence of at least 10 nucleotides adjoining the 3'-terminus of (second subsegment)t (second subsegment)t is a nucleic acid segment of 0 or more nucleotides, and (third subsegment)t is a nucleic acid segment of known sequence of at least 10 nucleotides adjoining the of (second subsegment)t, which method comprises: hybridizing to (first subsegment)t of said target segment a first primer, which is a single-stranded DNA which comprises a 3'-terminal subsegment of Formula II 5'-(promoter) -(variable subsegment) 1 (3'-primer subsegment)l-3'

II

wherein (promoter) 1 is a single-stranded DNA segment with the sequence of the nlus-strand of a bacteriophage DNA-dependent RNA polymerase-specific promoter, (variable subsegment)l is a single-stranded DNA segment of 0 to 100 nucleotides adjoining the 3'-terminal nucleotide of (promoter) 1 and the 5'-terminal nucleotide of (3'-primer subsegment) 1 and (3'-primer subsegment)l is a single-stranded DNA segment of at least 10 nucleotiies with a sequence which is complementary to the sequence of a subsegment of (first subsegment)t which terminates with the 3'-terminal nucleotide of (first subsegment)t, said (promoter) 1 adjoining the 5'-terminus of said (3'-primer subsegment) 1 if said (variable subsegment) 1 has 0 nucleotides; extending said first primer, hybridized in accordance with step in a reaction catalyzed by a -Lii- i WO 88'j10315 PCT/US88/02108 18 first DNA polymerase to make a first complementary DNA segment, which comprises a subsegment of Formula III (promoter) 1-(variable subsegment) (first subsegment) tc-(second subsegment)tc- (third subsegment)tc-3',

III

wherein (first subsegment)tc is the DNA segment with the sequence complementary to that of (first subsegment)t, (second subsegment)tc is the DNA segment 'ith the sequence complementary to that of (second subsegment)t, and (third subsegment)tc is the DNA segment with the sequence complementary to that of (third subsegment)t, provided that, if the target segment is an RNA segment, said first DNA polymerase is a reverse transcriptaserendering single-stranded the duplex formed in the reaction of step hybridizing to (third subsegment)tc of said first complementary DNA of Formula II1 a second primer, which is a single-stranded DNA of at least !0 nucleotides of Formula IV 3'-(5'-primer subsegment) 2 -(variable

IV

wherein (5'-primer subsegment) 2 has the sequence of a subsegment of (third subsegment)t which terminates with the 5'-terminal nucleptide of (third subsegment)t and wherein (variable subsegment) 2 is a segment of 0 to 100 nucleotides which adjoins the 5'-terminus of (5 f -primer subsegment) 2; extending said second primer segment, hybridized in accordance with step in a reaction catalyzed by a second DNA polymerase to form a second >ysieem n) mIne novel eennjque 1r Lne presen IAzs invention results in rapid increase in copy number of a selected target segment by making use of two properties of DNA-dependent RNA polymerase: appreciable iitiation of transcription from only a small number ,of sequences 4 S WO 88/10315 PCT/US88/02108 19 complementary DNA segment which comprises a subsegment of Formula V subsegment)2-(third subsegment)t- (second subsegment)t-(first subsegment)t- (variable subsegment) c-(promoter)ic-3',

V

wherein (variable subsegment)1c is the DNA segment with the sequence complementary to that of (variable subsegment) and (promoter)l c is the DNA segment with the sequence complementary to that of (promoter), provided that said second DNA polymerase is the same as or different from said first DNA polymerase; and employing the double-stranded product of step as the template in a reaction catalyzed by a first bacteriophage DNA-dependent RNA polymerase, which recognizes the promoter of which one strand is (promoter) 1 to make a first RNA product of Formula VI subsegment)ir-(first subsegment)tcr- (second subsegment)tcr-(third subsegment)tcr- (variable subsegment)2cr- 3

VI

wherein (variable subsegment l r is the RNA segment with the sequence of (variable subsegment) 1 (first subsegment)tcr is the RNA segment with the sequence complementary to that of (first subsegment)t, (second subsegment)tcr is the RNA segment with the sequence complementary to that of (second subsegment)t, (third subsegment)tcr is the RNA segment with the sequence complementary to that of (third subsegment)t, and (variable subsegment)2cr is the RNA segment with the sequence complementary to that of (variable subsegment) 2 In another of its aspects, the invention entails a i WO 88/10315 PCT/US88/02108 method wherein (first subsegment)t is at least nucleotides in length and is of Formula XIII 3'-(first subsegment)t 2 -(first

XIII

wherein (first subsegment)t2 has 0 or more nucleotides and, if more than 0, adjoins the 3'-terminus of (first subsegment)tl, and (first subsegment)t, is at least nucleotides in length; wherein (third subsegment)t is of Formula XIV 3'-(third subsegment)tl-(third

XIV

wherein (third subsegment)t2 has 0 or more nucleotides and, if more than 0, adjoins the 5'-terminus of (third subsegmont)tl, and (third subsegment)tl is at least nucleotides in length; wherein (3'-primer subsegment) 1 has the sequence complementary to that of a subsegment of target segment which consists of all of (first sub-egment)t 2 and 0 or more nucleotides of (first subsegment)tl; wherein (5'-primer subsegment) 2 is a subsegment which consists of all of (third subsegment)t 2 and 0 or more nucleotides of (third subsegment)t 1 and wherein, after steps to Supra, the RNA subsegment of Formula VII subsegment) ticr-(second subsegment)tcr- (third subsegment)tlcr-3'

VII

wherein (first subsegment)tlcr is the RNA segment with the sequence complementary to that of (first subsegment)t 1 and (third subsegment)tlcr is the RNA segment with the sequence complementary to that of (third subsegment)tl, is further amplified by a method which comprises: WO 88/10315 PCT/US88/02108 21 hybridizing to said first RNA product of Formula VI a third primer, which is a single-stranded DNA which comprises a 3'-terminal subsegment of FormVla VIII 5'-(promoter)3-(variable subsegment)3- (3'-primer subsegment) 3

VIII

wherein (promoter) 3 is a single-stranded DNA segment with the sequence of the plus-strand of a bacteriophage DNA-dependent RNA polymerase-'!pecific promoter, said sequence of (promoter), being the same as or different from that of (promoter) 1 (variable subsegment) 3 is a single-stranded DNA segment of 0 to 100 nucleotides which adjoins the 3'-terminal nucleotide of (promoter) 3 and the nucleotide of (3'-primer subsegment) 3 and (3'-primer subsegment) 3 is a single-stranded DNA segment which has the same sequence as (third subsegment)tl and adjoins the 3'-terminal nucleotide of (promoter) 3 if (variable subsegment) 3 has 0 nucleotides; extending said third primer hybridized in accordance with step in a reaction catalyzed by a third DNA polymerase, which is a reverse transcriptase and is the same as or different from said first and second DNA polymerase, to make a third complementary DNA segment which comprises 3'-terminal subsegment of Formula IX 5'-(promoter) 3 -(variable subsegment)3- (third subsegment)tl-(second subsegment)t- (first subsegment)tl-(first suibsegment)t2- (variable subsegient) c-3

IX

wherein (variable subsegment)ic is the DNA with the sequence complementary to that of (variable subsagment)l; WO 88/10315 PCT/US88/02108 22 rendering single-stranded the duplex formed in thqt reaction of step hybridizing to the third complementary DNA made in the reaction of step a fourth pri.mer of FormulaX subsegment) 4 -(5'-primer subsegment) 4 wherein (variable subsegment) 4 is a segment of 0 to 100 nuclectides, and (5 1-p, imer subsegment) 4 is a subsegment of known~ sequence which adjoins the 3'-nucleotide of (variable subsegment) 4 if (variable subsegment) 4 has more tii~m 0 nucleotides, and which comprises at least nuclootides of the segment of Formula XX subsegment), 1 -(first subsegment) t 2 c- (first subseginent) tlc- 3

XX

wherein (first subsegment)t 2 c is the DNA segment with the oeguence complementary to that of (first subsegmeht)t 2 and (first subsegment)tlc is the DNA segment with the sequence complementary to that of (first subsegment)tl, provided, that at least one of said at least 10 nucJ.potides is at or from the 5e-terminal nucleotide of (first subsegment) tic; (11) extending said fourth primer hybridized in, accordance with step (10) in a reaction catalyzed by a fourth DNA polymerase, which is the same as or different from said' first, second and third DNA polymerase, to form a fourth complementaty DNA which comprises a segment of Formula XI r ~1 WO 88/10315 PCTIUS88/021 08 23 subsegment)tlc-(second segment)tc-(third subsegment)t1c-(variable subsegment)3c-(promoter) 3c-3',

XI

wherein (second subsegment)t c is the DNA segment with the sequence complementary to that of (second subsegment)t (third subsegment)tlc is the DNA segment with the sequence complementary to that of (third subsegment)tl, (variable subsegment)3 c is the DNA segment with the sequence complementary to that of (variable segment) 3 and (promoter) 3 c is the DNA segment with the sequence complementary to that of (promoter) 3 and (12) employing the double-stranded product of step (11) as the template in a reaction catalyzed by a second bacteriophage DNA-dependent RNA polymerase, which is the same as or different from said first bacteriophage DNA-dependent RNA polymerase and which recognizes the promoter of which one strand is (promoter)3 to make a second RNA product with a 5'-termina'l subsegment of Formula XII 5'-(variable subsegment)3r-(third subsegment) tlr-(second subsegment)tr- (first subsegment) tlr-X 12 -(variable subsegment)4cr-3',

XII

wherein (variablet subsegment)3r is the RNA segment with the sequence of (variable subsegment) 3 (third subsegment)tlr is thie RNA segment with the sequence of (third subsegment)tl, (second subsegment)t r is the RNA segment with the sequence of (second subsegment)t, (first subsegment)tlr is the RNA segment with the sequerce of (first subsegment)tl (variable subsegment) 4 cr is the RNA segment with the sequence complementary to that of (variable subsegment)4 and X12 is the RNA segment with the sequence complementary to that of the subsegment of subsegment) 4 that is 5' from the 5'-terminus of (first subsegment)tlc.

i WO 88/10315 PCT/US88/02108 24 As noted supra, the invention also entails kits for carrying out the amplification methods of the invention. In the case of the method of the invention which entails making a first RNA product, a kit of the invention would comprise the two primers, the corresponding bacteriophage DNA-dependent RNA polymerase, and the DNA polymerase. A kit for carrying out a method in accordance with the invention which entails making both a first and a second RNA product would comprise four suitable primers (two sets of two) with the corresponding necessary poly-erases.

Methods and kits according to tne invention for carrying out nucleic acid hybridization probe assays which entail amplifying a target segment in accordance with the invention entail, in addition to the st ps and components, respectively, of the methods and kits for amplification, steps and components necessary for detecting the RNA product resulting from amplification according to the invention. The skilled understand the various additional steps and components, respectively, that are required to detect RNA from an amplification process by any of the numerous nucleic acid probe hybridization assay methods known in the art. One preferred nucleic acid probe hybridization assay method, involving bead-capture of labeled RNA amplification, is illustrated in Example II below.

It is preferred that (3'-primer subsegment) 1 subsegment) 2 (3'-primer subsegment) 3 and subsegment) 4 have between 20 and nucleotide*, and more preferably about 30 nucleotides to enhance the specificity with which the various primers bind to the enda of the target segments which are sought to be amplified.

It is also preferred that the target segment of a nucleic acid of interest which is selected for amplification (by virtue of selection of (first

L

WOf 881103155 PCT/US88/02108 subsegment)t and (third subsegment)t selected so that (second subsegment)tr have at least 30, and more preferably at least about 50, nucleotides. 2his permits the use of (second subsegment)tcr or (second subsegment)t r in the amplified RNA product to be targets for nucleic acid probes that will not have sequences that overlap those of any of the primer subsegments.

While the bacteriophage DNA-dopendent RNA polymerase can use templates longer than 1000 bp, their efficiency of transcription decreases as the template becomes lqnger, Thus, target segments less than 1000 bases long are favored.

It is preferred to use the identical set of primers used in the synthesis of RNA, to produce RNA in a second cycle of TAS, As stated in the section describing the production of RNA, the primer pair should not overlap, In carrying out the method of the invention in which the production of RNA is desired, it is possible that the subsegment of target segment with.sequence complementary to that of (5'-primer subsegment) 4 be in the from and not overlap the subsegment of target segment with sequence complementary to that of (3'-primer subsegment)i. Similarly it is preferred that the subsegment of target segment with sequence the same as that of (3'-primer subsegment) 3 be in the 3'-direction from and not overlap the subsegment of target segment with sequence the same as that of (5'-primer subsegment) 2 This strategy may be preferred because it reduces the competition of the different primers for the same sites and, thereby, may markedly enhances the efficiency of amplification according to the invention. Where there is some overlap between sets of nested primers, it is preferable to remove any unused first tst of primers, before employing the second set.

The focus of the present invention is on bacteriophage DNA-dependent RNA polymerase because of j L WO 88/10315 PCT/US88/02108 26 their high specificity for certain promoters. Other polymerase which have similarly high specificity for particular promoters could be employed in accordance with the invention in place of the bacteriophage polymerase and the invention is intended to cover such other polymerase as well.

The preferred of the bacteriophage DNA-dependent RNA polymerase are thoea from T7, T3 and SP6. Preferred promoters for use with these polymerase are described in the examples and claims, but numerous other promoters for these polymerase are known in the art and can be employed as well.

Further, polymerase from bacteriophages other than the preferred three, and promoters recognized by such other polymerase, can be employed in accordance with the invention.

It is preferred to employ for DNA polymerase in the methods of the invention reverse transcriptases and DNA polymerase which lack 5' to 3' exonuclease activity. It is most preferred that a single DNA polymerase be used for all steps ii, the methods. Most preferred of the DNA polymerase are the AMV reverse transcriptase and recombinant MMLV reverse transcriptase. Other polymerase are acceptable however, such as the heat-stable DNA polymerase from Thermus aquaticus (see Chien et al. J.

Bacteriol. 127, 1550 (1976)), SequenaseTM brand recombinant T7 DNA polymerase from the U. S. Biochemicals Corp., Cleveland, Ohio, and the well known Klenow Fragment of E. coli ONA polymerase I, and calf thymus DNA polymerase alpha.

The "variable subsegments" that are optionally included in the various primers serve three functions. In fact, they might be more aptly termed "multipurpose subsegments." Firstj for the first and third primers, which include promoters, the promoter subsegments preferably include transcription initiation sequences that WO 88/10315 PCT/US88/02108 27 are preferred by the RNA polymerase corresponding to the promoter. Second, for all of the primers, a variable subsegment can optionally contain a particular segment whereby RNA product from the amplification can be detected in a nucleic acid probe hybridization assay.

Indeed, amplification (and assay) can occur for several different target segments simultaneously by using sets of primers that differ in their recognition segments (3'-primer subsegment) 1 but include a common variable subsegment. The variable subsegment can also contain a polylinker sequence that conveniently contains a plurality of restriction sites for ease of cloning subsequently.

Finally, the variable subsegments can be used to incorporate into the RNA product from amplification sequences necessary to allow the RNA product to be (autocatalytically) replicated by a bacteriophage RNA-specific RNA polymerase, such as the Qp replicase, see Miele et al., supra, and BMV sequences from the RNA-3 chromosome of the plant virus that promotes the synthesis of coat mRNA. See Ahlquist et al., J. Mol. Biol. 172, 369 (1984); Ahlquist et al. Plant Mol. Biol. 3, 37 (1984); Ahlquist et al., Mol. Biol. 153, 23 (1981); Miller, et al Nature 313, 68 (1985); Miller et al., Viroloqy 125, 236 (1983); and Ou et al., PNAS 79, 5235 (1982).

In the case of the Qp replicase, which is known in the art, this is preferably carried out by including in (variable subsegment) 2 the sequence 5'-CGCGCTCTCCCAGGTGACGCCTCGAGAAGAGGCGCGACCTTCGTGC-3'and in (variable subsegment)l the sequence 5'-TGGGGAACCCCCCTTCGGGGGTCACCTCGCGCAGC-3', and then (autocatalytically) replicating the first RNA product RNA. I in Figure 1; see also Figure or including in (variable subsegment) 4 the sequence 5'-CGCGCTCTCCCAGGTGACGCCTCGAGAAGAGGCGCGACCTTCGTGC-3' and in (variable subsegment) 3 the seauence %GC-3' and then that has sufficient homology with the target sequence such that under suitable hybridization conditions it is capable of hybridizing, that is binding to, the target sequence.

A typical primer is at least about 10 nucleotides in WO 88/10315 PCT/US88/02108 28 (autocatalytically) replicating the second RNA product RNA II in Figure 1; see also Figure 4).

In the case of BMV replicase use, also known, this is preferably carried out by including the following sequence for BMV in variable sequence 2 (bases 1-25) (in the specific use of the HIV example infra): BMV promoter TCS 87-29 (HIV-specific) 20 30 5 -AAGATCTATGTCCTAATTCAGCGTA ACAGCATATGTATGTTCAGGGA AAGCTA-3' (54 bases), as a core sequence, additionally AU rich sequence 5'-thereof may be used to enhance the replicase activity from BMV. This oligo is to be used as the second primer. The first primer used with this second primer is a T7-based, HIV-specific primer.

T7 promoter TCS 87-34 +1S 20 30 40 TAATACGACTCACTATA GGGA CACCTAGGGCTAACTATGTGTCCTAATAAGG (52 bases).

Additional, non-limiting details to aid in understanding the invention are provided in the examples that follow: 0 SWO 88/10315 PCT/US88/02108 29 4. Examples EXAMPLE I of blood from a patient suspected of being infected with a human immunodeficiency virus-type 1 (HIV-1) is fractionated with a SepracellTM apparatus (Sepratech Corp., Oklahoma City, Oklahoma, or, alternatively, with a Ficoll gradient, to isolate lymphocytes. The lymphocytes are then lysed and nucleic acid from them is isolated with an extractor apparatus (Molecular Biosystems, Inc., San Diego, California, or, alternatively, the lymphocytes are lysed by standard sodium dodecyl sulfate (SDS)-enzyme treatment and tho nucleic acid isolated with reverse phase chromatography over DEAE cellulose. This is done as follows: Spin down cells: 5k rpm for 4' from 1 ml Tris-buffered saline (TBS), pH Draw off supernatant.

Resuspend pellet in: 500 pl 0.3 M NaCl/20 mM Tris, pH 7.5 Mix well, 100 pl 2% SDS then add 200 pl 5 mg/ml proteinase K to 200 pl 0.25 M EDTA pellet.

Vortex vigorously and incubate at 50 0 C for 45', vortexing for 10-15 seconds every Load onto drained extractor column and allow to enter.

Wash column ix 4 ml 0.3 M NaCl/20 mM Tris, pH Elute DNA/RNA with 4 ml 0.5 M NaCl/20 mM Tris pH In one of its aspects, the invention is a method for amplifying a target nucleic acid segment of Formula I 3'-(first subsegment)t-(second subsegment)t- WO 88/10315 PCT/US88/02108 To eluate add: 5 pl glycogen 400 pl 3 M NaOAc ml ice-cold EtOH mix well Precipitate at -20 0 C overnight. A dry ice/ethanol bath for one hour may be used.

Spin down rNA/RNA for 15' at 10,000 rpm in a swinging bucket rotor; pour off EtOH and drain dry on a kimwipe 2'.

Lyophilize for Resuspend pellet in 170 1 TE.

Incubate at 37 L for Pour a 1 ml spin column as follows: Plug a 1 ml syringe with a small amount of glass wool (autoclaved).

Fill syringe with TE (tris-EDTA) treated with diethyl pyrocarbonate (DEPC).

Quickly add sephadex G-50 fine (in TE; autoclaved).

Contii.ie to add until the solid matrix mounds over the top of the syringe.

Spin 30 seconds at 1000 rpm in an IEC table-top centrifuge.

Wash with 100 pl TE, spin. for 1' at medium speed (ca. 1000 rpm). Discard wash.

Load the extract onto the column. Spin again for 1'.

Rinse with 150 p1 TE, spin at '1000 rpm for Pool rinse and the first fraction. Samples may be split for multiple reactions at this step.

Add 1/10 volume 8 M LiC1. Mix with 2.5 x vol. 100% EtOH.

Vortex well. ppt. dry ice/EtOH for Spin d'.wn for 15' top speed in a microfuge at Dry pellet.

After the isolation, total nucleic acid from the sample is dissolved ii 100l of TE buffer (10mM Tris.Cl, 1mM EDTA, pH8). 1041 of the 100pl of total nucleic acid is dissolved to a final volume of 1004i containing: Tris.Cl, pHR NaCl j i i I nucleotides; extending said first primer, hybridized in accordance with step in a reaction catalyzed by a SWO 88/10315 PCT/US88/02108 31 MgC12 dithiolthreitol (DTT) 2mM spermidine 100g/ml bovine serum albumin 400mM each of dATP, dCTP, dGTP and TTP of the 101 base single-stranded DNA of sequence 5'-GAACGCGGCT ACAATTAATA CATAACCTTA TGTATCATAC ACATACGATT TAGGTGACAC TATA GAATAC TTTCGTAACA CTAGGCAAAG GTGGCTTTAT C primer A, is added. 30nM of the 29 base DNA of sequence 5'-ACACCATATG TATGTTTCAG GGAAAGCTA-3', primer B, is added. Primer B, which is the second primer, has the sequence of bases 5151-5179 in the SOR gene of the HIV-1. An alternative segment for the second primer, corresponding to bases 5357-5387 of the SOR gene of the HIV-1, has the sequence 5'-GCACACAAGT AGACCCTGAA CTAGCAGACC and, if used, is also present at 30 nM.

Primer A is the first primer; its 64 5'-nucleotides form the plus-strand of a promoter for the SP6 DNA-dependent RNA polymerase. Its segment of sequence 5'-GAATAC-3' is (alternative subsegment) 1 which includes the transcription start site (the and other bases apparently favored by the SP6 RNA polymerase at the of sequences transcribed by it. Finally, the 31 3'-terminal nucleotides form (primer subsegment) 1 and have a sequence complementary to that of bases 5577-5547 in the SOR gene of an HIV-1 isolate (See Ratner et al., Nature 313, 277 (1985) for complete sequence.) (The HIV isolate is referred to in these Examples as "the HIV-1")).

Note that all oligonucleotides employed in these examples'are made by solid-phase synthesis on an Applied Biosystems, Inc. (Foster City, California, U.S.A.) automated synthesizer (Model No. 380A) and are purified chromatographically essentially to homogeneity by HPLC using a C8 reverse phase column. Alternatively, other commercially available synthesizers and standard i i WO 88/10315 PCT/US88/02108 32 purification procedures could be used to prepare the oligonucleotides.

The 1001p of solution is heated at 65 0 C for 2 minutes and is then cooled to 420C over the course of 1 minute- This heating and then holding at 420C or above, in combination with the composition of the solution, provides conditions of stringency sufficient to provide hybridization of (3'-primer subsequent) 1 with sufficient stability to prime DNA synthesis, of high specificity to the sequence complementary to that of (3'-primer subsequent) 1 Then 10 units of avian myoblastosis virus (AMV) reverse transcriptase or 500 units of recombinant Moloney murine leukemia virus (MMLV) reverse transcriptase, as purchased from Life Sciences, Inc., St. Petersburg, Florida, are added to the solution and the solution is incubated for 10 minutes at 42 0 C. (One unit incorporates Inmole of TTP into acid-precipitable form in minutes at 37 0 C using poly(A).oligo(T) 1 2-18 as template-p:rimer as defined by Houts et al., J. Virol. 29, 517 '1979)). After the 10 minute incubation, the solution is placed in a boiling water bath for 1 minute. This heating causes strand-separation of the duplex formed by the reverse transcriptase.

Then the solution is iolled to 420C over 1 minute.

During this cooling, the second primer hybridizes to the 3'-terminal segment of the complement of target segment formed in the reaction catalyzed by the reverse transcriptase. Again, the hybridization conditions are sufficiently stringent for sufficiently specific hybridization between the second primer and sequence complementary to that of second primer.

After the cooling, 10 additional units of AMV reverse transcriptase or 500 additional units of cloned MMLV reverse transcriptase are added and further incubation for 10 minutes at 42 0 C is carried out.

WO 88/10315 PCT/US88/02108 33 Then RNasinR brand ribonuclease inhibitor from Promega Biotec, Madison, Wisconsin, U.S.A. is added (optionally) to a concentration of 1 unit per ml. (1 unit is the amount of inhibitor required to inhibit by 50% the activity of 5ng of ribonuclease A. Roth, Meth. Cancer Res. 3, 151 (1976)). Further, each of the ribonucleoside ATP, GTP, CTP and UTP, is added to 400mM. Finally between 10 and 20 units of SP6 DNA-dependent RNA polymerase, purchased from Promega Biotec, are added and the resulting solution is incubated at 42 0 C for 30 to 60 minutes. (1 unit is the amount of the RNA polymerase required to catalyze the incorporation of 1 nmole of ribonucleoside triphosphate into acid insoluble product in 60 minutes at 37 0 C under standard reaction conditions (40mM Tris.Cl, pH 7.9, 6mM MgC12, DTT, 2mM spermidine, 0.5 mM of each of ATP, GTP, CTP and UTP, 0.5Ci of 3 H-CTP, lg of SP6 DNA and the enzyme in a total volume of Then each of the following oligonucleotides is added to bring its concentration to third primer: ACAATTAATA CATAACCTTA TGTATCATAC ACATACGATT TAGGTGACAC TATA GAATAC ACTAATTCAT CTGTATTACT TTGACTGTTT TTC-3' fourth primer: TTATTAATGC TGCTAGTGCC-3' In the third primer, as in the first, the 64 bases are the promoter segment and the next 6 bases are the variable segment. The variable segment is the first six bases transcribed from the promoter by the SP6 RNA polymerase, and these bases are selected to enhance the level of such transcription. Finally, the (3'-primer subsegment) 3 portion of the third primer is the 3'-terminal 33 bases, in the same sequence as bases 5388-5420 in the short open reading frame (SOR) gene of the HIV-1. The fourth primer has the sequence WO 88/10315 PCT/US88/02108 34 complementary to that of bases 5546-5517 of the SOR gene of the HIV-1.

After addition of the third and fourth primers, the solution is incubated at 42 0 C for 1 minute. During this period, hybridization occurs between (3'-primer subsegment) 3 of third primer and the first RNA formed in the reaction catalyzed by the SP6 RNA polymerase. Because of the stringency of the hybridization conditions, the (3'-primer subsegment) 3 hybridizes, with stability sufficient for priming of DNA synthesis, with high specificity to the segment of complementary sequence in the first RNA.

After the incubation, 10 units of AMV reverse transcriptase or 500 units of cloned MMLV reverse transcriptase are added and the solution incubated for minutes at 42 0 C to form 'the third complementary DNA.

The solution is then suspended in a boiling water bath for 1 minute and cooled to 42 0 C over 1 minute to, first, render single-stranCed the duplex between third complementary DNA and first RNA and, second, allow hybridization between fourth primer and third complementary DNA. As with the other hybridizations, the conditions are sufficiently stringent that hybridization of fourth primer, with stability sufficient to prime DNA synthesis, occurs with high specificity to the segment of third complementary DNA of sequence complementary to that of fourth primer.

Then, again, 10 units of AMV reverse transcriptase or 500 units of cloned MMLV reverse transcriptase are added and the solution is incubated for 10 minutes at 42 0

C.

Then RNasinR brand ribonuclease inhibitor is added (optionally) to 1 unit per ml, followed by 10-20 units of SP6 RNA polymerase, and the solution is incubated for minutes to 1 hour at 42 0

C.

WO 88/10315 PCT/US88/02108 The resulting second RNA can then be detected by a nucleic acid probe hybridization technique.

EXAMPLE II The procedure of Example I is followed, with a modification noted below, with three samples: one of of human blood known to be free of HIV, one of of culture known to have about 103 HIV-l infected cells per ml, and one of 10ml of blood from a person suspected of being infected with an HIV-1. The modification of the procedure is that an alpha- 32 P-labeled ribonucleoside triphosphate is included as a substrate in the reaction catalyzed by the SP6 RN. pol'lmerase to make the second RNA. The second RNA is, consequently, 32 P-labeled.

Sephacryl-S500TM macroporous beads are derivatized with a carboxyl-group-terminated linker (of formula-C(=NH)NH(CH 2 5 C0 2 and then with 5'-(6-aminohexyl phosphoramidate)-derivatized oligonucleotide of sequence 5'-TGGTCTGCTA GTTCAGGGTC TACTTGTGTG C-3' which is the sequence complementary to that of bases 5357-5387 in the SOR gene of the HIV-1.

(The sequence of bases 4901-4932 of the SOR gene occurs in any second RNA produced during amplification of nucleic acid from a sample.) Preparation of the beads was according to procedures described in commonly assigned United States Patent App4i'-tion Serial No. 895,756, filed August 11, 1986, and incorporated herein by reference.

Briefly, the support materials are porous silicate glass or macroporous cross-linked dextran, derivatized with an amino-terminated linker, with substantially all of the amino groups that are not covalently joined through a phosphoramidate group to the terminal nucleoside of the capture probe being blocked from interactLng nonspecifically with nucleic acid having an aliphatic acyl WvO ,88/10315 PCT/US88/02108 36 group; macroporous cross-linked dext.:an activated with cyanogen bromide, which reacts with amines to link to aminoalkylphosphoramidate derivatized oligonucleotides; and porous silicate glass, macroporous cross-linked dextran or divinylbenzene-crosslinked polystyrene derivatized with a carboxyl-terminated or succinimideester-terminated linker, with a fraction of the carboxyl groups joined in an amide linkage to one amino group of a diaminoalkane, the other amino of which is part of a phosphoramidate which, in turn, is bonded directly to the terminal nucleoside of the capture probe. Preferred support materials are long chain alkylamine-,derivatized and carboxyl-derivatized controlled pore glass as sold by Pierce Chemical Co., Rockford, Illinois, USA, under product numbers 24875 and 23735, respectively, in the form of beads with nominal pore diameter of 500 Angstroms and particle diameters between about 125 and 177 microns, and Sephacryl S-500, sold by Pharmacia Inc., Piscataway, New Jersey, USA under Code No. 17-0475-01 in the form of beads with a wet diameter of about 40 to about 105 microns and an exclusion volume for dextrans of molecular weight above about 2x10 7 daltons, said Sephacryl to be derivatized with cyanogen bromide or with an amino-terminated or carboxylterminated linker. In one aspect the solid support is one of: porous silicate glass derivatized at silicons with groups of formula

-(CH

2 )n(NH)(CO)(CH 2 )cCH3 and

-(CH

2 )n(NH)(PO 2 )0-(Oligo), with substantially no silicon derivatized with a group terminated with an amino group, wherein c is 0 to 5, n is 2 to 8, -0-(Oligo) is the oligonucleotide probe, and the oxygen atom bonded to (Oligo) is the oligonucleotide probe, and the oxygen atom bonded to (Oligo) is the 'WO88/10315 PCT/US8,1/021o8 37 oxygen of the 5'-nucleoside or the 3'-oxygen of the 3'nucleoside of the probe; or groups of formula

(CH

2 n(NH) (CO) (Cl 2 mCO,4 H and -CH2) n(NH) (CO) (CH2) m(CO) (NH) (CH2) pNH (P0 2 0-(01igo), wherein m, n, and p are the same or different and are each 2 to 8; or a cross-linked dextran macroporous material derivatized at hydroxyl o~ygens, which are on carbons of the sugar moieties which have a least one neighboring carbon with an underivatized hydroxyl, with groups of formula

-C(NH)NH(CH

2 )q(NH) (CO) (CH 2 )cCH 3 and NH (CH 2 p(NH) (P0 2 )0O-Oligo) 1 with substantially no hydroxyl oxygen derivatized with a group terminated with an Amino group, or groug5s of formI2a

-C(=NH)NH(CH

2 )qCO 2 H and

-C(=NH)NH(CH

2 )q(CO) (NH) (CH 2 )pNfl(PO 2 0 'go), wherein c, q, and p are the same or different and q is 2 to 10; and divinylbenzene-crossl inked polystyrene derivatized at phenyl groups with groups of formula -(CH2)sCQ 2 H and

-CH

2 s(CO) (NH) (CH 2 pNH (P02)0- (Qligo), wherein s and p are the same or different and s is 2 to See Chosh etgl, Nucleic Acids Research 5353 (19871" Each of three batches of 50rmg of Sephacry). beads, derivatized as deiscribed above, is soaked for 4L5 minutes at 37 0 C C in 250pd of a prehiybridization solution containing 0.1% SDS, 10% dextran sulfate, 1mg/mi salmon sperm DNA, and 5X SSPt (0,75M4 NaClo 5mM NaH{ 2 PQ4o pH 7.4, and 5m.M EDTA) After the soaking, the bead moaterial is pelleted by centrifugation and prehybridization soltttion is removed by aspiration.

1L.ii WO 88/10315 PCT/US88/02108 38 The nucleic acid from the amplification procedure on each of the three samples, s ,solated by ethanol precipitation and is then dissolved to 250pl in solution that is the same as pre-hybridization solution but lacks the salmon sperm DNA, Each of the resulting nucleic acid solutions is then combined with one batch of prehybridized oligonucleotide-d;rivatized Sephacryl beads and the resulting mixture is incubated with gentle agitation at 42 0 C C for 90 minutes.

The Sephacryl beads are then pelleted by centri^ugation, hybridization solution is removed by aspiration, and the beads are then washed three times, minutes each at 370C C, in lml of a solution of 2 x SSC (0,,30M NaCl, 0.03M Na.Citrate, pH After each wash, the beads are pelleted by centrifugation and solution is removed by aspiration.

Immediately after the third wash, the beads are subjected to Cerenkov counting.

The beads with amplified nucleic acid from sample A produce a low level of counts, barely above background counts from scintillation fluid alone. The beads with amplified nucleic acid from sample B produce counts at a much higher level than that observed with beads associated with sample A. The beads with amplified nucleic acid from sample C, if they produce a level of counts significantly above tha level produced with beads associated with sample A, indicate that the person, whose blood was taken for sample C, is infected with HIV-1.

EXAMPLE III The procedurel of Examples I and XI are carried out with T7 RNA polymerase (Promega Biotec) in place of SP6 RNA polymerase, with each of the subsegment subsegment) 1 of first primer and the subsegment 5'-(promoter) 3 -(variable WO 88/10315 PCT/US88/02108 39 subsegment) 3 of third primer having the sequence 5'-TAATACGM?7' CACTATA GGGA-3', wherein the 17 bases are the promoter subsegment and the 4 31-bases are the variable subsegment. The variable segment corresponds to the 4 5'-terminal bases of the transcript from the promoter. RiJbonuclease inhibitor is not used in any step in this proces-,, at least with the polymerase preparation from P-:omega Biotec.

EXAM4PLE IV The procedures of Examples I and II are carried out with T3 RNA polymerase (from Stratagene, San Diego, California) in place, of the SPE RNA polymerase and with the following segment used as the (promoter)I- (variable subsagment), subsegment of first primer and the (promoter) 3 -(variable subsegmant) subsegmnent of thir-d primer: 5 -TATTAAqCCT CACTAAA GGGA-'3', where the 17 5'-term2,,;nal bases are the promoter segment and the 4 31-termin~al bases thp variable segment, including the 4 bases in transcripts from, the promoter, EXAM~PLE V Employin~g the T'7 RNA polymerase as in Example III, target segment in the genome of HIV from human blood samples is amplified using the following primers in place of the primers specified in Example I:* First Primer: 5'-TAATACGACT CACTATA GGGA TCTAATTACT ACCTCTTCTT CTGCTAGACT-3 t wherein the 31-terminal bases are complementary in sequence to bases 7076-7047 of the ENV gene of HIV-l.

Second Primer: 5'-ACAAGTTGTA ACACCTCAGT WCi. 88/10315 PCT/US88/02108 CATTACACAG-3' with the sequence of bases 6838-6866 in the ENV gene of HIV-1.

Third Primer: Same 21 5'-terminal bases as first primer of this Example adjoined to the following 27 3'-terminal bases: 5'-AAAGGTATCC TTTGAGCCAA TTCCCATA-3', which have the sequence of bases 6875-6902 in the ENV gene of the HIV-1.

Fourth Primer: 5'-AGTTGATACTACTGGCCTAATT-3', with the sequence complementary to that of bases 7033-7007 in the ENV gene of HIV-1.

EXAMPLE VI Employing the T7 RNA polymerase as in Example III, target segment in the genome of HIV from human blood samples is amplified using the following primers in place of the primers specified in Example I: First Primer: Same 21 5'-terminal nucleotides as thi first and third primers of Example V adjoined to the following 31 3'-terminal nucleotides: TAACTATGTG TCCTAATAAG whiCh have the sequence complementary to that of bases 5471-5441 of the SOR gene of HIV-1.

Second Primer: 5'ACACCATATG TATGTTTCAG GGAAAGCTA-3', which has the same sequence as bases 5151-5179 of the SOR gene of HIV-1.

Third Primer: Same 21 5'-terminal nucleotides as the first and third primers of Example V adjoined to the following 31 3'-terminal nucleotides: 5'-AAGAATAAGTTCAGAAGTACACATCCCACT-3', which have the same sequence as bases 5220-5249 of the SOR gene of the HIV.

1i. Lf ±'rlgure i; see a.iso rigure i) or including in (variable subsegment) 4 the sequence -CGCGCTCTCCCAGGTGACGCCTCGAGAAGAGGCGCGACCTTCGTGC-3' and in (variable subsegment) 3 the spmuence -TGGGGAACCCCCCTTCGGGGGTCACCTCL.c i GC-V', and then WO *W88/10315 PCT/US88/02108 41 Fourth Primer: 5' -TGGTCTGCTAGTTCAGGGTCTACTTGTGTCwhich has the sequence complementary to that of bases 5387-5357 in the SOR gene of HIV-1.

EXAMPLE VII The: procedure of Example I is followed for the isolation of nucleic acid from: 1) one~ sample containilbg 103 Hly-infected CEM cells mixed with lQ6 un,41nfected GEM cells (Cancer Center Research Foundation;' CCRI'CEM; ATCC No. CCL 119) and 2) one sample containing 106 uninfected GEM cells. These samples are resuspended in 100 Ail 10 inN Tris-HCl, pH 7.4f 1 mM EDTiA (TE) after the rethanol precipitation step to concentrate the gample obtained from the ExtractorTM column The resuspended samples are fractionate~d on a Sephadex G-50 fine spin column (Maniatis supra) eluting wjlx TE, The eluted samples are concentrated by ethanol precipitation (in 0.8 M LiCl, 3 volumes ethanol, in dry ice/ethanol bath for 15 minutes).

The precipitated sample is pelleted by centrifugation.

The pellet .s drained, dried, and then resuspended in 100 Al2 containing 40 mM Tris-HCl, pH 8.1, 8 MM McgC 2 25 MMN NaCl, 2 mM spermidine, 5 mM dithlothreitol, 100 /AM each dATP, dGTP, dCTP, and dTTP, 1 mM each rATP, rCTP, rGTP, and rUTP, 100 /pg/ml BSA (nuclease-free) and 250 nM each of DNA oligonucleotide pr-imer A (5'1 -AATTTAATACGACTCACTATAGGGACACCTAGGGCTAACTATG TCCTAA- TA.AGG-3) and primer B (5'1 -ACACCATATGTATGTTTCAGGGGAAAGCTA- As controls, duplicate samples of purified HIV RNA at a concentration of 0.01 fm were resuspended in 100 Al of the buffer described above. Finally, a 10-fm g-globin sequence contained in plasmid HA1l9A tWaVace et al., Nuci.

Acids Res. 3647 (191.) which had linearized by HindlII, was resuspendedin 100 Al of the buffer described WO 88/10315 PCT/US88/02108 42 above, except without the oligonucleotide primers.

Oligonucleotide primer D and primer C (5'-TAATACGACTCACTATAGGGACAAAGGACTCAAA-3'), which are specific for p-globin sequence, were added to the p-globin reaction at 250 nM each.

Except for the p-globin sample, the reactions are heated to 65 0 C for The p-globin sample is boiled for All samples are cooled to 42 0 C for 1 minute; then, units of AMV reverse transcriptase are added. The reactions are incubated for 15 minutes at 42 0 C, boiled for 1 minute, and then cooled at 42 0 C for 1 minute. Ten additional units of AMV reverse transcriptase are added, incubating at 42 0 C for 15 minutes. One hundred units of T7 RNA polymerase are added to the reactions, and -he reactionis are incubated at 37 0 C for 30 minutes.

The samples are boiled for 1 minute and cooled at 42 0 C for 1 minute, followed by the addition of 10 units AMV reverse transcriptase. The reactions are incubated at 42 0 C for 15 minutes, boiled for 1 minute and cooled to 42 0 C for 1 minute. An additional 10 units of reverse transcriptase are added, followed by incubation at 42°C for 15 minutes. One hundred units of T7 RNA polymerase are added, with a 30-minute incubation at 37 0 C. This cycle is repeated two additional times.

The amplified target is then detected using OligoBeadsTM as described below.

Sephacryl beads containing HIV-l-specific oligonucleotides were prepared as described and stored in TE at 4°C at a concentration such that 250pl of the suspension contains 50mg of Sephacryl beads.

Oligonucleotides were synthesized and HPLC purified as described.

Th; oligonucleotidaa employed for both attachment to the beads and detection are homologous to the SOR region of the HIV-1 genome. The oligonucleotides used in 6 WO 88/10315 PCT/US88/02108 43 these studies were 86-31 (detection oligo nucleotide) GCACACAAGTAGACCCTGAACTAGCAGACCA-3') and 87-83 (capture oligo nucleotide on the bead: 5'-ATGCTAGATTGGTAATAACAACATATT-3'), which are homologous to the nonsense strand of the SOR region, and separated by approximately 100 nucleotides. For detection, the oligonucleotides were end labeled with 3 2 -P according to a standard protocol. The unincorporated label was removed by gel filtration on a Sephadex G-50 fine column, and the oligonucleotide was stored at -20 0

C.

In a typical bead based sandwich hybridization system (BBSHS) experiment, the target and 3 2 P-labeled detection oligonucleotide are denatured in 10pl of TE containing 0.2% SDS at 65 0 C for 5 minutes in an Eppendorf tube. To this, 10il of 2X Solution Hybridization Mix Dextran Sulfate) are added, The solution is mixed, centrifuged 2 seconds, and incubated at 42 0 C for 1 hour.

During this time the Sephacryl beads are prehybridized. The stock suspension of beads is mixed well, and 2501l aliquots (50 mg of beads) are transferred to Eppendorf tubes with mixing between each removal to ensure a uniform suspension. The aliquots are centrifuged for seconds, and the TE is removed with a drawn-out Pasteur pipet. After 250pl of Hybridization Solution (5X SSPE [0.9M NaCI, 50 mM NaH 2

PO

4 pH 7.4, ImM EDTA] 10% Dextran Sulfate/0.1% SDS) is added, the beads are suspended by gentle shaking and incubated 37 0 C for 30-60 minutes with occasional mixing. Immediately prior to the capture step, the beads a,:e centrifuged for 10 seconds, the prehybridization solution is removed, 80il of Hybridization Solution at 37 0 C are added, and the beads are returned to 37 0

C.

The solution hybridization is centrifuged for 2 seconds and transferred to the beads and Hybridization Solution. The beads are suspended and incubated at 370C WO 88/10315 PCT/US88/02108 44 for 1 hour with frequent mixing to maintain the suspension.

Following the capture, the beads are centrifuged seconds and the Hybridization Solution, containing uncapture target and detection oligonucleotide, is transferred to a scintillation counter vial. The beads are then washed 5 times with 2X SSC at 37 0 C. The first 3 washes are rapid; 1 ml of wash is added, the beads are mixed well and centrifuged 10 seconds, and the wash is transferred to a counting vial. vor the final 2 vashes, 1 ml of wash is added and the beads are mixed and incubated at 37 0 C for 5 minutes before being centrifuged. Each wash is counted separately to monitor the procedure.

Cerenkov counts of the Hybridization Solution, washes, and beads are measured for 5-10 minutes. Counter background is subtracted from all samples. The fm target detected is calculated as follows: (CPM on Beads/Total CPM) X fm Oligonucleotide Where total CPM is the sum of the CPM for the Hybridization Solution, 5 washes, and beads.

DETECTION OF AMPLIFIED TARGETS BY BBSHS TARGET p1 USED (FM) DETECTED 15

MOLES

103 HIV infected cells; 0.33 0.06 106 CEM uninfected cells 0.66 0.13 106 uninfected CEM cell 0.07 0.01 0.14 0.015 0.7 0.01 0.008 0.01 fm HIV RNA 0.007 0.14 0.014 0.29 fm p-globin DNA 10 0.014 WO 88/10315 PCT/US88/02108 EXAMPLE VIII The procedure of Example I is followed for isolation of nucleic acids from two samples: a) 103 HIVinfected CEM cells with 10 6 uninfected CEM cells; and b) 106 uninfected cultured CEM cells to which 0.01 fmoles of purified HIV are added after extraction. The procedure is then modified by further purification through a Sephadex (fine) spin column (Maniatis supra) eluting with TE after the Extractor column step. This is followed by an ethanol precipitation of the nucleic acid (in 0.8 M Licl and 2.5 volume of EtOH). The nucleic acid is then resuspended in 100 1p containing: mM Tris-HCl, pH 8.1 8 mM MgC12 mM NaCI 2 mM spermidine mM DTT (dithiothreitol) 100 pM dATP, dTTP, dCTP, dGTP (each) 1 mM r;TP, rCTP, rGTP, rUTP 100 pg/ml BSA nuclease-free 250 nM 29 bp DNA oligonucleotide with the sequence 5'ACACCATATGTATGTTTCAGGGAAAGCTA-3' (Primer B) 250 nM 56 bp DNA oligonucleotide with the sequence

AATTTAATACGACTCACTATAGGGACACCTAGGGCTAACTATGTGTCCTAA

TAAGG-3') (Primer A) The 100 pl of solution are heated to 65 0 c for 1 minute and cooled to 420C over the course of 1 minute.

This heating and then holding at 42 0 C or above, in combination with the composition of the solution, provides conditions of stringency sufficient for hybridization of (3'-primer subsegment) 1 (see Figure 1) with sufficient stability to the sequence complementary to that of (3'-primer subsegment)I, in thet target HIV RNA with high specificity.

j WO 88/10315 PCT/US88/02108 46 Then, 10 units of avian myoblastosis virus (AMV) reverse transcriptase (Life Sciences, Inc.) are added to the solution, and the solution is incubated for 10 minutes at 42 0 C. [One unit incorporates 1 nmole of TTP into acidprecipitable form in 10 minutes at 37 0 C using poly(A)oligo (T)12-18 as template-primer, as defined by Houts et al. J. Virol. 29, 517, (1979)]. After a 10 minute incubation, the solution is placed in a boiling water bath for one minute. This heating causes strand separation of the duplex formed by reverse transcriptase and inactivation of the reverse transcriptase.

Then, the solution is cooled to 42 0 C over one minute. During this cooling, the second primer, primer B, hybridizes to the 3'-terminal end (third subsegment)t 2 c, see Figure 1, of the target complement of DNA strand formed in the reaction catalyzed by the reverse transcriptase in the previous step. Again, the hybridization conditions are sufficiently stringent for specific hybridization between the second primer and the sequence complementary to that of the second primer.

After cooling, 10 additional units of AMV reverse transcriptase are added and further incubation is carried out for 10 minutes at 42 0

C.

One hundred units of T7 polyrerase are added to the reaction. The reaction is then incubated at 37 0 C for minutes. An RNA transcript is synthesized which is complementary to the original target HIV RNA starting from the T7 promoter present at the 5' end of the duplex DNA template newly synthesized by the reverse transcriptase. The transcript has the sequence then continues with sequence which is complementary to the second subsegment of the target sequence second subsegmenttcr, see Figure Since the reverse transcriptase has not been inactivated prior to this step, and since primer B is present, as well as the deoxynucleotide triphosphates, a secondary synthesis SWO 8/110315 PCT/US88/02108 47 occurs at this stage. Primer B hybridizes to the 3' region of the newly synthesized RNA transcript (third subsegment)t2cr, see Figure 1, which is complementary to B primer. The reverse transcriptase which had been added at the previous step) synthesizes a DNA strand using the newly synthesized RNA transcript (made by T7 polymerase) as a template, and using oligonucleotide B as the primer at the 5' end.

The reaction is then boiled for one minute to denature the RNA:DNA duplex. The reaction is then cooled to 42 0 C over one minute. During this cooling step, the target complementary segment of primer A hybridizes to the DNA strand synthesized during the previous step. At the same time, primer B hybridizes to its complementary sequence on the RNA transcript synthesized in the previous step.

After cooling, 10 additional units of AMV reverse transcriptase are added, and further incubation for minutes at 42 0 C is carried out. Reverse transcriptase synthesizes DNA complementary to the HIV target using oligonucleotide A as a primer at the 5' end and DNA made in the previous step as a template. A second DNA strand is synthesized using oligonucleotide B as a primer and the RNA transcript made in the previous step as a template.

The reaction is boiled for one minute to denature the DNA duplex and the RNA:DNA duplex. Ine reaction is cooled to 42 0 C over one minute. Primer A hybridizes to the cDNA made using the RNA transcript as template in the previous step. (If the 3' end of the template strand is used as a reverse transcriptase primer also, a product identical to the end product of the next reaction is produced.) Primer B hybridizes to the DNA strand which is complementary to the target HIV RNA. This second synthesis produces a product which is a duplex DNA containing a T7 promoter sequence at its 5' end, as well as a 4 bp "variable" segment, 5'-GCGA-3'.

II11111 i ii WO 88/10315 PCT/US88/02108 48 One hundred units of T7 polymerase are added to the reaction. The reaction is incubated for 30 minutes at 37 0 C. The T7 RNA polymerase uses the double-stranded duplex DNA containing a duplex T7 promoter as a template to transcribe an RNA which is complementary to the target second subsegment containing the additional sequence GGGA-3' at its 5' end.

This cycle (boil 1' at 42 0 C, RT 10' at 42 0 C, RT 42 0 C, T7 polymerase 37 0 C 30') can be repeated as many times as needed to achieve the desired amplification of target sequence. The resulting product can then be detected by a nucleic acid probe hybridization technique.

EXAMPLE IX Purified HIV RNA at a concentration of 1 fmole was resuspended in: mM Tris-HC1, pH 8.1 8 mM MgC12 mM NaCl 2 mM spermidine 5 mM dithiothreitol 100 pM dATP, dTTP, dCTP, dGTP (each) 1 mM each rATP, rCTP, rGTP, rUTP 100 pg/ml BSA nuclease-free 250 nM 29 bp DNA oligonucleotide with the sequence 5'-ACACCATATGTATGTTTCAGGGAAAGCTA-3' (primer B) 250 nM 56 bp DNA oligonucleotide with the sequence TGTGTCCTAATAAGG-3' (primer A) The 100 pl of solution are heated to 650C for 1 minute and cooled to 42 0 C over the course of 1 minute.

This heating and cooling step, in combination with the composition of the solution, provides conditions of stringency sufficient for the hybridization of primer A with sufficient stability to the sequence complementary to WO 88/10315 PCT/US88/02108 49 that of the primer A region in the target HIV RNA ((first subsegment)t2, in Figure 1] with high specificity.

Then, 10 units of avian myoblastosis virus (AMV) reverse transcriptase (Life Science, Inc.) are added to the solution, and the solution is incubated for 10 minutes at 42 0 C. After a ten-minute incubation, the solution is placed in a boiling water bath for one minute. This heating causes strand separation of the duplex formed by reverse transcriptase and inactivation of the reverse transcriptase.

The solution is cooled to 42 0 C over one minute.

During this cooling, the second primer B hybridizes to the 3' end of the newly synthesized target-complementary strand [or (third subsegment)t2c in Figure Again, the hybridization conditions are sufficiently stringent for specific hybridization between the second primer and the sequence complementary to that of the second primer, After cooling, 10 additional units of AMV reverse transcriptase are added and further incubation is carried out for ten minutes at 42 0

C.

One hundred units of T7 RNA polymerase (New England Biolabs) are added to the reaction. The reaction is then incubated at 37 0 C for 30 minutes. An RNA transcript is synthesized which is complementary to the original target HIV RNA starting from the T7 promoter present at the end of the duplex DNA template synthesized by the reverse transcriptase. The RNA transcript has the sequshue GGGA, then continues with the sequence which is complementary to the second subsegment of the target sequence second subsegmenttcr, Figure Since the reverse transcriptase has not been inactivated prior to this step, and since primer B is present as well as the deoxynucleotide triphosphates, a secondary synthesis occurs at this stage. Primer B hybridizes to the 3' region of the newly synthesized RNA transcript [(third subsegment)t2cr, see Figure 11 which is complementary to WO 88/10315 PCT/US88/02108 primer B. The reverse transcript:se (which had been added at the previous step) synthesizes a DNA strand using the newly synthesized RNA transcript (made by the T7 RNA polymerase) as a template, and oligonucleotide B as a primex at the 5' end.

The reaction is then boiled for one minute to denature the RNA:DNA duplex. The reaction is then cooled to 42°C over one minute. During this cooling step, the target-complementary segment of primer A hybridizes to the DNA strand synthesized in the previous step. At the same time, primer B hybridizes to its complementary sequence on the RNA transcript synthesized in the previous step.

After cooling, 10 units of AMV reverse transcriptase are added, and further incubation for minutes at 42°C is carried out. Reverse transcriptase synthesizes DNA complementary to the HIV target using oligonucleotide A as a primer at the 5' end and DNA made in the previous step as a template. The. 3' end of the template strand is used as a primer to produce a final duplex DNA with a double-stranded, T7 promoter site at its end. A second DNA strand is synthesized using oligonucleotide B as a primer and the RNA transcript made in the previous step as a template.

One hundred units of T7 polymerase are addedl to the reaction. The reaction is incubated for 30 minutem at 37°C. The T7 RNA polymerase synthesizes an RNA transcript using the duplex DNA containing the polymerase binding site (T7 promoter) at its 5' end as a template. The RNA transcript is complementary to the target second subsegment, see Figure 1, containing the additional sequence 5'-GGGA-3' at its 5' end. Further cycles may be performed to increase the amplification. The resulting products can be detected by a nucleic acid hybridization protocol.

I i WO 88/10315 W088/0315PCT/US88/02108 51 EXAMPLE X Protocol for Last-Round Labeling of a TAS Amplified Product A. column to remove unincorporated cold nucleotides 1. The TAS reaction (100 ul) is taken after the cDNA synthesis in the final cycle of TAS. Add 400 p1 of R~eagent A (0.1 M Tris-HCl, pH 7.71 10 m' triethylamnine,1 mM~ disodium EDTA) to the reaction, 2, Equilibrate a NENSORB 2 0 TM (Dupont*) prepac column by wet,'ting the. column maeilin 100?% methanol (HPLC grade), then equilibrating with 2 ml R~eagent A.

3. Toad sample on column.

4. wash the column 3 ttmes with 1 ml Reagent A.

Wash the coluimn 3 times with I ml water.

6. Elute the nucleiO, acids with- 50%4 methanol, collecting 250-300 pl. fraction~s up to 1 il total volume.

(The first two fractions will contain. the majority of the nucleic acid.) 7. Dry the fractions in the speed-vac or lyophilizer.

IB Protocol to Label, trne Amplified Product 1. Resusriend the fractions from the NENSORB 2 0TM column (the first two fractions may be combin-,4) in 40 mM Tris-HCl, p8 8.1, 8 MM MgC1 2 1 25 inN NaCl, 2 inN spermidine- (tICl)3 5 mM dithiothr.eitol, 400 pM each rATP, rCTP, and rGTP, 12 pM rUTP, and 25 pCi a-32p--rUTP (800 Ci/inmol) 2. Add 50 units T7 RNA polymerase (New England Biolabs) for each 50 paI, of sample. incubate at 37 0 C for minutes.

3. The Unincorporated label may be removpd by a spin column (Maniatis gjrra), 4. The labeled sample may be run on a sequencing polyacrylamide 9e1 to determine the size of the amplified WO88/10315 PCT/US88/02108 52 product. The labeled product may also be detected using the Oligo-BeadsTM.

EXAMPLE XI Use of an Internal Standard During TAS Amplification Samples were prepared having: 1 0.1 fm HIV RNA and 0.1 fm p-globin DNA sequences (Hpl9A cleaved with PstI); 2) 0.01 fm "IV RNA and 0.1 fm P-globin DNA (Hp19A cleaved with PstI); 3) 0.1 fn HIV RNA; or 4) 0.1 fm fglobin DNA (Hpl9A cleaved with PstI in 100 pl containing 40 mM Tris-HCl, pH 8.1, 8 mM MgC12, 25 mM NaCI, 2 mM spermi6ine-(H1Cl) 3 5 mM dithiothreitol, 100 pl each dATP, dTTP, dCTP and dGTP, 1 mM each rATP, rUTP, rCTP, and rGTP, 100 pg/mnl BSA (nuclease-free), and 250 nM primer A

AATTTAATACGACTCACTATAGGGACACCTAGGGCTAACTATGTGTCCTAATAAGG-

250 nM primer B -ACACCATATGTATGTTTCAGGGAAAGCTA-3'), 250 nM primer C -TAATACGACTCACTATAGGGAACTAAAGGACCGAGACTTTTTGCC-3), and 250 nM primer D (5'-ACATTGCTTCTGACACAACTGTGTTCA-3').

A sample having 1/20th the starting nucleic acids was placed into denaturing solution 7.4% formaldehyde, 10x SSQ M NaCl, 0.15 M Na citrate, pH 7,4) for the zero time-point samples.

The samples were boiled for 1 minute and cooled at 42QC for 1 minute, followed by the addition of 10 units of ANV reverse transcriptase. The reactions are incubated at 42 0 C for 15 minutes, boiled for 1 minute, and cooled at 42dC for 1 minute. An additional 10 units of AMV reverse transcriptase are added, followed by incubation at 429C for 15 minutes. One hundred units of T7 RNA polymerase are added, followed by incubation at 37 0 C for 30 minutes.

This cycle is repeated a second time. (More cycles can be done, depending on the amplification neded.) Two samples containing 1/20th the starting target nucleic acids were placed into denaturing solution for the second transcription tia-point samples.

WO 8810315 PCT/US88/2108 53 All samples were heated to 55 0 C for 30 minutes prior to filtering onto two nitrocellulose membranes. The nucleic acids were fixed to the membrane by irradiation with 254 nm UV light for 4 minutes. As controls, 1, 0.1, and 0.01 fm of plasmid pARV7/2 [Luciw et al., Nature 312, 760 (1984)), which contains the entire HIV genome cDNA as well as plasmid Hp19A [Wallace et al., Nucl. Acids Res. 9 3647 (1981)], were denatured in 0.2 N NaOH, neutralized with an equal volume of 2 M ammonium acetate, and then filtered onto the nitrocellulose membrane. One membrane was hybridized with 3 2 p-labeled oligonucleotide 86-311, which is specific for the amplified product of HIV. The other membrane was hybridiLed to 32 p-labeled oligonucleotide 87-4592, which will hybridize to the amplified p-globin product, well as to the target pglobin plasmid.

The hybridizations were in 1- SDS, 6.9 M NaCl, nM NaH 2

PO

4 (pH 5 mM EDTA (pH and 106 cpm/m.

3 2 p-labeled oligonucleotide for 1 hour at 55 0 C. The membranes were washed 3 times i)i 1% SDS, 0.18 M N4Cl, mMA 2aH 2 Po 4 (pH and 1 mM EDTA at room temperature, followed by 1 wash in the same luffer at 5o5 0

C,

The membranes were then kutoradiographed for 16 hours on Nodak XAR film at -70 0 C with one intensifying screen.

Notes 1. 86-31; 5'-GCACACAAGTAGACCCTGAACTAGCAGACCA-3' 2. 87-459: 5' -AGGTTTAAGGAGACCA)ATAGAAACT-3' The autoradiograph in Figure 3 shows the amount of HIV and -globin sequences detected aO:r two cycles of '2AS carried out simultaneously on HTV and p-globin nucleic acid. The starting amount of f-globin was kept constant while thv amount of HIV target was varied.

WO 88/10315 PCT/US88/02108 While the invention has been described with some specificity in the present specification, persons of ordinary skill in the pertinent arts will recognize variations and modifications of the invention as described that are within the spirit of the invention. Such variations and modifications are also within the scope of the invention as described and claimed herein.

_1

Claims (20)

1. A method of preparing an RNA containing a sequence corresponding to a target sequence, said method comprising: providing a first nucleic acid primer, containing a promoter sequence operably linked to a sequence corresponding to a first segment of the target sequence, said first segment including the 3'-end of the target sequence, and a second nucleic acid primer, which is hybridizable to a sequence complementary to that of a second segment of the target sequence, said second segment including the 5'-end of the target sequence; hybridizing under suitable conditions said first nucleic acid primer with target sequence in a sample containing nucleic acid; extending said hybridized first nucleic acid primer in a polymerase extension reaction complementarily to the target sequence to form a corresponding, first duplex nucleic acid; 15 separating the strands of said first duplex; hybridizing under suitable conditions to the resulting, separated promoter-sequence-containing strand said second nucleic acid primer; extending said hybridized second nucleic acid primer in a polymerase extension reaction complementarily to said promoter-sequence- S" containing strand to form a second duplex nucleic acid; and G) prior to separatn the strands of said second duplex, employing said second duplex as a template for the preparation of a plurality of RNA transcripts therefrom in a reaction catalyzed by an RNA 25 polymerase that recognizes the promoter corresponding to the promoter sequence of the first primer, each of said transcripts bearing an RNA sequence corresponding to said target sequence. 2, The method according to Claim 1 comprising additionally: hybridizing under suitable conditions, to RNA transcripts prepared from the second duplex, the second nucleic acid primer; extending said hybridized second nucleic acid primer in a polymerase extension reaction, catalyzed by an RNA-dependent DNA polymerase, complementarily to said RNA transcripts to form a third duplex nucleic acid; separating the strands of said third duplex; prenybridization solution is removea, oup Hybridization Solution at 37 0 C are added, and the beads are returned to 37°C. The solution hybridization is centrifuged for 2 seconds and transferred to the beads and Hybridization Solution. The beads are suspended and incubated at 37 0 C 56 hybridizing under suitable conditions with first primer second-primer-containing strands from separation of the strands of third duplex; extending both first primer and second-primer-containing strands, in the hybrides of first primer with second-primer-containing strands from separation of the strands of third duplex, in polymerase extension reactions to form a fourth duplex nucleic acid; prior to separating the strands of said fourth duplex, employing said fourth duplex as a template for the preparation of a plurality of RNA transcripts therefrom, in a reaction catalyzed by an RNA polymerase that recognizes the promoter correLponding to the promoter sequence of the first primer, each of said transcripts bearing an RNA sequence corresponding to said target seauence; and optionally, repeating steps of this claim at least 15 once, employing, in step each time steps are carried out, hybridization of second primer to RNA transcript prepared in step of the immediately preceding time steps are carried out.

3. A method useful for the detection of at least one specific nucleic acid target sequence in a sample containing nucleic acid comprising preparing, according to Claim 1 or Claim 2, RNA transcripts, which contain a sequence corresponding to said target sequence, and detecting the presence of said RNA sequence.

4. A method according to Claim 1 or Claim 2 wherein, in the first primer, the sequence corresponding to a first segment of the target 25 sequence is complementary to said first segment; the ,econd primer has a sequence identical to that of a second segment of the target sequence; said first and second segments of the target sequence do not overlap; and all polymerase extension reactions are catalyzed by the same reverse transcriptase.

5. A method according to Claim 4 further comprising detecting RNA transcripts prepared in accordance with Claim 4.

6. The method according to Claim 3 or Claim 5 wherein said transcripts contain replicase recognition site for replication of said transcripts by replicase Induction.

7. The method according to any one of Claims 3, 5 or 6 wherein the detected RNA sequence of said RNA transcripts are measured in a standardized manner sc as to measure the amount of target sequence A I 385u i i lli 57 contained in a sample of nucleic acid used in preparing the double-stranded nucleic acid template.

8. The method according to Claim 7 wherein the detected RNA sequence of said RMA tr'a:scripts is measured in a manner internally standardized with the presence of a known copy number of nucleic acid also contained in said sample.

9. The method according to Claim 3 wherein said target sequence is disposed within a nucleic acid sequence associated with the characteristics of a genetic or pathogenic disease or condition.

10. The method according to Claim 9 wherein said nucleic acid sequence is a segment of a human immnodeficiency virus.

11. A method according to Claim 10 wherein said nucleic acid sequence is a segment of a defective gene.

12. A method according to Claim 5 wherein the promoter 15 corresponding to the promoter sequence of the first primer is a bacteriophage T7 promoter and the RNA transcripts are produced using T7 RNA polymerase.

13. A method according to Claim 5 wherein the promoter corresponding to the promoter sequence of the first primer is a bacteriophage SP6 promoter and the RNA transcripts are produced using SP6 RNA polymerase.

14. A method according to Claim 1 wherein the target sequence is a DNA sequence and the polymerase extension reaction is catalyzed by E. coli DNA polymerase I. 25 15. A method according to Claim 1 whereir the target sequence is a DNA sequence and the polymerase extension reaction is catalyzed by Klenow Fragment of E. coli DNA polymerase I.

16. A method according to Claim 1 wherein the target sequence is a DNA sequence and the polymerase extension reaction is catalyzed by T4 DNA polymerase.

17. A method according to Claim 1 or Claim 2 wherein all polymerase extension reactions are catalyzed by a reverse transcrlptase.

18. A met'Ilod according to Claim 3 or Claim 5 wherein said RNA transcripts are labelled prior to detection.

19. A method according to Claim 18 wherein said RNA transcripts are radio-labelled. 58 A method according to Claim 18 wherein said RNA transcripts are chromophore labelled.

21. A method for amplifying a target nucleic acid segment of Formula I 3'-(first subsegment)t-(second subsegment)t-(thlrd I wherein (first subsegment) t is a nucleic acid segment of known sequence of at least 10 nucleotides adjoining the 3'-terminus of (second subsegment) t (zecond subsegment) t is a nucleic acid segment of 0 or more nucleotides, and (third subsegment)t is a nucleic acid segment of known sequence of at least 10 nucleotides adjoining the 5'-terminus of (second subsegment) t which method comprises: 15 hybridizing to (first subsegment) t of said target segment a first primer, which is a single-stranded DNA which comprises a 3'-terminal subsegment of Formula II e .i -(variable subsegment) 1 -(3'-pri mer subsegment) 1 -3' a 21 A mehdfrapiyn are uecadsgeto Formia 59 II wherein (prbmoter) 1 is a s ngle-stranded DNA segment with the sequence of the plus-strand of a bacteriophage DNA- dependent RNA polymerase-specific promoter, (variable subsegment), is a single-stranded DNA segment of a 0 to 100 nucleotides adjoining the 3'-terminal nucleotide of (promoter), and the 5'-terminal nucleotdide of (3'-primer subsegment) 1 and (3'-primer subsegment), is a single- stranded DNA segment of at least 10 nucleotides with a sequence which is complementary to the sequence of a subsegment of (first subsegment)t which terminates with the 3'-terminal nucleotide of (first subsegment)t extending said first primer, hybridized in accordance with step in reaction catalyzed by a first DNA polymerase to make a first complementary DNA segment, which comprises a subsegment of Formula III 5'-(promoter) -(variable subsegment) 1 'first subsegment) t- (second subsegment)t c (third subsegmenc)) III wherein (first subsegment)t, is the DNA segment with the sequence complementary to that of (first subsegment) (second subsegment) t is the DNA segment with the sequence complementary to that of (second subs,.. .ment)t, and (third subserment)t, is the DNA segment with e a sequence co n, *,Ientary to that of (third subsegment)t provided that, if the target segment is an RNA segment, said first DNA polymerase is a reverse transcriptase; SU _STITUTTE SH4 SWO 88/10315 PCT/US88/02108 rendering single-stranded the duplex formed in the reaction of step hybridizing to (third subsegment)tc of said first complementary DNA of Formula III a second primer, which is a single-stranded DNA of at least 10 nucleotides of Formula IV subsegment)2-(variable IV wherein (5'-primer subsegment) 2 has the sequence of a subsegment of (third subsegment)t which terminates with the 5'-terminal nucleotide of (third subsegment)t and wherein (variable subsegment) 2 is a segment of 0 to 100 nucleotides which adjoins the 5'-terminus of subsegment) 2; extending said second pri-aer segment, hybridized in accordance with step in a reaction catalyzed by a second DNA polymerase to form a second complementary DNA segment which comprises a subsegment of Formula V 5'-(variable subsegment) 2 -(third subsegment)t- (second subsegment)t-(first subsegment)t- (variable subsegment)lc-(promoter) c- 3 V wherein (Variable subsegmerlt) 1 c is the DNA segment with the sequence complementary to that of (variable subsegment) 1 and (promoter)ic is the DNA segment'with the sequence complementary to that of (promoter)l provided that said second DNA polymerase is the same as or different from Said first DNA polymerase; and employing the double-stranded product of step as the template in a reaction catalyzed by a first WO 88/10315 W088/0315PCT/US88/02108 61 bacteriophage DNA-dependent RNA polymerase, which recognizes the promoter of which one strand is (promoter- to make a first RNA product of Formula VI subsegment) ir-(first subsegment)tcr- (second subseginent)tcr.-(third subsegment)tcr- (variable subsegment) 2cr- 3 VI wherein (variable subsegment)lr is the RNA segment with the sequence of (variable subsegment) 1 (first subseg- ment)tcr is the RNA segment with the sequence complemeni- tary to that of (first subsegment)t, (second subseg- ient)tcr is the RNA segment with the sequence compleman- tary to that of (second subsegment)t, (third subsegient)tcr is the RNA segment with the sequence complementary to that of (third subsegment)t, and (variable subsegment) 2 cr is the RNA segment with the sequence complementary to that of (variable subsegment)2 2*L A method according to Claim 2Qwherein (first subsegment)t is at least 10 nucleotides in length and is of Formula XIII 3'-(first sUbsegment)t 2 -(first XIII wherein (first subsegment)t 2 has 0 or more nucleotides and, if more than 0, adjoins the 3'-terminus of (first subsegment)t 1 and (first subsegment)t, is at least nucleotides in length; wherein (third subsegment)t is of Formula XIV subsegment)tl-.(third subsegment) t2- 5 1, XIV wherein (third subsegnent)t 2 has 0 or more nucleotidez and, if mnore than 0, adjoins the 5'-terminus of,(thi~rd ,LA, subsegment)tl and (third subsegment)t 1 is atleast, 7,C-, i nucleotides in length; wherein (3 '-primer subsegment) 1 has the sequence complementary to that of a subsegment of target segment which consists of all (first subsegment)t, and nucleotides of (first subsegment)tl; wherein (51-primer subsegment), is a subsegment which consists of all of (third subsegment) and nucleotides of (third subsegment)t 1 wherein each of (variable subsegment) 1 and (variable subsegment), has 0 nucleotides; and wherein, after steps to of Claim 22, the RNA product of Formula VII subsegment)ir-(first subsegment)t 2 cr-(first subsegment) tic (second subsegment) tc- (third subsegment) tp-3 Orri VII wherein (first subsegment) O1cr is the RNA segment with the s3equence complementary to that of (first subsegment) and (third subsegment) 1 c, is the RNA segment with the sequence complementary to that of (third subsegment)t 1 is further amplified by a method which comprises: hybridizing ti said first RNA product of Formula VII a third primer, %hich is a single-stranded DNA which comprises a 30terminal subsegment of Formula VIII (promoter) (variable subsegment) (3 'primer subsegment) 3 -3,1 Vill wherein (prtomoter) 3 is a singe-stranded DNA segment with the sequence of the plus-strand of a bacteriophage DNA- dependent RNA polymerase-specific promoter, said sequence of (promoter) 3 being the same as or different from that of (promoter) 1' (variable subsegment) 3 is a single-stranded DNA segment of 0 to 100 nucleotides which adjoins the 3'-terminal nucleotide of (promoter) 3 and the 5'-termihal nucle tide T*T* WO 88/10315 PCT/US88/02108 63 of (3'-primer subsegment) 3 and (3'-primer subsegment) 3 is a single-stranded DNA segment which has the same sequence as (third subsegment)t. and adjoins the 3'-terminal nucleotide of (promoter)3 if (variable subsegment)3 has 0 nucleotides; extending said third primer hybridized in accordance with step in a reaction catalyzed by a third DNA polymerase, which i9 a reverse transcriptase and is the same as or different from said first and second DNA pqlymirae, to make a third complementary DNA segment which Comprises a 3'-terminal subsegment of Formula IX 5'-(promoter)3-(variable subsegment)3- (third subsegment)tl- (fecond subsegment)t- (first subsegment)t1-(firit subsegment)t2- (variable subsegment) c- 3 1 IX wherein (variable subsegment) 1' the DNA with the sequence complementary to that of (variable subsegment) 1; rendering single-stranded the duplex formed in the reaction of step (8) hybridizing to the third complementary DNA made in the reaction of step a fourth primer of Formula X subsecment) 4 -(5'1-primer subsegment) 4 3' wherein (variable subsegment) 4 is a segment of 0 to 100 nucleotides, and (5'-primer subsegment) 4 is a subsegment of kolown sequence which adjoins the 3-nucleotide of (variable subsegment) 4 if (variable subsegment) 4 has more WO 88/10315 PCT/US88/02108 64 than 0 nucleotides, and which comprises at least nucleotides of the segment of Formula XX

51-(variable subsegment)l-(first subsegment)t2c- (first subsegment)tl 1 XX wherein (first subsegment)t2c is the DNA segment with the sequence complementary to that of (first subsegment)t2 and (first subsegment)tl 1 is the DNA segment with the sequence complementary to that of (first subsegment)t 1 provided that at least one of said at least nucleotides is at or from the 5'-terminal nucleotide of (first subsegment)t1c; (11) extending said fourth primer hybridized in ascordance with step (10) in a reaction catalyzed by a fourth DNA polymerase, which is the same as or different from said first, second and third DNA polymerase, to form a fourlth complementary DNA which comprisez a segment of Formula XI subsegment tlc- (second segment)tc- (third subsegment)tc-(variable subsegment) 3 c- (promoter)3c-3Vi XI wherein (second subsegment)tc is the DNA segment with the sequence complementary to that of (second subsegment)t, (third subsegment)tlc is the DNA segment with the sequence complementary *o that of (third subsegment)tl, (variable subsegment) 3 e is the DNA segment with the sequence complementary to that of (variable segment) 3 and (\romoter) 3 c is the DNA segment with the sequence complementary to that of (promoter) 3 and r L WO 88/10315 PCT/US88/02108 (12) employing the double-stranded product of step (11) as the template in a reaction catalyzed by a second bacteriophage DNA-dependent RNA polymerase, which is the same as or different from said first be teriophage DNA-dependent RNA polymerase ad which recognizes the promoter of which one strand is (promoter) 3 to make a second RNA product with a 5'-terminal subsegment of Formula XII subsegment)3r- (third subsegment)tlr- (second subsegment)tr-(first subsegment)tlr- X 1 2 -(variable subsegment)4cr- 3 XII wherein (variable subsegment)3r is the RNA segment with the sequence of (variable subsegment) 3 (third subsegment)tlr is the R1PA segment with the sequence of (third subsegment)tl, (second subsegment)t r is the RNA segment with the sequence of (second subsegment)t (first subsegment)tlr is the RNA segment with the sequence of (first subsegment)t 1 'variable subsegment)4cr is the RNA segment with the sequence complementary to that of (variable subsegment) 4 and X 12 is the 1RNA segment with the sequence complementary to that of the subsegment of (5'-primei subsegment) 4 that is 5' from the 5'-terminus of (first subsegment)t 1 c. ,3 2-4. A method according to Claim 2-2-wherein each of (variable subsegment) 1 and (variable subsegment) 2 has 0 to nucleotides, each of (3'-primer subsegment) 1 and (5'-primer subsegment) 2 has 15 to 45 nucleotides, (second subsegment)t has at least 30 nucleotides, and target segment has 1000 or fewer nucleotides. Lt A method according to Claim wherein the target segment is a DNA segment, wherein the product of step(2) is rendered single-strar.ded by thermal WO 88/10315 PCT/US88/0210 8 66 denaturation, wherein (variable subsegjment) 2 has 0 nucleotides, wherein each of the first and s.Iecond DNA polymerase is selected from the group consisting of Klenow Fragment of E. coil DNA polymerase I, AXy reverse transcriptase, cloned MvflL\ reverse transcriptase, calf thymus DNA polymerase alpha, Thermus aquaticus heat-stable DNA polymerase, and SequenaseTM brand cloned T7 DNA polymerase, and wherein the bacteriophage DNA-dependent RNA polymerase is selected from the group consisting of T7 RN'. polymerase, T3 RNA polymerase and SP6 RNA polymerase. 5 A method according to Claim L4 wherein each of the first and second DNA polymerase is selected from the group consisting of Klenow Fragment of E. coli DNA polymerase I, AMV reverse transcriptase, and cloned MMLV reverse transcriptase. a-1 S2-v-. A method according to Claim 24\wherein, the bacteriophage DNA-dependent RNTA polymerase is T7 RNA polymerase, (promoter),1 is 5 '-TAATACGACTCACTATA-31 and (variable subsegmont) 1 has a dinucleotide of sequence 5'-GG-31 at its 5'-terminus; or the bacteriophage DNA-dependent RNA polymerase is T3 RNA polymerase, (promoter),1 is 5 '-TATTAACCCTCACTAAA-3' and (variable su. 1 segment) 1 has a tetranucl.eotide of sequence 5'-GGGA-3'at its 5'-terminus7 or the bacteriophage DNA-dependent RNA polymerase SP6 RNA polymerase, (promoter) 1 is 5' -GAACGCGGCTACAATTAATACATAAC- CTTATGTATCATACACATACGATTTAGGTGACACTATA-3', and (variable subsegment) 1 has a hexanucleotide of sequence 51-GAATAC-3' at its A method according to Claim %2\4wherein the of the first primer is the 5'-nucleotide of (promoter) A method according to Claim wherein, i~f the bacteriophage DNA-dependent RNTA polymerase is the T7 or T3 IINA polymerase, (variable subsegment) 1 has the sequence IV -GGGATGGGGAACCCCCCTTCGGGGGTCACCTCGCGCAGC-3' and, if WO088/10315 PCT/US88/02 108 67 bacteriophage DNA-dependent RNA polyinerase is the SP6 RNA polymerase, (variable subsegment) 1 has the sequence -GAATACTGGGGAA.CCCCCCTTCGGGGGTCACCTCGCGCAGC-3'. A method according to Claim 2*Xwherein the target segment is a RNA segment, wherein the product of step(2) is rendered single-stranded by thermal denatura- tion, wherein (variable subsegment) 2 has 0 nucleotides, wherein the first DNA polymerase is selected from the group consisting of AMY reverse transcriptase and cloned MMhLV reverse tzanscriptase, wherein the second DNA polymerase is selected from the group consisting of Klenow Fragment of E. coli L'NA polymerase I, AMV reverse transcriptase, cloned MMLV reverse transcriptase, calf thymus DNA polymerase alpha, Thermus aquaticus heat-stable DNA polymerase, and SequenaseTM brand cloned T7 DNA polymerase, and wherein the bacteriophage DNA-dependent RNA polymerase is selected from th1 group consisting of T7 RNA polymerase, T3 RNA polyinerase and SP6 RNA polymerase. A method according to claim 3-\wherein the second DNA polymerase is selected from the group consisting of Klenow Fragment of Ei coli DNA polymerase I, AI.!V reverse transcriptase, and cloned MMLV rev'erse transcriptase. n -346, A method according to Claim -3-1wherein the bacteriophage DNA-dependent RNA polymerase Is T7 RNA polymerase, (promoter) 1 is 5 '-TAATACGACTCACT,TA-3' and (variable subsegment) 1 has a dinucleotide of sequence 5f-GG-3' at its 5'-terminus; or the bacteriophage DNA -dependent RNA polymerase is T3 RN~A polymerase, (promoter) 1 is 5'-TATTAACCCTCACTAAA-3' and (variable silbsegment)la has a tt,,tranucleotide of sequence 5'-GGGA-3'at its 51-terminus; or the bacteriophage DNA-dependent RNA polymerase is SPE R.NA polymerase, (promoter) 1 is U-4A CTTATGTATCATACACATACGATTTAGGTGACACTATA-3', and (varabje, 7rV.,E I WO 88/10315 PCT/US88/02108 68 subsegment) 1 has a hexanucleotide of sequence 5'v4AATAC-3' at its 5'-teirmintis. a i5-913 A method according to Claim 3-\whe:'-ein the of the first primer is the 5'-nucleotide of (promoter),. S3-4-. A method according to Claim-%.\hri each of the first and second DNA polymerase is selected from AMV reverse transcriptase and cloned MI4LV reverse transcriptase. A method according to Claim 34)\herein, if the bacteriophage DNA-dependent RNA polyinerase is the T7 or T3 RNA polymerase, (variable subsegment) 1 has the sequence -GGGATGGGGAACCCCCCTTCGGGGGTCACC ,TCGCGCAGC-3' and, if the bac,:teriophage DNA-dependent TINA polymerase is the SP6 RNA polymerase, (variable subsegs ent) 1 has the sequence -GAATACTQ SGGAACCCCCCTTCG4 GGGTCACCTCGCLCAGC-3'.

56..A method according Swherein the target segment is a segment of the genome of a human immunodeficiency virus that is an HIV- virus and wherein (3'-primer subsegment) 1 has tha sequence 5' -TCTAATTACTACCTCTTCTTCTGCTAGACT-3' and primer CCTCTTCTTCTGCTAGACT-31 and (5'-primer subsegment) 2 has the sequence 5'-ACAAGTTGTZkACACCTCAGTCATTACACAG-3' or (3'-primer subsegment) 1 has the sequence 5'-TTTCGTAACACTAGGCAAAGGTGGCTTTATC-3' and -pvimer subsegment) 2 has a sequence selected from the group consisting of 5 '-GCACACAAGTAGACCCTGAACTAGCAGACCA-3' I and 5' -ACACCATATGTATGTTTCAGGGAA.AGCTA-3'. method according to claim B-3.)wherein each of (variable subsegment) 1 and (variable subsegment) 3 has 0 to nualectides, wherein each of (30-primer subsegment) 1 (5'1'primer subsegment) 2' (3V-primer subsegment) 3, and, subsegment) 4 has 15 to 45 nucleotides, (second subsegiment)t has at least 30 nucleotides, and target segment has 1000 or fewer niucleotides. SWO 88/10315 PCT/US88/02108 69 S1 3-89 A method according to Claim -3+wherein if the target segment is a DNA segment, the product of step is rendered single-stranded by thermal denaturation and the product of step is rendered single-stranded by thermal denaturation; each of the )irst, second and fourth DNA polymerase is selected from the group consisting of Klenow Fragment of E. coli DNA polymerase I, AMV reverse transcriptase, cloned MMLV reverse transcriptase, calf thymus DNA polymerase alpha, Thermus aquaticus heat-stable DNA polymerase, and SequenaseTM brand cloned T7 DNA polymerase; the third DNA polymerase is selected from the group consisting of AMV reverse transcriptase and cloned MMLV reverse transcriptase; and the each of the first and second bacteriophage DNA-dependent RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase; and if the target segment is a RNA segment, each of the product of step and the product of step is rendered single-stranded by thermal denaturation; each of the second and fourth DNA polymerase is selected from the group consisting of Klenow Fragment of E. coli DNA polymerase I, AMV reverse transcriptase, cloned MMLV reverse transcriptase, calf thymus DNA polymerase alpha, Thermus aquaticus heat-stable DNA polymerase, and SequenaseTM brand cloned T7 DNA polymerase; each of the first and third DNA polymerase is selected from the group consisting of AMV reverse transcriptase and cloned MMLV reverse transcriptase; and the each of the first and second bacteriophage DNA-dependent RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA pclymerase and SP6 RNA polymerase. 8 A method according to Claim s wherein the targe2 aegment is a RNA segment and wherein each of the second aid fOirth DNA polymerase is selected from the group consisting of Klenow Fragment of E. coli DNA polymerase I, AMV reverse transcriptase and cloned MMLV A I WO 88/10315 PCT/US88/02108 reverse transcriptase and wherein each of the first and third DNA polymerase is selected from the group consisting of AMV reverse transcriptase and cloned MMLV reverse transcriptase. S A method according to Claim 3a-wherein the subsegment of target segment which has the sequence complementary to that of (3'-primer subsegment) 1 is in the 3'-direction from and does not overlap the subsegment of target segment which has the sequence complementary to that of (5'-primer subsegment) 4 and wherein the subsegment of target segment which has the sequence of subsegment) 2 does not overlap the subsegment of target segment which has the sequence of (3'-primer subsegment)3. 21c) uo. A ethod according to Claim -4-wherein the subsegment of tiird complementary DNA with the sequence complementary to that of (3'-primer subsegment) 1 is in the from and does not overlap the subsegment of third complementary DNA which has the sequence complementary to that of (5'-primer subsegment) 4 and wherein the subsegment of target segment which has the sequence of subsegment) 2 des no't overlap the subsegment of target segment which has the sequence of (3'-primer subsegment) 3 4A A method according to Claim 4-\wherein, in the third complementary DNA, (variable subsegment)lc has more than 0 nucleotides and wherein, in the duplex between subsegment) 4 and third complementary DNA, at least a subsegment of (5'-primer subsqgment) 4 is hybridized to (variable subsegment) 1 c. A method according to Claim 4-wherein the subsegment of target etgment which has the sequence complementary to that of (3'-primer subsegment)l is in the 5s 3'-direction from and does not overlap the subsegment of /j target segment which has the sequence complementary to B A" r t oy WO 88/10315 PCT/US88/02108 71 that of (5'-primer subsegment) 4 and wherein the subsegment of target segment which has the sequence of subsegment) 2 does not overlap the subsegment of target segment which has the sequence of (3'-primer subsegment)3. l3S 44- A method according to Claim 4+ wherein the subsegment of third complementary DNA with the sequence complementary to that of (3'-primer subsegment)l is in the from and does not overlap the subsegment of third complementary DNA which has the sequent complementary to that of (5'-primer subsegment) 4 and wherein the subsegment of target segment which has the sequence of (5'-primer subsegment)2 does not overlap the subsegment of target segment which has the sequence of (3'-primer subsegment) 3 4.4, 5-4- A method according to Claim 44 wherein, in the third complementary DNA, (variable subsegment)lc has more than 0 nucleotides and wherein, in the duplex between subsegment) 4 and third complementary DNA, at least a subaegment of (5'-primer subsegment) 4 is hybridized to (variable subsegment)lc. S. 4-6. A method according to e-ef wherein if T7 RNA polymerase is employed in step or step (promoter) 1 if the step is step or (promoter) 3 if the step is step has the sequence 5'-TAATACGACTCACTATA-3' and (variable subsegment)l, if the step is step or (variable subsegment) 3 if the step is step has a dinucleotide of sequence 5'-GG-3' at its 5'-terminus; or if T3 RNA polymerase is employed in step or step (promoter) 1 if the step is step or (promoter) 3 if the step is step has the sequence 5'-TATTAACCCTCACTAAA-3' and (variable subsegment) 1 if the step is step or (variable subsegment) 3 if the step is step has a tetranucleotide of sequence 5'-GGGA-3'at its or if the SP6 RNA polymerase is employed in step (6) WO 88/10315 PCT/US88/02108. 72 or step (promoter) 1 if the step is step or (promoter) 3 if the step is step has the sequence TTAGGTGACACTATA-3', and (variable subsegment) 1 if the step is step or (variable subsement) 3 if the step is step (12) has a hexanucleotide of sequence 5'-GAATAC-3' at its 5'-terminus; and wherein the 5'-terminus of the first primer is the 5'-nucleotide of (promoter), and the 51-terminus of the third primer is the S'-nucleotide of (promoter)3- 5 ':9E A 7; A method according to\li.Gi-1 F.I. Is wherein if the first bacteriophag( DNA-dependent RNA polymerase is the T7 or T3 RNA polymerase, (variable subsegment)1 has the sequence 5'-GGGATGGGGAACCCCCCTTCGGGGGTCACCTCGCGCAGC-3' and second primer (variable subsegment) 2 has the sequence GTGACGCCTCGAGAAGAGGCGCGACCTTCGTGC-3'; or (ii) if the first bacteriophage DNA-dependent rZNA polymerase is the 5P6 RNA polynerase, (variable subseg- ment)l has the sequence GGGTCACCTCGCGCAGC-3'and second primer (variable subsegment) 2 has the sequence 5'-CGCGCTCTCCCAGGTGACGCCTCGAGAAGAGCGCGACCTTCGTGC-3' if the second bacteriophage NA-dependent RNA polymerase is the T7 or T3 RNA polymerase, (variable subsegment) 3 has the sequence 5'-GGGATGGGGAACCCCCCTTCGGGGGTCACCTCGCGCAGC-3'; (ii) if the second bacteriophage DNA-dependent RNA polymerase is che SP6 RNA polymerase, (x~,ish)e subsegment) 3 has the sequence 5' TACGG AACLCCCTTCGGGGGTCAC 3' and the fourth primer has as the 51terminal nualeotides CGCGCTCTCCCAGGTGACGCCTCGAGAGAGGCGCGACCTTCGTGC-3'; and after step the second RNA is autocatalytically replicated with QP replicae. _I_~i WO 88/10315 PCT/US88/02108 73 LC, A method according toM 4 1 1 aFP\I wherein the target segment is a segment of the genome of a human immunodeficiency virus that is an HIV-1 virus and wherein (3'-primer subsegment) 1 has the sequence 5'-TCTAATTACTACCTCTTCTTCTGCTAGACT-3', subsegment) 2 has the sequence 5'-ACAAGTTGTAACACCTCAGTCATTACACAG-3', (3'-primer subsegment) 3 has the sequence 5'-AAAGGTATCCTTTGAGCCAATTCCCATA-3', and fourth primer has the sequence 5'-AGTTGATACTACTGGCCTAATT-3'; or (2\ (3'-primer subsegment) 1 has the sequence 5'-TTTCGTAACACTAGGCAAAGGTGGCTTTATC-3', subsegment) 2 has a sequence selected from the group consisting of 5' -GCACACAAGTAGACCCTGAACTAGCAGACCA-3, and 5'-ACACCATAVGTATGTTTCAGGAAAGCTA-3' (3'-primer subsegment) 3 has the sequence -ACTAATTCATCTGTATTACTTTGACTGTTTTTC-3, and fourth primer has the sequence 5'-TTTTTTGGTGTTATTAATGCTGCTAGTGCC-3'. L 4-9. A method according to Claim 24-wherein if the target segment is a DNA segment, the product of step is rendered single-stranded by thermal denaturation; each of the first and second DNA polymerase is selected from the group consisting of Klenow Fragment of E. coli DNA polymerase I, AMY reverse transcriptase, cloned MMLV reverse transcriptase, calf thymus DNA polymerase alpha, Thermus aquaticus heat-stable DNA polymerase, and SequenasePT brand cloned T7 DNA polymerase; and the first bacteriophage DNA-dependent RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase; and if the target segment is a RNA segment, the product of step is rendered single-stranded by thermal denaturation; the setzond DNA polymerase is selected from the group consisting of Klenow Vragment of E. coli DNA olymerase I, AMV reverse transcriptase, cloned MMLV reverse transcriptase, calf thymus DNA polymerase alpha, Thermus O I C 74 aquaticus heat-stable DNA polymerase, and SequenaseTM brand cloned T7 DNA polymerase the first DNA polymerase is selected from the group consisting of AMV reverse transcriptase and cloned MMLV reverse transcriptase; and the first bacteriophage DNA-dependent RNA polymerase is selected from the T7 RNA polymerase, the T3 RNA polymerase and the SP6 RNA polymerase. 49. A method according to Claim 48 wherein each of the first and second DNA polymerase is selected from the group consisting of AMV reverse transcriptase and cloned MMLV reverse transcriptase. A method according to Claim 49 wherein the 5'-terminus of the first primer is the 5'-nucleotide of (promoter) 1 wherein, if the bacteriophage DNA-dependent RNA polymerase is the T7 RNA polymerase, the subsegment (promoter) 1 -(variable subsegment) 1 of the first primer has the sequence 5'-TAATACGACTCACTA TAGGGACGCGCTCTCCCAGGTGACGCCTCGAGAA- GAGGCGCGACCTTCGTGC-3'; wherein, if the bacteriophage DNA-dependent RNA polymerase is the T3 RNA polymerase, the subsegment (promoter) 1 (variable subsegment) 1 of the first primer has the sequence -TATTAACCCTCACTAAAGGGACGCGCTCTCCCAGGTGACGCCTCGAGAAGAGGCGCG- ACCTTCGTGC-3' wherein, if the bacteriophage DNA-dependent RNA polymerase Sis the SP6 RNA polymerase, the subsegment (promoter) I-(variable subsegment) of the first primer has the squence *AATACATAACCTTATGTATCATACACATACGATTTAGGTGACACTATAGAATACCGCGCTCTCCCAGGTGA- CGCCTCGAGAAGAGGCGCGACCTTCGTGC-3 wherein (variable subsegment) 2 has 25 the sequence 5 -TGGGGAACCCCCCTTCGGGGGTCACCTCGCGCAGC-3 kind wherein, after step the first RNA is autocatalytically replicated with replicase. 51. A method of preparing an RNA containing a sequeice corresponding to a target sequence, said method as set out in claim 1 and 30 substantially as hereinbefore described with reference to any one of the Examples. 52. A method for amplifying a target nuaclee acid segment of Formula I as set out in claim 21, said rethod substantially as hereinbefore described with refererice to any one of the Examples, and/or Figures 2 and Z In the Drawings, DATED this THIRTIETH day of DECEMBER 1991 Siska Diagnostics Inc Patent Attorneys for the Applicant SPRUSON FERGUSON 1385U b( J II