CA2136764A1 - Ligase chain reaction starting with rna sequences - Google Patents

Abstract

The present invention involves a method of amplifying RNA by producing complementary DNA (cDNA) by reverse transcription of RNA, and amplification of the cDNA sequences. The analysis of the amplified material facilitates the detection of pathogens and disease states associated with the presence of particular nucleic acid sequences, so the present invention is important in medical diagnostic procedures. A method of producing cDNA of predetermined length is also disclosed.

Description

W093/~46~6 ~ 1 3 6 7 6 'I PCI/US93/04863 j .
~Ligase chain reaction starting with RNA sequences .

s This application is a continua~ion-in-part of U.S. Application Serial Number 07/891,543 filed May 29, 1992, the whole of which is incorporated by reference.
Teçhnical Field The present invention relates to methods and kits ~for amplifying and detecting 10 speci~lc RN~ sequences. In particular, the invention relates to methods for producing complementa~y DNA (cDNA) by reve~se transcliption of RNA, and amplification of the DNA sequences. llle analysis of the amplified matenal facili~ates the detecuon of pathogens and disease states associated with~ the presence of par~cular nucleic acid ;
sequences, so the present inven~on is important in medical diagnostic procedures.

Back~nd ~-Nucldc acid amplification techniques are~established as powerful tools for :' detec~ing small amounts of DNA or RNA which Feviously were undetectable by standard nucleic acid hybridization methods. DNA amplifica~on most commonly 20 employs the polymerase chain reacdon (PCR) as~described in U.S. Patents 4,683,195 and 4,683,202 Or the ligase chain reaction (LCR)~ as described in EP-A-320 308 and E~A439 182. The en~ire disclosure of each of these publicatlons is incoIporated he~rein by reference.
When ~coupled with~reverse transcQtion, PCR permits the ampllfication and 5~ ~ detection o~.rninute amounts~of l~A as desc~i~d in PCR ProtocQI~. A Guide tQ
Methods and~ AmDIifiçà~ion~, Acadernic Press, Inc., (1990). The PCR process~is discussed fi~rher in WO 91/0594, which des~bes a one-enzyrne system that Gan ; amplify~NA.~ A~dlcr~ble~DNA~polyraerase~having~reYerse~transcriptase activity is reported.`~ The~reverse ~rans~riptase activity~ malces a cDNA copy;Qf the RNA and the ` 30 cDINA is ~nplified by PCR, using the same enzyme~and reagents.
Efforts t:o avoid amplifying contamina~ng DNA are disclosed by Shuldiner e at.,~in~publishe U.S~ntapplication~04,591;;(~S~pubIished~Mayl4,1991;
for RNA eemplate-specific~PCR. ~ .
~e~present~invention prondes a me~hod to~amplify RNA using the LCR.~ It 35~ ~ udlizos a Gombination~of~cligo ucleotido prob~s and ampbfication~methods which nhancc the sensidviy and reliability~of RNA~asnpli'dcadon and dctecdon~with LCR. ~' WO 93/246~6 PCI`/US93/~3 2136~76~ 2 Summarv of the Invention The present invention relates to methods and kits useful for amplifying and detecting ribonucleic acid (RNA) from a multitude of sources. In a f~t aspect, the invention provides a method of amplifying a known RNA target sequence present in a .l , biological sample, said method comprising: , (a) treating RNA in the sample under hybridizing conditions wi~h a first oligonucleotide probe which is hybridizable to a first segment of the known target ~NA;
(b) extending a 3' terminus of said first pro~ by reverse transcription of the RNA target so that a cDNA segrnent is produced ha~ing at its 5' end said first probe and - -at its extended 3' end a nucleotide sequence complementary to a second segment of t'ne target RNA, said reverse transcription being limi~ed to the addition of not more than --about 30 nucleotides;
(c) dissociating the extended f rst probe from the RNA target;
(d) hybridizing a~second oligonucleotide probe to said extended first probe, said second probe ha~ring a 3' end hybridlzable to the extended cDNA segment of the first probe, but substan8ally not hybridizable to said nrst probe when it is unextended;
(e) forming at least one of:
(i) an elongated second probe complex by covalently ligating a third DNA probe to the 3' terminus of said second probe, with the proviso that if said second or third probe is modified, it is corrected prior to ligation of the third probe to the second probe; and (ii) an elongated first probe complex by forrning a fourth DNA segment ; covalently attached to the 3' terrninus of said first probe and complementary to said second probe; and (f) amplifying at least one of said elongated second pro~e complex and said .
elongated first probe complex.~
; Preferably, the length of the cDNA extension of the first probe is limited to a predetermined length by providing a pool of less than all four nucleoside triphosphate types. In this way, extension is tenninated at the stopbase which calls for an omitted 30 "nucleotide.
An irnp~rtan~part of this invention is the fo~nation of a DNA copy from the J `
; ~ RNA that is long enough to support amplification. The method of the inven~on ~ ~:
ptovides several ways of accomplishing this, it being partiGularly important that ~e second p~be hybridize with the first probe substantially only when the first probe has ;~ ~ 35 been exter~ded on the RNA target. Ideally, the second and extended first probes hybridiæ together for `only a relatively short portion at th~r respective 3' ends, leaving relatively large S' overhangs. The S' overhangs are then used to complete the forma~ion of a fi}ll length DNA product . This is done by 1) polymerization extension of the ~ wo g~/24656 2 1 ~ 6 7 6 4 PCI/US93/04863 ~"'' ' second (or first) probe using the first (or second) --nbe as template; or 2) by ligating third or fourth probes (complementary to the~ 5' ~ang portions of the f~t and second probest respectively) to: the second or firs, l.robes, respectively.
Qnce the full length DNA copy is rnade? it may be amplified by several 5 techniques, the most useful being LCR using the same four probes mentioned already.
Thus, the method further comprises amp!ifying by at least one repeated cycle of forming both : ~ :
(i) an ~elongation complex of a third oligonucleotide probe covalently ligated to~the 3' tenrinus of said second probe and complementary to : at least::a por~ion of said first probe, with the proviso that if said second ~or: third probe is ri~dified, it is corrected prior to: ligation of the third probe:~to the;second probe; and ::: : (ii) an elongation~ oligonucleotide complex covalently attached to the 3' terminus~ of said first probe: and complementary to at least a portion of said second probe.
In a second aspect~the ~invenhon provides~a method of forming cDNA of a ptedetcnninèd length fr~m a:known RN~ target~sequence present in a sample, :;
comprising the~ s~eps :of~
s~ (a) trea~ng ~A~u~d~ h~ng con~s with a~first oligonucleohde probe 20~ which is~hy~dizablcto~a~f~t~s ~ent~of~the~RNA;and (b) ~extending~a~:3' tetminus of:said~probe;~:reverse transcript n~of the ~A ~ ~ c ` ~ under con~hons inc~g less:than~ four nùcleoside t~phosphate;~t~es, so that a : cDNA seg~l nt of ~a j~lmined~length~ ~s~produced, wherein~su~ch ex~enslon is ;
termina~at said p~ ined~length~when~said:~NA~emplate:reqùires a:nucleoside~

lnfinal~?sp~ theinvontion~ides~dlagnosticl~tfordetecting~anRNA
~ge~ p~nt in~a~àl ~ c~ompnsing ~in~G~mbination~
(à)~afir tolig nuc ~`w his nt ~ oa~p~lonof~t e::

`~ 30~ o a ext gr agèntcapable~dfrève s~ ns~ptonoft :é!~A t rgetih :t~e~pr s nceofa~t ~nuc~sid~e~hat sc mplementa t the~A
~et `~re~on ~:3 ' ~:of ~ p~ c, ~usi g ~è ~t ~ ~; as: a ;
) a:sec~
~ucleot dc:~substa ;daDy~y~when~said~st~ has een~extended by ` `35 ~s` t s pdo~
::(d) a~ least:one;of - r~
a third ol~uc~ ~ c~n~ ~to a ~on of said ~st saîd~ ~pto~:haying a~5' terminus ligatable to the 3' te~nus of W0 93/2~6S6 ~ PCI`/US93/04~3 ' ~-. .. ,213676~ ~ ~ , ' ).~;,.;, . ..
said second probe and complementary to a pomon of said first probe~
w!th the prwiso~that;if said second or third pro~e is corrected prior to ligadon, the second pt~be is ligatable to the third; probe in theLr ;;
concctéd fo~:~ereby~to form~ an elongated second probe complex; ~ :

(ii) `a fot~ oligonucleotide:prolx~whi~h is:covaiently ligatable to the 3 terminus of saiAd~first~obé once~lt ls eYtended~and complementary to :a po:~don~ ?f said second~:probe, thèreby to fonn an elongated first 10 ~ (e)~;anasse~ng~reagentfor~fo ' g~t~ngate secondpro e:complex,t e ~ -elongàt~::f~st`~be~`or .`

Dc~n~ of ~c wc~ zed RNA amplification ~
15 method~employing elong~d~first ànd sècond oligonuclcotide probes. In this~Figure, dongation:is~ depicted by~"squlg~":anes w represent extenslons, and "shaded'l bars t o 2 1 3 6 7 6 ~ PCr/US93/04863 Detailed ~escription D~nitions "Oligonucleodde" refers to a m~lecule consisdng of two or more deoxyribonucleoddes, preferably more than three. The exact size depends on many 5 factors such as the ultimate function or use of the oligonucleotide. By definidon, an oligonucleotdde (so~etimes shortened to "oligo") has polarity and 3' and 5' ends. As used herein, "terminus" refers to the endpoint of an oligonucleodde. Usually this is a 5' phosphate or a 3' hydroxyl, but in some cases the termini are modified, either to prevent unwanted target-independent ligation (e.g. with internal termini), or to attach a label or 10 reporter group (e.g. external termini). By contrast, the "end" of an oligonucleotide refers to a terminal por~ion or segment, not the actual terminus. Typically, an -oligonucleotide is a 2'-deoxyribo oligonuc!eotide, but it may also be a mixed ribo/deoxyribo-oligonucleotide. ~ ~
A "probe" is an oligonucleotide. ~ The exact length of the probe according to the 15 invention depends on many~factors, including temperature, source of probe, and how it is used in the method. Probes as short as 6 or 7 nucleotides (hence the commonly used terminology "6-7 mer") and~ as long` as several hundred-mers have been used for other purposes, but more typlcally~probes for LCR are in the range of 10 to 40 or 50-mers.
For example, depending on the complexity of the target RNA sequence, a probe 20 ~ typically co ains 15-40 nucleotidesj a1though it~may contain more or fewer nucleotides.
Probes may be derived from a natural; source, as in a purified restriction digest.
Alternadvely, it is ? routine~nuner to synthesize the desired probes using conventional nucleotide phosphcram~dite (or phosphonate) chemistry and instruments available from Applied~Biosystems, Inc., ff~?ster City, CA); ~DuPont, (Wilmington, DE); or Milligen, ~-; 25~ ~ (Bedford, MA). Phosphorvlation of the 5' tennini~of the probes, which is necessa~y for ligation by ligase,~may be accomp!ished by a kinaso or by chemical synthesis (e.g.
Phosphate-On~, Clontech, Palo;AIto,~CA) as is hlown in the art.
As described belo~, a probe i~ capable of acting as a point of ligation to second probe or as a point of inidation~ of polylnerizadon extension (e.g. a pnmer). When used -30 as à primer, the probe~must be sufficiently long to permit the synthesis of extension ploducts~in the'prèsencè of `a polymenzation agent. "Polymerization" and "extension~
refer to the addition, one by~one, of nucleoside ~triphosphate monomers to a p.: ;ner using a~ten~late~as~is well~known-in jthe~art. By contrast,~"elonga~ion" refers to a process which resuIts in a covalendy attached longer~probe regàrdless of the mechanism.
35~ ~ Specifically, elongation;includes~the~ligadon of another short oligonucleotide to form an "elonga~on" product~or complex. ~EIongation also encompasses a "colTec~on" (e.g. an extension) step prior to; a ligation~step as is known from the incorporated EP-A-439 182.

:

WO 93/24657 6 4 ~ ~ Pcr/us93/0~63 The ~erms "modified" and "corrected", with regard tO probes, have the meanings specified in IJS Serial No. 07/634,771 filed January 9, 1991 ~published as EP-A-439 182) and US Serial No. 07/925,402 filed August 3, 1~92, each of which is incoIporated herein by reference. Briefly, a modified probe is one that cannv~ be ligated S to its same-sense partner probe because it ~ails to present the proper substrate for ligase.
Ligase requires the following as subs~ate: 1) adjacent or abut~ing probes, presenting 2) a 5' phosphate terminus and 3) a 3' hydroxyl term~nus. Ligase exhibits a marked preference for probes meeting these requirernent when they are hybridized to a template whi¢h dictates adjacency. Generally, a probe is modified by leaving a gap, an overlap (to destroy the adjacency requirement); by changing the 3' hydroxyl to a phosphate, ribonucleotide or other bloclcing moiety; or by altering ~e 5' phosphate or inser~ing a 5' rnismatch. Other "modifications" that fall within the general definition are also contemplated by the present invention. As is described in detail in the above-noted US
S~al No. 07/634,771 and US Serial No. 07/925,402, the modif1cations are "corrected"
in template-dependent fashion to yield ligatable probes; but this correction process takes place substantially only when the probes are hybridized to target (or amplicons made from targe~).
It is also to be understood that the term "all 4 nucleoside triphosphate types"
shall refer to Guanine (G), Cytosine (C), Adenine (A~ and Thymine (T) when the context is that of DNA; or Guanine (G), Cytosine (C), Adenine ~A) and Uracil (U) in the context of RNA. The term also includes analogs and derivanves of the bases named above. Accordingly, "less than all four nucleoside triphosphate types" refers tO an omission of at least one of the four nucleoside triphosph~tes.
Mefhod Pha~es In general, the amplificadon method of the invention proceeds in three stages orphases: I) initial hybridization and reverse transcr~ption; Il) separation and folma~ion of at least one strand of full length DNA; and III) amplification of the full length DNA
strand(s). Division of the method into phases is not rigid however, and is intended to facilitate discussion, not to limit the invention.
I In the firs~ phase, a first probe hyblidizes with target RNA if present in thesample, and is extended by reverse transcliption using the RNA as template. Thisextension may proceed with reverse transcnptase or, Imder certain conditions, with DNA polymerase. For DNA polymerases tested by applicants~ the presence of a divalent ca~ion is required, typically at a concentration of about 0.5 mM to about 30 mM.
It is presently believed that manganese is necessary for the reverse transcriptiorl activity of h~own DNA polymerases; a concentraaon of 0.5 rnM to about S mM is suitable.
This iniaal extension to make cDNA is preferably limited so that no more than about 30 nucleotides are added to the first probe. Extension may include all four W~ 93/246~6 2 1 3 6 7 fi ~ P~r/US93/04863 nucleotide triphosphates, as is conventional in the art for PCR amplification of RNA, but more preferably, it include less than all four nucleoside triphosphate ~ypes since cDNA of a predetermined length is usually desired. By limiting the reagents so that less than all four nucleoside triphosphate types are present, extension will cease when the S RNA template dictates that an omitted nucleoside tIiphosphate be added to the growing primer. In this situation, the template base which calls for the addition of an omitted base is referred to herein as a "stopbase". ;~
While a single round of first phase cDNA extension is generally suff1cient to support subsequent arnplification, it may be desirable to cycle the fIrst phase to make add}rional cDNA copies from each RNA targe~ molecule. Cycling requires the application of sufficient heat (or otherwise altering the stringency conditions) to separate the RNA:DNA duplexes. This is followed by cooling, to reanneal unextended first probes to the RNA target molecules, and extending the first probes again. Cycling the first phase requires that the reverse transcTiption acti~ity be relatively the~mostable, or that precise stringency condi~ons be used so as to pen ~it separation of the RNA:DNA
duplex without loss of the enzyme activity, or that the enzyrne be re-added at each cycle.
A polymerase enzyme having thermoac~ive reverse transcTiptase acti~r~ty has beenreported by Gelfand, e~ al. in WO 91/09944. The first phase is completed by separating -the RNA:DNA duplex, usually by heating. By hea~ing to about 100 C, the reverse ~anscriptase enzyme can also destroyed.
In the second phase, the extended ~rst probe is combined with one or more other probes to syn~hesize at least one strand of full length DNA. A "full length" DNA strand is one which is long enough to support further amplifieation, typically by LCR. It can ~-range rom about 40 to over 100 nucleotides in lengtll, usually from 40 to 60. Although `-it is only necessary to synthesize one full length strand, the preferred method utilizes ' three additional pr~bes and synthesizes both full length sh-ands. These same four ~.
probes are then used for a further LCR amplification phase.
In the simplest case, a full length strand of DNA is made by using all four ~ `
nucleoside ~phosphate types in the extension reaction. In this case, extension is not 30 limited to a predetermined lenj~h by a stopbase, and phases I and II merge.
Accordihg to the present inven~aon, extension is limited to a predetenni~ed length not exceeding about 30 nuc!eotides. For targets, probe designs and ~111 criteria that ~ --pennit it, a full length DNA can be made using only extension, even if less than all four nucleoside triphosphate types are used. More usually, a full length DNA is made using 35 addinonal probes which can be ligated to ~onn elongation complexes, a process which can take place by several mechanisms. In all eases a second probe is utilized under condi~ons such that it hybridi~es to the ex~ended first probe, but does not hybridize tO i the unextended first probe und r the conditions used. More specifically, the 3' end of the second probe is complementary to some or all of the extended pornon of the first ~ -WO 93/24656 PCrJUS93/0~.~63 2~ 6e4This complementary region is referred to herein as the "overlap" region, and it must be long enough for the first and second probes to form a stable hybridization cornplex under the reaction conditions. Its exact length is dependent on the stnngency conditions (particularly temperature) and on the specific probe configurations. For 5 example, at 25 C an overlap of about S-lS nucleotides is sufficient and preferred. At higher temperatures, a longer overlap region is required, and at cooler temperanlres a shorter overlap may be used. The overlap length is also dependent on target constraints as is discussed in detail in the section titled "Probe Design", below.
Once a sta~le duplex of first and second probes is formed, one or the other (and10 preferably both) is filled in using the other as a template to synthesize a full length DNA
(and for subsequent amplification~ Several mechanisms are possible and are surnrnarized in Table 1 below:
Table 1 _ ~
Whether or not reverse transcnption was lim~ted Extension of first probe in the first phase, all four nucleoside triphcsphate types may be added in the second phase to make an elongated first probe complex by polymerization using the second probe as tem~late All four nucleoside triphosphate types may be Extenston of second probe added in the second phase to make an elongated second probe complex by polymerization using the first probe as template .
Where reverse transcrption was limited in the Elongatlon of first probe first phase, an elongated first probe complex can be formed by ligating a fourth probe to the 3' terminus of the extended first pro~e. While possible, in the amplification phase, to use a fourth probe which is directly ligatable to the 3' term~nus of the first probe, it is preferred to use a fourth probe that is ligated to the 3' terrninus of ~e extended first probe, and to perforrn extension in the second ~hase also.
. . ~
Elongation of second probe Where reverse transcription was lirnited in the first phase, an elongated second probe complex can be formed by ligating a third probe to the 3' tenninus of the second probe. It is possible to use a third probe which is directly ligatable to 7; -the 3' tem~inus of the second probe. . r~
Optiona}ly, the 3' ter~inus of the second probe may be "corrected", such as by extension or cleavage of a blocking moiety, prior to ligation to the third probe; or the third probe may be "co~r~cted", such as by exonuclease cleavage of a S' mismatched base or a S' non-phosphate.
. . ~

~ WO 93/24656 2 1 3 6 7 6 4 PCr/lJS93/~4863 ~ . .

It is reiterated that the modifications and corrections mentioned in this summary table are like those discussed in EP-A-439 182, and in copending US Serial No.
07/925,402, and they need not be discussed in detail here. In the preferred four probe-double gap filling version, elongation complexes are made in both the top and bottom 5 strands by gap filling-extension and liga~ion. The lengths of the two gaps are preferably diffe~en~; the length of the gap between ~rst and fourth pr~bes being about 5-15 bases, so that a stable duplex can be forrned between the extended first probe and the second probe. By contrast, the gap between the second and third probes, when present, is typically much smaller, for example, from 1 to about 5-10 bases, usually defining an 10 "asymmetric" gap~ The second gap may be non-existent, which amounts to direct or abumng alignment of second and third probes. Gaps between probes that forrn elongation complexes may be characterized then by a gap ratio: a first number specifying the length of the gap between first and fourth probes (bot~om strand), and a second number specifying the length of the gap between second and third probes ~top strand).
15 I'hus, embodiments according to the invention include, but are not limited to, probe configurations having gap ratios like: l5:û or 5:5 and several intermediate ra~os, for example, 10:2, 12:3, B:0, 8:1 or9:3.
Although these methods may seem burdensome at first for creating full length cDNA, their true utility is seen in the amplification phase (phase III), where the same 20 probes and nucleotide reagents that were used to create the full length DNA in the first place, are now used to arnplify it.
Amplification Phase It is possible, once the stable duplex of f~st and second probes is forrned, tO
simply extend each over the other to obtain ampli~led DNA. I his is similar to a PCR
25 reacdon, depending on the exac~ probe configuration. In most cases, however, first and second probes will be selected with 3' ends so dlat they are slightly overlapping, or essentially vertically aligned. For simple extension amplification, a polymerase is used, preferably a therrnostable DNA polymerase. Several publications have descIibed suitable thermostable polymerases, including U.S. Patent Nos. 4,889,818 and 30; 5,079,352, and published applica~ions WO 91/09950 and WO 92/~3556.
It is prefeIred, however, to synthesize at least one elongation complex by liga~on of a third or fourth probe. In this case, amplification proceeds much like the LCR or the modified ends LCR described in EP-A-439 182. Of course it is possible, and within the scope of this invention, to u~lize a hybrid amplifica~on scheme, 35 perf~ning an extension reaction on one strand and an elongation/ligation reaction (optionally, modified and wrrected) on the other strand~ Hybrid amplifica~ion is less preferred when it requires addi~onal reagents (e~g. endonuclease IV or ribonuclease).

WO 93/246~6 PCI/US93/0~.63 ?.,~ 36rl 6 ~ ' The conditions and reagents which make possible the preferred enzymatic ligation step are generally known to those of ordinaTy skill in the art and are disclosed in the references mentioned in the background. Ligating reagents useful in the present invention include prokaryotic ligases such as E coli ligase, T4 ligase and Thennlls thermophilus ligase (e.g., AT(: C 27634) as taught in EP-320 308. This latter ligase is presendy preferred for its ability to maintain activity dunng the thermal cycling of LCR.
Other suitable thermally stable ligases are commercially available from New England Biolabs, Inc. (Beverly, MA), Epicentre Technologies~ Inc.(Madison, WI) and Molecular Biology Resources (Milwaukee, WI). Abs~nt a thermally stable ligase, the ligase must be added again each time the cycle is repeàted. Also useful are eukaryotic ligases, including DNA ligase of Drosophilia, reported by Rabin, et al., J. Biol. Chem.
261:10637-10647 (1986).
Once ligated, the fused probe is dissociated (e.g. melted) from the target and, as with conventional LCR, the process is repeated for several cycles. The number ofrepeat cycles may vary from 1 to about 100, although from about 15 to about 70 are preferred presently.
Probe Design For purposes of this invention, the target RNA may be any RNA selected from visal, nuclear or cytoplasmic sources including, for example, genomic RNA, mRNA,tRNA, hnRNA and rRNA, and any combination thereof.
The probes used herein are selected to be substantially complementaly to the different strands of each specific sequence to be amplified. The term "selected" in this context is taken to mean that a target sequence having the desired characterisdcs is located and probes are constructed around an appropriate segment(s) of the target sequence. It will be understood that those of ordinaly skill in the art will know how to search for and idèntify specific target sequences meeting ~he requirements of this invention. For example, many databases contain sequence inforrnation (e.g.
GENBANK, NBRF, EMBL or Swiss-Prot) which can be searched by readily available computer software (e.g. MacVector, MacMolly or Geneworks). For convenience an 30i IRNA sequence in the database may readily be converted to its corresponding DNA
sequence for searching using DNA search c~iteria, e.g. as described in Example 3. It will also be realized that in any organism's known genome, multiple locations meeting the requirements will generally be found. For example, a search of the HCV genome ~GENBANR Acc. No.~ M58335; (see Examples 1-2, and Appendix A)], which contains approximately 9.4 kilobases, reveals over 1486 possible locations poten~ially suitable for practicing this invention.
Probes #1 and #3 need not reflect the exact sequence (or complement) of the template, but must be~ sufficiendy complementary to hybridize with sequences of the :

WO 93/~4656 ~ 1 3 6 7 6 ~ PCI/U~;93/04863 11 `
target. Probe #2 need be complementary to the extended portion of probe #1 and can even overlap Probe #1, provided it does not substantially hybndize thereto prior to extension of probe ~1. The remaining 5' portion of probe #2 may be any sequence at all and need not be specif1c for the target. Probe #4, when used, need only be 5 complementary to the 5' portion of probe #2. Once the elongated first or second probe complexes are formed, the probes will typically be perfect complements, regardless of whether or not the original target was.
One possible exception to perfect complementarity could be the creation of a stopbase in the DNA amplification phase that was non-existent in the initial cDNA
10 extension phase. This nL;,ht be done, for example, by designing probe #2 to include a mismatch in the region of overlap between probes ~1 and #2. The rnismatched basecalls for a dNTP not provided in the pool, whereas the original template called for a dNTP that is provided. This has the effect of perrnitting a longer cDNA extension - product while limiting the gap fill in arnplification to a smaller size. A disadvantage 15 would be t'ne destabilizing effect of the mismatch in the overlapping area of extended probe #1 and probe #2.
Although more detailed examples of probe design are provided at the end of the specification, a typical exarnple will now be described. This exa~lnple illustrates the identification of one type of potential 9:3 asymrnetric gap LCR site t'nat has on the top 20 strand one base (G) followed by a 9 base sequence of T or C followed by a 3 base sequence of G ~r A followed by one base (T); (that is, a search is conducted for the sequence RYYYYYYYYYRRRY, wherein R = A or G and ~ = C vr T). Such a typical example is demonstratéd by sequences found in the Hepatitis C 5' untranslated region, virus core, matrix, envelope and non-structural protein RNA ~Takamizawa,A.,et al., J.
Virology 65:1105-1113 (1991), designated "CHIJMR").
SF,Q ID I~o, S' -AAWGCCAGGACGACCGGGUCCUUUCWGGAUCAACCCGCUCAAUGCCUGG-3' 29 CCTAGTTGGGCGAGTTACGGACC-5' 3'-TTAACGGTCCTGCTGGCCC 4 5'-AATTGCCAGGACGACCGGGTCCTTTCTT 2 TCAACCCGCTCAATGCCTGG-3' 3 `
1 i ~ ~ '.
Probes 2 and 4 are chosen such that a sufficient number of complementary bases exist between the two~probes toresult in a thermal melt temperature (Tm) of ~ ~-approximately 5~80 C. This is usually 15-30 bases of complementarity. Similarly, pr~bes 1 and 3 are typically chosen to have a Tm that is close to that of probes 2 and 4.
In the exampls above, probes 2 and 4 have 19 complementary bases and probes 1 and 3 have 20 complementary bases. Probe 2 has a s~ing of 9 bases (5' TCCmCI~ 3') that do not hybridize with probe 4, but which serve as a template for the addi~on of G
and A to probe 1 (by DNA polymerase, after probes 2 and 3 are ligated) during the WO93/7~ 64 PCI/US93/~4~3 amplification phase of the reaction. Probe 1 has a string of three bases (5' TCC 3') that do not hybridize tO probe 3 but serve as template for the addition G and A to probe 2 (by ~, DNA polymerase, with or without liga~on of extended probe 1 to probe 4) dunng phases II or m of the reaction.
S Additionally, it should be noted that the initial extension of probe 1 by reverse transcription reswlts in an extended first probe having a predeterm~ned length if the nucleoside t~iphosphate C is unavailable to the reverse transcriptase (i.e. the corresponding G on the RNA target strand serves as a stopbase). The predetermined length is the sum of original probe 1 plus the 9 added As and Gs. This 9 base added section provides an overlap between probes 1 and 2 that is sufficient to form a duplex at about 25~C. Thus, a full leng~ DNA can be formed on the bottom strand by adding probe 4 and ligating it to extended pro~e 1; and full length DNA can be formed on the top strand by adding probe 3, extending probe 2, and ligating extended probe 2 to probe - 3.
For the amplification phase, the probes are added in approximately equimolar concentration since they are expected to react stoichiometrically. Each probe is present in a concentration ranging from about 0.5 nanomolar ~nM) to about 1000 nM; preferably ~rom a~out 1 nM to about 100 nM for LCR. The optimum quantity of probe used for each reaction also varies depending on the number of cycles which must be perforrned.
Other probe designs and optimum concentrations can readily be determined by one of ordinary skill in this art.
Another facet of pro~e design is related to the par~cular amplification method employed. If a method is used which requires only polymerization extension of probes to make elongated probe complexes, then only two probes would be required. If, on the other hand, any part of the first or second elongation probe complex is formed by lig~tion, one or two additional probes are needed. Thus, the invention describes three embodiments: a two probe version, a three probe version and a four probe version. In the three probe version, it is simpler if the ~lrst probe elongation product is forrned by extension, while the second probe elongation complex is formed by ligation, with or without a correction mechanism ~e.g. extension). In the four probe version, bothelongation complexes require ligation; correction is optional, though preferred.It should be mentioned that probes capable of detecting RNA targets may very i well also be capable of detecting corresponding DNA targets of the same organism, and vice versa.
Detec~n Following amplification, the arnplified sequences can be aetected by a number ofconventional ways h~own in ~e art. No particular detection mechanism is essential to the present invention. In a par~cularly preferred mechanism, hooks are attached at the WO 93~24656 2 1 3 6 7 6 ~ PCr/US93/04863 available outside ends of at least two probes (opposite ends of elongated probe complexes), and preferably to the outside ends of all four probes. A "hook" is any moiety having a specific ligand-receptor affinity. Typically, the hook(s) at one end comprises an antigen or hapten capable of being immobilized by a reagent (such as S antibody or avidin) coated onto a solid phase. The hook(s) at the other end contains a different antigen or hapten capable of being recognized by a label or a label system such as an antibody-enzyme conjugate. Using this approach, the arnplified product can be det~cted in any sandwich irnrnunoassay format. A substrate ls then added which is converted by the enzyme to a detectable produc~
Many different haptens are known in the art, and virtually any hapten can be used with the present invention. Some illustrative haptens include many dmgs (e.g digoxin, theophylline, phencyclidine (PCP), salicylale, etc.), T3, biotin, fluorescein (FITC), dansyl, 2,4-dinitrophenol (DNP); and modified nucleotides such as bromouracil and bases modified by incorporation of a N-acetyl-7-iodo-2-fluorenylamino (AIF) group; as well as many others. Certain haptens described herein are disclosed in co-pending, co-owned paten~ applications U.S. 07/808,508 (adamantaneacetic acids), U.S. 07/8~8,839 (carbazoles and dibenzofurans), both filed December 17, 1991; U.S.
07i858,929 (acridines), and U.S. 07/ 858,820 (quinolines), both filed March 27, 1992;
and continuations-in-part of each of these four applications filed respectively on April 21, 1993, , March 26, 1993, and March 26, 1993~ respectively (collectively referred to herein as the "hapten applica~ons"). The entire disclosure of each of the above-mentioned-previously filed hapten applications is incorporated herein by reference.
Many methods of adding haptens to probes are known in the literature. Enzo Biochemical (New York) and Clontech (Palo Alto) both have described and commercialiæd probe labeling techniques. For example, a primary amine can be attached to a 3' oligo telminus using 3'-Amine-ON CPGIM (Clontech, Palo Alto, CA).
Similarly, a primary amine can be attachecl to a 5' oligo term~nus using Aminomodifier Il~ (Clontech). 'I~e aII~ines ean be reacted to various haptens us~ng conven~ional activation and linking chemistries. In addition, copending applications U.S. Serial Nos.
625,S66, filed December 11, 1990 and 630,908, filed December 20, 1990 teach methods ~or labeling probeis at ~heir 5' and 3' ~ermir~i respectively. Both the aorementioned copending appl*ations are inco~porated by reference. ~}
Publica~ons WO92/10505, published 25 June 1992 and WO 92/11388 publîshed 9 July 1992 teach methods for labeling probes at dleir 5' and 3' ends respectively. According to one known method for labeL~ng an oligonucleotide, a label-phosphoramidite reagent is prepared and used to add ~he label to the oligonucleotide during its synthesis. For example, see Thuong, N. T. et al., Tet. Letters, WO g3J246~6 PCr/US93/1)48~63 2~3(46) 5905 5908 (1988); or Cohen, J.S et al., U.S. Patent Applicaoon 07/246,688 ~NTIS ORDER No. PAT-APPl -7-246,688) (1989).
Of course, other detection mechanisms, such as specific probe cap~ure and/or detection, are also useful with the invention.
Those sldlled in the art recognize that the present method can be used in a variety of contexts where amplification of RNA is desired. The following examples are provided merely to illustrate the invention and not to lirnit the scope of the accompanying claims. Probes are generally written in the 5"to 3' sense left to right~ as is conventional;
but wherl shown as target ~or as aligned on target), one strand (or two probes) are 10 shown in reverse.

Examples - ln each of the following examples the DNA ligase is a thermostable ligase purified from ThRrmus lhennophilus and the thermostable polymerase is obtained from 15 Molecular Biology Resources (MBR), Milwaukee, WI, derived from a Thermus species. Quantities of polymerase are expressed in units, defined (e.g.., by MBR) as follows: 1 unit of enzyme equals the amount of enzyme required to incorporate 10nanomoles total nucleotides înto acid-insoluble material in 30 min at 70C. Units of ligase enzyme are defined ~internally by Abbott Laborat~nes) as: 1 mg of 95% purified Thenn~s ther7nophilus DNA ligase has a specific activi~y of about 1 x 108 units. While this is not precisely standardized and may vary by as much as 20%, optimization is within the skill of the routine pr~ctitioner.
E~ .
The following probe set was designed to detect HCV target RNA as the target sequence~ The underlined bases become stopbases, as will be seen. The probes were synthesized using a model 380B DNA Synthesizer (Applied Biosystems, Inc., FosterCity, CA) and were la~led with biotin (bio) and fluorescein ~fl) on the respective ends using commercially available (Clontech, Palo Alto, CA) biotin and fluorescein phosphoramidite reagents.
First ~obe bio-CCAGGCATTGAGCGGGTTG~TCC SEQDD No.l Second Probe fl-AATTGCCACGACGACCGG~TCCTTTCTT SEQ ID No. 2 Th}rd Probe pTCAACCCGCTCAATGCCTGG-bio SEQ ID No. 3 35 Fou~h Probe pCCC~GTCGTCGTGGCAATT-~ SEQnD No.4 RNA target was a RNA transcript of approximately 350 bases prepared by using D~A dependene RNA polymerase and Hindm linearized DNA plasrnid containing a bacterial promoter upstream of a segment of a S' untranslated region of HCV ("HCV
40 5' UTR"). Detection of target RNA was accomplished by a two step reaction. The first WO ~3/24656 2 1 3 6 7 6 4 PCr/US93/048~3 step was a reverse transcription step to extend the first probe in the presence of only dATP and dGTP and generate a 9 base sticky end of 3'-AGGAAAGAA. Reverse trans~iption proceeded lmder the following conditions:
Table 2 Fin~l Conc.
M~l2 ~ r~
PCR buffer II 50 mM KCl 10mMTris pH8.3 dGTP and dATP 50 ~LM each Probe 1 0.332 ~lM (4 x 1012) Reverse Transcriptase 50 units RNase inhi~itor 20 units RNA Target in dH20 as desired The reaction was initiated by incubation at 42 C for one hour to allow extension of the first probe. The sample was then boiled for S min to inactivate the reverse transcriptase and denature the RNA:DNA hybrid.
To 20 ',IL of the reac~on mixture above, 180 ,~ of the following mixture was added:
Table 3 concentra~ion/1 8Q ,uL
LCR buffer 50 mM Epps pH 7.7, 30 mM
MgCl2, 19.2 mM K~
NAD 111 ',lM
Oligos 2, 3 and 4 0.111 ~LM (12x1012) each DNALigase 8925 units DNAPolvmerase 1 unit ~.

The extended first probe was then hybridized to the second and third probes. Using 30 GAP LCR under the conditions set forth below, the second and third probes were extendcd by DNA polymerase and ligated. Similarly, the fourth probe and the extended first probe were ligated when hybridized to the second probe.
The following complex was ~ormed, wherein nucleotides shown in lowercase -(all g or a) result ~rom the extension steps, and the underlined bases are stopbases.
SEO lD Nos.

fl-AATTGCCACGACGACCGG~TCCTTTCTTggaTC~ACCCGCTCAATGCCTGG-bio 30 fl-TTAACGGTGCTGCTGGCCCaggaaagaaCCT~GTTGGGCGAGTTACGGACC-bio 30' ~-.

4~
.
Gap LCR extension was performed by incuba~ion at 85 ~C for 90 seconds followed by 25 C for 30 minutes. The ampliilca~ion procedur~ was perfo~med for 45 cycles, each cycle consis~ing of an 85 second incuba~on at 85 C, followed by a 56 C
45 incubation for 6Q seconds.

.:
;

W0 93/~ ~S~ PCr/US93/0~863 Following amplification, the double-labeled LCR amplification products were detected in replicates via a sandwich immunoassay performed on the Abbott IMx(~ !
system with results shown in Table 4 below. The method for detection on the IMx is described in the literatu~e.
S Table 4 H20 negative control 39.3 - 25.7 48.6 104 HCV RMA i 43.0 153.9 105HCV RNA 1269.4 1227.0 7x107 ~globin RNA 14.4 45.0 7x101 B-globin RN~ 83.4 71.3 - - - --- :

l~mulQ~ ' ''25 ~--~
The following probe set was designed to detect HCV target RNA as ~he target sequence. The underliried bases become stopbases, as will be seen. I'he probes were synthesized and labeled as descri d in Example 1.

First Probe bio-AccGTTTcTGcGTGAAGAcAG~AG SEQ ID No. S
Second Probe fl-CACCATAGATCACTCCCCTGTGAGGAA SEQ nD No.6 -;
Third P~be pACTGTCTTCACGCAGAAACGGT-bio SEQ ~D No.7 FourthProbe pACAGGGGAGTGAT5TATGGTG~fl SEQ ID No. 8 .
RNA target was the sasne as in example 1. Detection of target RNA was accomplished by a two step reaction as described in Exarnple 1, above. The fLrst step was a reverse transcription step to extend the first probe in the presence of only dCIP
and dl~P and generate a 6 base sticky end of S'-TrCCI'C. Reverse ~anscriphon proceeded as desc~ibed in Example 1, Step 1, except that dCI P and dl-IP were substituted for dGTP and dATP. The reaction was initiated by incubation at 42 C for fifteen ~nutes. ~e s~nple was th:en incubat~d at 99 (: for ~ min to inactivate the reverse ~ansc~iptase and den~ure ~he RNA:DNA hybrid. The sample was then incubated at 5 C for ~ min.
Using C~AP LCR unde~ the condi~ons set forth below, the secvnd probe was e~ctended by DNA polymerase and ligated to ~he third probe. Simila¢ly, the fourth probe W0 93/24656 21 36;7~ ~ PCr/VS93/04863 ;,:

and the extended first probe were ligated when hybndized to the second probe. To 2û
of the step 1 reac~on, 80 ~L of ~he following~ mixture was added.
Table S
Final concentranonl8Q Uk S LCR buffer 50 mM Epps pH 7.7, 3Q mM MgC12, 19.2 mM K+
NAD (lOmM) 62.5,uM
Oligos 2, 3 and 4 0.021 ~M ~lx1012) each DNA Ligase (1.53E5UI,lL) 4500 units DNA Poly~erase (4U/~L) 0.5 unit The following complex was ~ormed, wherein nucleotides shown in lowercase (all c or t) result ~rom the extension steps, and the underlined bases are stopbases.
SEO ID Nos.

fl-CACCATAGATCACTCCCCTG~GAGGAActACTGTCTTCACGCAGAAACGGT-bio 31 ~l-GTGGTATCTAGTGAGGGGACActccttGA~GACAGAAGTGCGTCTTTGCCA-bio 31' Gap LCR extension was performed by incubation at 85 C for 60 seconds followed by 25 ~C for 30 minutes. The amplification procedure was performed for 47 cycles, each cycle consisting of a 30 second incubation at 85 C, followed by a 64 ~C
incubation for 30 seconds.
Following amplification, the double hapten labeled LC~ amplification products detected in triplicates via a sandwich immunoassay perfo~ned on the Abbott IMx(~system with results as follows:
Table 6 ....~
Number or Molecules IMx Rate (c/s!s) H2O negative control........ 9.0 8.9 8.5 ol HCV RNA................. 9.2 9.1 .
8.8 1 o2 HCV RNA................ 8.4 g 8 103 H~V RNA................. 9.0 f 9.1 j~
4~ 8-7 104 HCV RNA ...................... 1076.3 778.1 ix107 J~-globin RNA.......................... 8.4 8.7 8.7 . . , . _ . _ . . ~ , Thus, 104 HCV target molecules could be distinguished from no targeL.

WO 9~/24656 PCr/~'S93/04~63 ~36~64 18 It is well understood by those s~cilled in the art that 100% homology is not essential to achieve hybridization. Depending on the precise conditions, as little as 60%
homolojgy may be sufficient to achieve hybridization, albeit under lower stringency 5 conditions. For thermally cycled procedures such as LCR and PCR, homology in the range of 75-100% is thought necessary, preferably at least 80%. Thus, a 20-mer probe may vary from target in aQ.7 many as 4 nucleotides. Of course, the position of any mismatches in the probe may be crucial. Mismatches at the juncture of extension or ligation, if tolerated at all, will have a much more deleterious effect than an equivalent 10 number of mismatches at the outside ends of the probes.
~sam~
Location of HCY tar~et regions com~atible with other Drobe d~si~ns:
The invention is useful with any known RNA target. For any DNA regions known to code for RNA (e.g. mRNA or rRNA), or for any known RNA sequences, the 15 sequences can be searched for target regions meeting the requirements of this invention.
Of course, it rnay 'oe desirable to convert a RNA sequence to itS corresponding DNA
sequence first. A generalized search methodology is to look ~or regions that meet the criteria of Table 8 below, wherein the symbols have the following meanings:
'~;
Table 7 `
~ymbol__ Meani~ ~ I SYm'OOI Meaning ~
A Ademne ~ ~ R A or G only B any base but adenine (not A) S C or G only ~C Cytosine T Thymine ;
D any base but cytosine (not C) U Uracil G Guanine V any base but thymine/uracil H any base but guanine (not G) W A or TIU only K G or T/U only ~ Y C or T/U only M A or C only N any base h an integer from about 5 to about 15 k an integer from 0 to about 5 , . . , :, .
, 1 . I , '` I ~ i ' : , : ., , ., ,...
~., - .
~ . ~

! - WO 93/24656 21 3 ~ 7 ~ ~ PCl`/US93/04863 Table 8 EXEMPL~RY ASYMMETRIC GAP TARGETS
e do~ "."serves only ~ aligTI sequences in the Table and to divide between right and lef~ j S probe s~ All targe~s are wriUen with their S' end to the left. ) 1, r ~ SEQUENCES TO SEAR CH FOR
. . . _ ~
Filling with just one v (T) h . (A) kB A fills nucleoside triphosphate type H (G) h~ (C) kD C fills D (C) h~ (G) kH G fills ~ ~ B (A) h (T) kV T fills Fillillg with two s (w) h~ (S) kW C and G fill complementary nucleoside w ~s) h~ (W) kS A and T fill triphosPhate t~es _ . ~ . _. ., . .. . .. .. ~ . .. ~ _ _ Fillingwithtwonon- M(~C)h. ~M~kK A and C fill complement~ nucleoside R(Y)h.~R)kY A and G fill ~iphosphate types Y (R) h. (Y) kR C and T fill K(M)h. (X)kM G and T fill . ~ , , , , . _ .
Fillingwithallthree T(v)h. (s)kA c, G and T fill nucleoside triphosphate G (H)h.(D)kC A, G and T fill types C(D)h~ (H)kG A, C and T fill AtB)h. (V)kT A, C and G fill _ , , .

Integers are selected for h and k and the search is conducted. It may be przdicted that few ~arget re~ions will be found which support single nucleoside ~iphosphate 10 filling. If such regions do exist,,they may not work well due to melting temperature constraints or secondary structure constraints. By contrast, one might expect to find ~ven grea~er numbers of locations when ~hree different nucleoside triphosphates are used to fill the gap. To stril;e a balance b~tween the number of available locations and a desire to limit the amount of reagents added to each reaction, it seems reasonable to 15 search for regions which can be filled with two nucleo~ide types.
The attached Appendix A shows jus such tw~base-fill loca~ons in the CHUMR
HCV Sequence, using co~Dventional nomenclature understood by ~hose skilled in the art~ ~, ~a~* 4 20 ~ _ ~
An assay is performed for the detection of rabbit B-globin mRNA according to the procedures and condi~ons of Examples 1 and 2. The mRNA sequence and numbering system is that publishe~ by GENBANK. ver. 69 Acc NO. J00659 and the W(:~ 93/246~6 PCr/US93/04~63 ~, ~36~ 6 4 ~
selected pl~bes hybridize in the region between 532 and 584. The following probes were synthesized and labeled as described in Example 1, the stop bases being underlined:

5 FirstPro~e biotin-ACCAGCAGCCTGCCCAGGGCCT SEQID No. 9 Second Probe fluor-GCAAGGT~AATGTGGAAGA~GTTGGTGGTG SEQ DDNo.10 Third ~be pGGCCCTGGGCAGGCTGCTGG-bio~n SEQ ~D No.ll Fourth Probe pTTCTTCCACATTCACCTTGCC~fluor SEQ ID No. 12 The probes are designed in this case to have a 10 base gap between the first and10 fourth probes, and a 1 base gap between the second and third probes. They align on target (SEQ ID No 32) as ~ollows, the gaps being filled by C and A only:

~EO ID Nos.
5'- GCAAGGUGAAUGUGGAAGAAG W GGUGGUGAGGCCCUGGGCAGGCUGCUGGU-3' 32 lS T~CGGGACCCGTCCGACGACCA-5' 9 3'- CCGTTCCACTTACACCTTCTT 12 :
S'- GCAAGGTGAATGTGGAAGAaGTTGGTGGTG 10 GGCCCTGGGCAGGCTGCTGGT-3' 11 The outside termini of the probes are labeled with differentiable haptens biotinand fluorescein as before and the amplified product is detected in the Abbott IMx~ -system as before. :
It is noted that two o~er potential asymrnetric gap LCR loca~ons have been 25 identified in the rabbit ~-globin mRNA as shown in Table 9 below Note that the gap in the top s~and is nonexistent in these cases.
Table 9 Position G ratio Fillin nucleotides :
__ ap _ _ 467-477 11:0 G and T
~ _ _ 1645-1653 9:0 A and T . .

Of course, as discussed above, one would expect to find several other locations in the genome if the search were expanded to include shorter gaps, or to include three nucleotide types in filling the gap. ~, . i The following probes were synthesized as described in Example 1. The probe ends indica~ed were labeled ~nth reporter haptens designated "crb'~ (~or a carbazole . .-deriva~ive~ and "adam'~ (for an adamantane derivative) using hapten-phosphoramidi~es as `~
is known in the art (see section above on Detec~on~. ;-WO 93/~4656 2 ~ 3 6 76 4 PCI/US93/~4863 ,; .

esignation 5'-Sequence-39 Sequence ID No.
1 adam-CTAGTGTAGCTGCTGGTCCCAATG 13 2 crb-CGAACCCAGATTGTAAGACTATTTTAAAAG 14 3 pGGGACCAGCAGCTACACTAG-adam 15 4 pGTCTTACAATCTGGGTTCG-crb 16 The probes are specific for positions 1773-1826 of the C;AG region of HIY-1 and were selected from HIY SF2CG GenBank release 71 Accession number K02007.
10 HIV RNA target was a RNA ~anscript of approximately 675 bases prepared as in example 1 using an insert of HIV DNA in place of the HCV DMA and lineariæd with EcoR1. Probes align on the target (SEQ ID No 33) as shown below, such th~ only A, T and C are needed to fill. The nucleotides underlined and in lower case are deliberate mismatches introduced to relieve secondary s~ucture; underlined nucleotides in upper 15 case are stopbases.
SEQ ID Nos.
5'- CGAACCCAGAUUGUAAGACUAUUUUAAAAGCA W GGGACCAGCAGCUACACUAG-3' 33 GTAACCCTGGTCGTCGATGTGATC-S'adam 13 20 crb3' GQTTGGGTCTAACATTCTG 16 crbS' C~AACCCAGATTGTAAGA~TATTTTAAAAG 14 GGGACCAGCAGCTACACTAG-3'adam lS
The HIV target was diluted in Sngt~LL of E. coli 16s23s ribosomal RNA and the 25 diluent E. coli 1 6s23s ribosomal RNA alone served as a nega~ive control.
The detection of HIV RNA was accomplished in phases. The first step was to synthesize a cDNA ~rom the target and probe #1, providing only dATP, dCIP, and d~ to generate an 11 base limited length extension. This creates a sticky end vs.
probe #2. cDNA was formed under incubation conditions of 99C for 1 second, 62C30 for 15 minutes, arld 4C for 2 minutes in the following mixeure.

IlL per r~ae~on Fina} concentra~on Mine~loil ~ -- 1 drop 10 mM MnC12 1.8 1 mM MnCl2 100 mM Tris /900 mM KCl 1.8 10 mM Tris pH8.3 pH~ 8.3 90 n~M KCl Therrnus sp. Polymerase .125 0.5 Units 4U/~
L~TP, dClP & dlTP (lmM) 0.18 10 IlM e~ch H20 12.93 Olig~#l lx1012/~ 0 8xlOll oligos The second step was ~ormation of a full length DNA product by separaeing 45 ex~ended probe 1 from the template RNA and hybridizing ies sticky end with probe 2, and liga~ng probe 4 to extended probe 1, using probe 2 as the template. Step three, which is really done concurrently with step 2, was (:;ap LCR (GLCR~ as described in :

W{~ g3/~4656 PCI /US93/04~3 ?,;~36164 22 `~
- EP-A-439 182. This process takes advantage of the sticky ended probe #1 generated in the firse step. Probes 2 and 3 can now both hybridize to the extended probe #1. Probe 2 is also extended in the presence of only dAl P, dCI P and dl~ and is ligated to probe #3 to form a first DNA target strand for subsequent GLCR cycles of amplification.
5 Sirrularly, in subsequent cycles, probe #4 and the extended probe #1 can be ligated while hyb~idized to probe #2 to forrn a second DNA target strand for ampli~lca~ion.
To 20 ~L of the above reaction rnixture, 180 ~L of the following rnixture was added. The 200 ~IL reaction rnixture was cycled 40 times at 97C for 1 second, 55C for 1 second, and 62C for 50 seconds.
1 0 _ _ _ _ _ _ _ per reac~on Final conc. l 200 H20 130.28 LCR buffer 40.050mM Epps pH 7.7 1 8.8 mM K+
lS Oligos 2, 3 & 4 0.88X1011 oligos each DNA Ligase 0.05268942 Units Thennus sp. Polymerase 0.125 0.5 Units 5% EGTA/lM KOH 1.14 0.75 mM EGTA
lM MgCl2 _ 6.0 30 mM
Following amplification, the double hapten labeled LCR amplification products were detected in tnplicates via a sandwich imrnunoassay performed on the Abbott IMx~
system with results as follows:
Table 10 Number or Mol~cules IMx Rate (c/s!s) rRNA (negadvecontrol) 7 9 6.9 10 HIV EaNA ~ 7.3 9.0 18.0 :
102 HIV RNA 29.9 37.7 22 . 3 103 HIV RNA 105.9 43.6 141.9 .
104 HIV RNA 496.6 525.3 655.4 This shows detection sensitivity of at least 103 molecules and potentially 10 molecules of target HIV RNA.

::

WO 93/24656 2 1 3 6 7 6 ~ PCI`/US93/04863 ~ 23 ~L-~
The following probes were synthesized as described in Example 1. The probe ends indicated were labeled wi~ reporter haptens designated "crb" (for a carbazole S derivative) and "adam" (for an adamantane derivative) using hapten-phosphoramidites as in Example ~.
esignatiQn 5'-Se~uence-3' Seg~ence ID No.
0 1 adam-GTATTGCTACTTGTGATTGCTCCA 17 2 crb-GAGCAGTATCTGGAGACCTGGAAAAACA 18 3 pAGCAATCACAAGTAGCAATAC-adam 19 4 pAGGTCTCCAGATACTGCTC-crb 20 The probes are specific for positions 8905-8956 of the NEF region of HIV-1 and were selected from HIV SF2CG GenBank release 71 Accession number K02007.
- HIV target was CsCl purified total cellular RNA from HIV infected H9 IIIB cells.
Probes align on the target (SEQ ID No 34) as shown below, such that only G, T and C
are needed to fill. The nucleo~des underlined and in lower case are deli~erate mismatches introduced to relieve secondary s~ucture; underlined nucleo~des in upper case are stopbases.
SEQ ~D Nos~
5'- GAGCAG~AUCUGGAGACCUGGAAAAACAUGGAGCAAUCACAAGUAGCAAUAC-3' 34 ACC~CGTTAGTGTTCATCGTTATG-5'adam 17 c~b3' CTCGTCATAGA~CTCTGGA 20 crb5' GAGCAGTATCT~GAGACC~GGAAAAACA 18 . AGCAATCACAAGTAGCAATAC-3'adam 19 The HIV target was diluted in 5ng/~L of E. coli 16s23s nbosomal RNA and the diluent E. coli 16s23s ribosomal RNA alone served as a negad~e con~ol.
The detection of H[V RNA was accomplished in phases as described in example S except that dGTP replaced dATP for the extension steps and a 9 base lirnited length extension resulted, having a sticky end with respect to the second probe.
Following amplification, the double hapten labeled LCR amplification products were detected in replicates via a sandwich immunoassay performed on the Abbott IMx~
system with results as follows:

WO 93/246~6 PCr/VS93/0~63 ~6~6 24 ;s Table 1 1 Number of Molecul~s IMx Rate (c/sls) Ribosomal RNA (Negative control)~............ 71 37.6 ', 43.4 7.3 7~
approximately 102 molecules ........... 71 ~4.8 30.8 ~` . 5.8 lS `~; 22.8 approximately 103 molecules ........... 272.5 190.5 -20 262.g 27~.8 409.2 approximately 104 molecules ... 1163.8 829.7 1 128.6 ~96.8 151~.9 1206.7 This shows detection sensitivity of about 103 molecules of target RNA.

The ~ollowing probes were synthesized as described in Example 1. The probe ends indicated were labeled with reporter haptens designated "crb" (for a carbazole derivahve) and "adam" (for an adamantane derivative) using hapten-phosphora~irLidites as in Example 5.
40Desi~ation S'-Sequence 3' Se~ence ID No.
1 adam-AGATTTTTAA~TGGCTCTTGATAAA 21 2 crb-GCAGGGGCAAGGCCAATGGACATATCAAA 22 3 pCAAGAGCCATTTAAAAATCT-adam 23 ~ pCCATTGGCCTTGCCCCTGC-crb 24 . .
The probes are speci~lc for positions 3549-3603 of the pol region of HIV-1 and were selected from ~ SF2CG GenBank release 71 Accession number K02007. HIV
RNA target was the CsC1 pun~Sed total cellul~ RNA prepara~ion as in Example 6.
Sû Probes align on the target (SEQ lD No 35) as shown below, such that only dATP, dGTP, and ~IP are needed to fill. Underlined nucleo~ides are stopbases.

kJ _ WO 93/246~6 2 ~ 3 6 7 6 ~ PCl /US93/04863 SF.Q ID Nos.
5'- GCAGGGGCAAGGCCAAUGGACAUAUCAAAUUUAUCAAGAGCCAUUUAAAAAUCU-3' 35 . AAATA~TTCTCGGTAAATTTTTAGA~5'adam 21 crb3' CGTCCCCGTTCCGGTTACC 24 ~5 crb5' GCAGGGGCAAGGCCAATG~CATATCAAA 22 '" CAAGAGCCATTTAAAAATCT-3'adam 23 Extension and amplification phases were performed as in Example S except ~, lx1012 of each probe was used per reaci~on and dATP, dl~IP and dGTP (at 10 IlM) were used to ~111. Following ampli~lcation, the double hapten labeled LCR amplification products were detected in replicates via a sandwich in~nunoassay performed on ~he Abbott IMx~) system with results as follows:
Table 12 t . ~
Num~er or Mole&ulç~ IMx Rate (&l~L~
Negative Control 7 2 !5 7.4 7.3 ', 7.0 approximately 103 targe~ molecules 472.5 352.5 680.4 208.1 16.4 :: ~
The following probes were synthesized as described in Example 1. The probe ends indicated and were labeled with biotin (bio) and fluorescein (fl) on the respec~ve ends using commercially available (Clontech, Palo Alto, CA) biotin and fluorescein phosphoramidite reagents.
Desi~nation 5'-Se~uence-3' Sequence ID No.
1 fl-TCGCA~GCACCCTATCAGGCAGT25 2 bt-CGAGTAGTGTTGGGTTGCGAAAGGCCTTGTGGT 26 3 pTGCCTGATAGGGTGCTTGCGAG-fl27 4 pTTTCGCAACCCAACACTACTCGG-bt 28 The probes are specific for the 5' UTR at posi~ions 246-302 of the HPCHUMR
Sequence as found in GenBanlc release 71 Accession num~er M~8335. HCV RNA
ta~get was ~hat used in Example 1. Probes align on the target (SEQ ID No 36) as shown 45 below, such th~t only A, C and G are needed ~o fill. Stopbases are underlined.

,. .
W093/246~ 26 PClIU593/Og~i3 SEQ ID Nos.
i S'- CGAGUAGUG W GGGUUGCGAAAGGCC W GUGGUACUGCCUGAUAGGGUGC W GCGAG-3' 36 TG~CGGACTATCCCACGAACGCT-5'fl 25 bt3' GGCTCATCACAACCCAACGCTTT 28 I S bt5' CGAGTAGTGTTGGGTTGCGAAaGGCCTTGTGGT 26 I TGCCTGATAGGGTGCTTGCGAG-3'fl 27 The HCV target was diluted Ln 2ng/~L of E. coli 16s23s ribosomal RNA and the diluent E. coli 16s23s ribosomal RNA alone se~ved as a negative control.
The detection of HCV RNA was accomplished in phases. The first step was to synthesize a cDNA from the RNA target and probe #1, providing only dATP, dCIP, and dGTP to generate an 11 base limited length extension. This creates a sticky end vs.
pro~e #2. cDNA was f~rmed under incubation conditions of 99C for i second, 62Cfor lS minutes, and 4C for 2 minutes in the following rnixture.
- IlL~erreac~n ~concçntra~i~n Mineral oil -- 1 drop 10 mM MnC12 1.8 1 mM MnC12 100 mM Tris t900 mM KCl 1.8 10 mM Tris pH8.3 pH 8.3 90 mM KCl Thennus sp. Polymerase .125 O.S Units 4U/~L
dATP, dCTP & dGTP 0.18 10 ,uM (each) H20 4.93 Oligo#1 lx1012/~,lL 0.80 8xlOll oligos RNA Tar~et 2.0 variable '~e second step was formation of a full length DNA product by separating extended probe 1 from the template RNA and hybridizing its sticky end with probe 2, and ligating probe 4 to extended probe 1, using probe 2 as the template. Step three, which is really done concuIrently with step 2, was Gap LCR (GLCR) as described in EP-A-439 182. This process takes advantage of the sticky ended probe #1 generated in tlie first step. Probes 2 and 3 can now both hybridize to the extended probe #1. Probe 2 is also extended in the presence of only dATP, dCIP and dl-rP ~nd is ligated to probe #3 to form a first DNA target s~and ~or subsequent GLCR cycles of arnplification.
I~ ~ , ; Slmilarly~ in subsequeht cycles, probe #4 and the extended probe ~1 can be ligated while hybridized to probe #2 to forrn a second DNA target s~and for amplification. The following reaction mixhlre was cycled 4V ames at 97C for 1 second, 55C for 1 second, and 62C for 50 seconds.
To 20 ~L of the above reaction mixture, 1 8û IlL of the following mixture was added. Ihe 200 ~L reacaon mixture was cycled 40 times at 97C for 1 second, 55C for 1 second, and 62C for 50 seconds.

:;~

q WO93/246~6 213676~ PCI`/US93/04863 :~! 27 ~E reaction Final conc ~ 200 H20 1340 Q8 50mM Epps pH 7.7 18.8 mM K+
~! DNA Ligase 0 0526 8942 Units Thernus sp. Polymerase 0.125 0.5 Umts 5% EGTAllM KOH 1.14 0.75 mM E
lM MgC12 _ 6.0 _ 30 mM _ _ Following arnplification, the double hapten labeled LCR amplification products were detected in ~iplicates via a sandwich immunoassay performed on the Abbott IMx(~
system with results as follows:
_ _ _ Table 13_ Number or Molecules IMx Rate (cls/s) r RNA (negative control) 19.9 102 HCV E~NA 383 2 692.2 1321.7 Ihis shows detection sensitivity of about 102 molecules of target HCV RNA.
The foregoing examples serve to illus~a~e the invention, not to limit it. The invention is defined by the appended claims.
, ~ I ! , ;
~. :
:`
' ~ ' 1;:~ ` . .

~: WO 93/24656 P~r/US93/04~3 . . ~ , ~,~36rt 6 Appendix A
: . Positions ~nd Types of HCV RNA Targets .. Supporting Asymlmetric Gap I,CR
(Posi~ioll numbering based on GENBANK, Acc. No. M58335) ;.~ No. ~f Length and ,, ,.~
l 1 10~MK 4128 i~ 1 11-0 MK 470 ~j 1 11-O YR 6066 ~ 0 SW 8830 i~l 1 9-0 SW 2880 - 4 11-0 WS 762 1386 3378 591g ~ 4 12~0 WS 1968 2036 3987 7206 `I 4 12-0 YR 4499 7326 3047 9141 ' 5 9~ RY 190 3797 4458 75Q5 8139 ! 6 11-0 KM 369 2067 5253 5558 5745 9189 7 ~0 SW 688 935 6227 6358 8308 9183 9272 7 8~ Y~ 1503 2238 2282 3784 4822 7851 9265 3398 3447 3897 4798 493~

8~ RY 1041 1190 1632 2468 2960 3263 3541 3982 5085 5481 ~801 6125 6179 6~3 6700 ~754 6870 69~4 7439 8639 24 7-0 KM 1358 166& 1804 2886 3187 3436 3982 5134 5261 6127 6244 6455 6~32 6548 6791 72t 6 7503 7944 798~ 8014 8036 8466 ~579 9~77 ; WO 93/246~6 2 1 3 6 7 6 ~ PC~r/~93/~4863 6384~397 7240 7566 7800 7963 8106 29 7-0 MK 10 278 612 655 692 76g 1134 ~3745583 6027 6296 7265 8319 9070 48765050 5g57 5984 6515 6937 7027 81588~42 8386 90~5 9329 9356 33463416 34~3 4359 4495 4904 4955 41 6~ YR 45 66 269 314 515 769 811 835 50745405 5429 549~ 552~ 6268 6599 6-0 KM 23 200 3~1 523 532 633 661 669 49955032 53~3 5546 5687 5790 6~17 64966563 6873 6920 7023 7309 76~0 , 73337701 78g4 7958 8023 8125 8295 53 ~0 WS 124 783 844 1553 1923 2313 2325 26572690 2718 3076 3198 3619 3877; ~
40574130 4546 4671 4856 5131 5194 :~i 532~5689 ~733 ~839 5893 5946 60~7 61546374 6478 6576 6585 6592 6618 ~ . .
67406920 7035 7062 71~0 7~97 74~0 : 91019115 9211 9222 WO 93J24656 . PCI/US93/M~63 ?,~36164 30 - .
,~ Appendix B Sequence Listing ¦ SEQUENCE LISTING
(1) GENERAL INFORMATION: ~
~i)APPLICANT: Ronald L. Marshall L
, John J. Carrino I Joann C. Sustachek ABBOTT LABOR~TORIES
.ii)TITLE OF INVENTION: AMPLIFICATION OF RNA SEQUENCES USING
THE LIGASE CH~IN REACTION
~iii) NUMBER OF SEQUENCES: 36 CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Abbott Laboratories I IB~ ST~EET: One Abbott Park Road j - ~C) CITY: Abbott Park ~D) STATE: Illinois (E) COUNTRY: USA
(F) ZIP: 60064-3500 ~v) COMPUTER READABLE FORM:
I ~A) ~DIUM TYPE: Floppy diskette ~B) COMPUTER: I~M PC compatible ~C) OPERATING SYSTEM: PC-DOS~MS-DOS
~D) SOFTWARE: Wordperfect ~ ~vi) CURRENT APPLICATION DATA:
I ~A) APPLICATION NUMBER:
~B) FILING D~TE:
tC) CLASSIFIC~TION:
i ~vii) PRIOR APPLICATION DATA: ~:~A) APPLICATION NUMBER: US 07/891,543 ~) FILING DATE: 29 MAY 1992 ~viii) A'rTORNEY/AGENT INFORMATION:
~A) NAME: Thomas D. Brainard (B~ REGISTRATION NUMBER: 32,459 ~C) REFERENC~/DOCKET NUMBER: 5172.PC.01 ~ix) TELECOMMUNICATION INFORMATION:
~A) TELEPHONE: 70B-937-4884 ~B) TE~EFAX: 708-938-2623 i ~2) INFORM~TION FOR SEQ ID NO: l:
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 23 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: singl~
~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Other nucleic acid ~synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CCAGGCATTG AGCGGGTTGA rcc 23 ~3) INFORMATION FOR SEQ ID NO: 2:

WO ~3/24656 2 1 3 6 7 6 ll P~/US93/0~863 ~:~ 3 . (i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 28 ~ ) TYPE: nucleic acid '.. (C) STRANDEDNESS: single ~ ~D) TOPOLOGY: linear '~, tii) MOLECULE TYPE: Other nucleic acid ~synthetic DNA) xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2:

~ 4) INFORMATION FOR SEQ ID NO: 3:
i~ ~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 ~ ) TYPE: nucleic acid '1 (C) STRANDEDNESS: single ; ~D) TOPOLOGY: linear ,;~ tii) MOLECULE TYPE: Other nucleic acid ~synthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

$

~1 - (5) INFORMATION FOR SF.Q ID NO: 4:
(i) SEQUENCE CHARACTSRISTICS:
(A) LENGTH: l9 i~ (B) TYPE: nucleic acid -~ ~C) STR~NDEDNESS: single 9 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CCCGGTCGTC GTGGCAATT l9 (6) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid tsynthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

(7) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(8) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 22 ~: (B) TYPE: nucleic acid ~ (C): STRANDEDNESS: single ~ (Dj TOPOhOGY: linear (ii) MOLECULE TYP~: Other nucleic acid (synthe~ic DNA) (xi) SEQUEUCE DESCRIPTION: SEQ ID NO: 7:
ACTGTCTTCA CGCAG~A~CG GT 22 ! . ~ ` ' ;l . .
WC~ 93/24656 PCr/US93/n4~63 '36~6~ 32 ~, ~9) INFORMATION FOR SEQ ID NO: 8:
~ (i) SEQUENCE CHARACTERISTICS:
'~ ~A) LENGTH: 21 ~ ~B~ TYPE: nucleic acid ., ~C) STRANDEDNESS: single ` ~D~ TOPOLOGY: linear ~ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
ACAGGGGAGT GATCTATGGT G . ^ 21 1 ~10) INFORMATION FOR SEQ ID NO: 9:
3 ~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 22 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single tD) TOPOLOGY: linear ~ii) MOLECULE TYPE: O~her nucleic acid (synthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

fi ~11) INFORMATION FOR SEQ ID NO: 10:
l ~i) SEQUENCE CHARACTERISTICS:
.1 (A) LENGTH: 30 I (B) TYPE: nucleic acid il ~C) STRANDEDNESS: single I ~D) TOPOLOGY: linear ¦ ~ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) I (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
~ GCAAGGTGAA TGTGGAAGAA GTTGGTGGTG 30 'l ~12) INFORMATION FOR SEQ ID NO: 11:
~7 ~i~ SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

~13) INFORMATION FOR SEQ ID NO: 12:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: ~1 (B) TYPE: nucleic acid ' I ~C) ST~ANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) : - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

(14~ INFORMATION FOR SEQ ID NO:13:
~i) SEQUENCE CHAR~CTERISTICS:
. (A) LENGTH: 24 ~B) TYPE: nu~leic acid (C) STRANDEDNESS: single ~I ~D) TOPOLOGY: linear I (ii~ MOLECULE TYPE: Other nucleic acid (synthetic DNA) j ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

WO ~3/24656 2 1 3 6 7 6 ~ PCI`/U~;93~04$63 ;, `,:

,,.5,~1 j tl5) INFORMATION FOR SEQ ID NO:14:
i) SEQUENCS CHARACTERISTICS:
~ (A) LENGTH: 30 :*,~, tB) TYPE: nucleic acid ~C) STRANDEVNESS: single ~, ~D~ TOPOLOGY: linear ~ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
,~

~16) INFORMATION FOR SEQ ID NO:15:
~ (i) 5EQUENCE CHARACTERISTICS:
:~ ~A) LENGTH: 20 (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear (ii) MOLEULE TYPE,: Other nucleic acid (synthetic DNA~
_ ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

.
tl7) INFORMATION FOR SEQ ID NO:16:
i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 ~B) TYRE: nucleic acid (C)~ STRANDEDNESS: single : ~D) TOPOLOGY: linear ~ MOLECUI.E TYPE: Other nucleic acid (synthetic DNA) : ~xi) S~QUENCE DESCRIPTION: SE~ ID NO:16:
GTCTTACAAT CTGGGTTCG : ~ 19 : :
(I8) INFORMATION FOR SEQ ID NO:17:
~i) SEQUEN~E CHARACT~RISTICS:
A) LENGTH: 24 ~B) :TYPE: nucleic acid ~C) STRANDEDNESS: single ~) TOPOLOGY: linear : ~ii) MOLECULE TYPE: Other nucleic acid ~synthetic DNA) xi) SEQUENCE:DESCRIPTION: SEQ ID NO:17:
~GTATTGCTAC TTGTGATTGC TCCA ~ 24 :
~1~) INFORMATION~FOR SEQ ID NO:18:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 ~B) TYPE: nucleic acid : ~ ~ ~C) STRAND~DNESS: single : : (D): TOPOLOGY: linear MOLECULE TYPE: Qther nucleic acid ~synthetic DNA) xi) SEQUE~CE DESCRIPTION: SEQ ID NO:18:
GAGCAGTATC~TGGAGACCTG GAAAAACA ~ 28 ~ : (20)~ INFO~M~TION FOR~SEQ ID NO:19:
: :~ i) :SEQUENCE CHARACTERISTICS:
: : (A)~ :LENGTH:~21 B):~TYPE:~ :nucleic acid C):~ STR~NDEDNESS: single D) TOPOLOGY:` linear :` ~

W O 93/24656 ' ` PcT/~s93/o~x63 6~36~6~ 34 - ~ii) MOLECULE TYPE: Other nucleic acid (synthetlc DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

~21) INFORMATION FOR SEQ ID NO:20:
~i) SEQUENCE CHAR~CTERISTICS:
~A) LENGTH: 19 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~.
~D~ TOPOLOGY: linear ~ii) MOL~CULE TYPE: Other nucleic acid (synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

~22) INFORMATION FOR SEQ ID NO:21:
~i) SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 25 ~B) TYPE: nucleic acid (C) STRANDEDNESS: single - (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

(23) INFORMATION FOR SEQ ID NO:22:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 29 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(24) INFORM~TION FOR SEQ ID NO:23:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear : (ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
C~AGAGCCAT TTAAAA~TCT 20 1 ~
(25) INFORMATION FOR SEQ ID NO:24:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 19 ~B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: O~her nucleic acid (synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

(26) ~NFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 B) TYPE: nucleic acid ~ W O 93/24656 2 1 3 6 7 6 4 PCT/US93/n4~63 ;~ 35 (C) STRANDEDNESS: single tD) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2S:
TCGC.~AGCAC CCTATCAGGC AGT 23 (27) INFORMATION FOR SEQ ID No:26:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: Other nucleic acid (synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID No:26:

~2~) INFORMATION FOR SEQ ID NO:27:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: singlc ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid tsynthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27 (29) INFO~MATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid ~synthetic DNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 a ~30) INFORMATION FOR SEQ ID NO:29:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 53 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: :linear ~ii) MOLECUhE TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Ii GGAAWGCCA GGACGACCGG GUCCWUCUU GGAUCAACCC GCUCAAUGCC UGG. 53 ~31) INFORMATION F ~ SEQ ID NO:30:
~i) SEQUEN -~ARACTERISTICS:
hENGTH: 51 ~) TYPE: nucleic a~id ~C) STRANDEDNESS: double ~D): TOPOLOGY: linear ~ ii) MOLECULE TYPE: Other nucleic acid ~synthetic DNA) : ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

(32) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE:CHAR~CTERISTICS:
~.:.

WO 93/2~65~ . PCI'~US~3/04~63 ~36~l64 36 (A) LENGTH: 51 (B) TYPE: nucleic acid ~C) STRANDEDNESS: double ~D) TOPOLOGY: linear ~ii) MOLECULE TY~E: Other nucleic acid (synthetic DNA) ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

~33) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: S3 ~) TYPE: nucleic acid ~C) STRANDEDNESS: ~ingle ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: RNA
~xi) SEQUENCE DESCRIPT}ON: SEQ ID NO:32:

~34) INFORMATION FOR SEQ ID No:33:
~i) SEQUENCE CHARACTERISTICS:
~A) 1ENGTH: 54 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: RNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

~35) INFORMATION FOR SEQ ID NO:34:
~i) SEQIJENCE CHARACTERISTICS:
~A) LENGTH: 52 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D~ TOPOLOGY: linear ~ii) MOLECULE TYPE: RNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
GAGCAGUAUC UGGAGACCUG GAA~AACAUG GAGCAAUCAC AAGUAGCAAU AC 52 ~36) INFORMATIGN FOR SEQ ID NO:35:
~i) SEQU5NCE CHARACTERISTICS:
~A) LENGTH: 54 ~B) TYPE: nucleic acid tC) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE ;TYPE: RNA
' I ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
GCAGGGGCAA GGCCAAUGGA CAU~UCAAAU W AUCAAGAG CCAUUUAAAA AUCU 54 ~37) INFORM~TION FOR SEQ ID N~:36:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 57 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) :TOPOLOGY: linear ~ii) MOLECULE TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Claims (34)

What is claimed is:

1. A method of amplifying a known RNA target sequence present in a biological sample, said method comprising:
(a) treating RNA in the sample under hybridizing conditions with a first oligonucleotide probe which is hybridizable to a first segment of the known target RNA
under specific hybridizing conditions;
(b) extending a 3' terminus of said first probe by reverse transcription of the RNA target so that a cDNA segment is produced having at its 5' end said first probe and at its extended 3' end which, under hybridizing conditions, is a nucleotide sequence complementary to a second segment of the target RNA, said reverse transcription being limited to the addition of not more than about 30 nucleotides;
(c) dissociating the extended first probe from the RNA target;
(d) hybridizing a second oligonucleotide probe to said extended first probe, said second probe having a 3' end which, under specific hybridizing conditions, is hybridizable to the extended cDNA segment of the first probe, but substantially not hybridizable to said first probe when it is unextended;
(e) forming at least one of:
(i) an elongated second probe complex by covalently ligating a third oligonucleotide probe to the 3' terminus of said second probe, with the proviso that if said second or third probe is modified, it is corrected prior to ligation of the third probe to the second probe; or (ii) an elongated first probe complex by forming a fourth oligonucleotide segment covalently attached to the 3' terminus of said first probe and complementary to said second probe; and (f) amplifying at least one of said elongated second probe complex and said elongated first probe complex.

2. The method of Claim 1, wherein said elongated first probe complex is formed by the polymerization of nucleotide triphosphates to the 3' terminus of the extended first probe.

3. The method of Claim 1, wherein said elongated first probe complex is formed by covalently ligating a fourth oligonucleotide probe to the 3' terminus of the extended first probe.

4. The method of Claim 3, wherein said first probe is extended by polymerization of nucleotide triphosphates to its 3' end, and wherein said elongated first probe complex is formed by covalently ligating the 5' terminus of a fourth oligonucleotide probe to the 3' terminus of the extended first probe.

5. The method of Claim 4, wherein said first probe is extended by polymerization of between about 5 and about 15 nucleotides.

6. The method of Claim 1, wherein said elongated second probe complex is formed by covalently ligating the 5' end of a third oligonucleotide probe directly to the 3' terminus of the second probe.

7. The method of Claim 1, wherein said second probe is corrected by polymerization extension, adding nucleotide triphosphates to its 3' end, and wherein said elongated second probe complex is formed by covalently ligating the 5' terminus of a third oligonucleotide probe to the 3' terminus of the extended second probe.

8. The method of Claim 7, wherein said second probe is extended by polymerization of between 1 and 5 nucleotides.

9. The method of Claim 1, wherein said second probe is corrected by cleavage of a blocking moiety present on its 3' end, and wherein said elongated second probe complex is formed by covalently ligating the 5' terminus of a third oligonucleotide probe to the 3' terminus of the corrected second probe.

10. The method of Claim 1, wherein said reverse transcription comprises the use of a reverse transcriptase or a DNA polymerase.

11. The method of Claim 1 wherein the length of the cDNA extension of the first probe is limited to a predetermined length by providing a combination of less than all four nucleoside triphosphate types complementary to a selected segment of target RNA.

12. The method of Claim 11, wherein the length of the cDNA extension comprises a sufficient number of bases complementary to the target RNA so that said second and first probes form a stable hybridization complex under the reaction conditions.

13. The method of Claim 11, wherein the length of the cDNA extension from the first probe is from about 5 to about 15 bases.

14. The method of Claim 1, wherein said target RNA sequence is selected from genomic RNA, mRNA, tRNA, rRNA, nuclear RNA, cytoplasmic RNA, total RNA, and viral RNA and any combination thereof.

15. The method according to Claim 1, wherein said amplifying step comprises at least one repeated cycle of forming both:

PCT/US 93/048??

(i) an elongation complex of a third oligonucleotide probe covalently ligated to the 3' terminus of said second probe and complementary to at least a portion of said first probe, with the proviso that if said second or third probe is modified, it is corrected prior to ligation of the third probe to the second probe; and (ii) an elongation oligonucleotide complex covalenely attached to the 3' terminus of said first probe and complementary to at least a portion of said second probe.

16. The method according to Claim 15, wherein said first, second and third oligonucleotide probes used in the amplifying step are identical to those used in step (e) to form the elongation probe complex.

17. The method according to Claim 15, wherein said fourth oligonucleotide segment is formed by covalently ligating a fourth oligonucleotide probe to the 3' terminus of said first probe, with the proviso that said first probe is extended prior to ligation, whereby said amplification step becomes a variation of LCR.

18. The method according to Claim 15, wherein said elongation oligonucleotide complex is formed solely by extension of said first probe, whereby said amplification step becomes a hybrid amplification reaction with elongation forming one elongation complex and polymerization extension forming the other elongation complex.

19. A method of amplifying a known RNA target sequence present in a biological sample, said method comprising:
(a) treating RNA in the sample under specific hybridizing conditions with a first oligonucleotide probe which is hybridizable to a first segment of the known target RNA
under said hybridizing conditions;
(b) extending a 3' terminus of said probe by reverse transcription of the RNA
target so that a cDNA segment is produced having at its 5' end said first probe acid at its extended 3' end a nucleotide sequence complementary to a second segment of the target RNA, wherein the length of the cDNA extension of the first probe is limited to apredetermined length by providing a combination of less than all four nucleosidetriphosphate types complementary to a selected segment of target RNA;
(c) dissociating the extended first probe from the RNA target;
(d) hybridizing a second oligonucleotide probe to said extended first probe, said second probe having a 3' end which, under hybridizing conditions, is hybridizable to the extended cDNA segment of the first probe but substantially not hybridizable to said first probe when the first probe is unextended;
(e) ligating a third oligonucleotide probe, complementary to said first probe, to the 3' terminus of said second probe, with the proviso that if said second probe is extended prior to ligation, the third probe is ligated to the 3' terminus of the extended second probe, thereby to form an extended second probe complex;
(f) ligating a fourth oligonucleotide probe, complementary to said second probe,to the 3' terminus of said first probe, with the proviso that if said first probe is extended prior to ligation, the fourth probe is ligated to the 3' terminus of the extended first probe, thereby to form an extended first probe complex; and (g) amplifying at least one of said extended second probe complex and said extended first probe complex by the ligase chain reaction, using the first, second, third and fourth probes as reactants, with the proviso that if said reactant probes are modified, they are corrected prior to ligation.

20. A method of forming cDNA of a predetermined length from a known RNA target sequence present in a sample, comprising the steps of:
(a) treating RNA under specific hybridizing conditions with a first oligonucleotide probe which is hybridizable to a first segment of the target RNA under said hybridizing conditions; and (b) extending a 3' terminus of said probe by reverse transcription of the RNA
under conditions including less than all four nucleoside triphosphate types, so that a cDNA segment of a predetermined length is produced, wherein such extension is terminated at said predetermined length when said RNA template requires a nucleoside triphosphate which is not present.

21. A method according to claim 20, further comprising dissociating the extended first cDNA probe from the RNA target and detecting said cDNA probe.

22. A method according to claim 21, further comprising amplifying said cDNA prior to said detecting step.

23. A diagnostic kit for detecting a nucleic acid target present in a biologicalsample, comprising in combination:
(a) a first oligonucleotide probe which is complementary to a portion of the target;
(b) an extending reagent for reverse transcription of an RNA target and/or extension of the first probe on a DNA target in the presence of a supply of the nucleoside triphosphatescomplementary to the target region 3' of the first probe, using the first probe as a primer;
(c) a second oligonucleotide probe capable of hybridizing to said first oligonucleotide probe under hybridizing conditions substantially only when said first probe has been extended by reverse transcription;
(d) at least one of (i) a third oligonucleotide probe complementary to a portion of said first probe, said probe having a 5' terminus ligatable to the 3' terminus of said second probe and complementary to a portion of said first probe, with the proviso that if said second or third probe is corrected prior to ligation, the second probe is ligatable to the third probe in their corrected form, thereby to form an elongated second probe complex;
or (ii) a fourth oligonucleotide probe which is covalently ligatable to the 3' terminus of said first probe once it is extended, and complementary to a portion of said second probe, thereby to form an elongated first probe complex; and (e) an assembling reagent for forming the elongated second probe complex, the elongated first probe complex, or both.

24. The kit of Claim 23 wherein said extending reagent comprises reverse transcriptase or DNA polymerase.

25. The kit of Claim 23 wherein said assembling reagent comprises a ligase and optionally a DNA polymerase.

26. The kit of Claim 23 wherein in step (d) a third oligonucleotide probe is provided.

27. The kit of Claim 23 wherein in step (d) a fourth oligonucleotide probe is provided.

28. The kit of Claim 26 wherein in step (d) a fourth oligonucleotide probe is also provided.

29. A kit according to Claim 23 for use in detecting HCV, wherein said first probe is about 15 to about 30 nucleotides in length and at least 80% homologous with the oligonucleotide (SEQ ID No 1) and said second probe is about 15 to about 40 nucleotides in length and at least 80%homologous with the oligonucleotide (SEQ ID No 2).

30. A kit according to Claim 23 for use in detecting HCV, wherein said first probe is about 15 to about 30 nucleotides in length and at least 80% homologous with the oligonucleotide and said second probe is about 15 to about 40 nucleotides in length and at least 80%homologous with the oligonucleotide .

31. A kit according to Claim 23 for use in detecting HCV, wherein said first probe is about 15 to about 30 nucleotides in length and at least 80% homologous with the oligonucleotide and said second probe is about 15 to about 40 nucleotides in length and at least 80%homologous with the oligonucleotide .

32. A kit according to Claim 23 for use in detecting HIV, wherein said first probe is about 15 to about 30 nucleotides in length and at least 80% homologous with the oligonucleotide and said second probe is about 15 to about 40 nucleotides in length and at least 80%homologous with the oligonucleotide .

33. A kit according to Claim 23 for use in detecting HIV, wherein said first probe is about 15 to about 30 nucleotides in length and at least 80% homologous with the oligonucleotide and said second probe is about 15 to about 40 nucleotides in length and at least 80%homologous with the oligonucleotide .

34. A kit according to Claim 23 for use in detecting HIV, wherein said first probe is about 15 to about 30 nucleotides in length and at least 80% homologous with the oligonucleotide and said second probe is about 15 to about 40 nucleotides in length and at least 80%homologous with the oligonucleotide .