Real time homogeneous nucleotide assay

A method for performing real time, homogeneous assay of a target nucleic acid comprising annealing a labeled, ribo-oligonucleotide probe to the target DNA sequence and degrading a portion of the probe with RNase H to release labeled fragments. Sequential measurements of the released fragments permits kinetic characterization of the presence of the target sequence. Preferably, the assay is integrated into a polymerase chain reaction so that target amplification can be detected in real time.

BACKGROUND OF THE INVENTION 
Recent advances in the general field of molecular biology have made it 
possible to detect specific genes of clinical and commercial importance. 
The use of nucleic acid hybridization assays as a research tool for the 
detection and identification of a unique deoxyribonucleic acid (DNA) 
sequence or a specific gene in a complete DNA, a mixture of DNA's, or a 
mixture of DNA fragments have made it possible to diagnose human disease 
at the genetic level. 
The most common techniques for detecting a specific gene sequence are 
hybridization-based assays. A specific nucleotide sequence or probe is 
marked with a detectable label, typically a radioactive label (isotopic) 
or chemical modification (non-isotopic). The detectable label is combined 
with the nucleic acid sample of interest, either in situ as part of intact 
cells or as isolated DNA or RNA fragments. The hybridization conditions 
should be those which allow the probe to form a specific hybrid with its 
complementary DNA or RNA target while not becoming bound to 
non-complementary DNA or RNA molecules. The target sample sequence can be 
either free in solution or immobilized on a solid substrate. The probe's 
detectable label provides a means for determining whether hybridization 
has occurred and, thus, for detecting the DNA or RNA target. 
Detection methods employed in nucleic acid hybridization based assay 
systems allow for the distinction of hybridized probe to the target 
nucleic acid sequence from the unhybridized probe. One of the oldest and 
most widely used procedures is called the Southern blot filter 
hybridization assay. This assay is carried out by isolation and 
immobilization of the nucleic acid target to a solid membrane 
support(nylon, nitrocellulose, etc.). The membrane bound nucleic acid 
target is subject to denaturation conditions(heating, or alkaline 
treatment) and subsequently treated with a solution containing a labeled 
probe and allowed to hybridize under conditions which reinforce the 
specificity of the labeled probe to its complementary target sequence. 
Unhybridized labeled probe is then washed away and the hybridized label 
probe is detected by the means specific for that label type, i.e., 
isotopic labeled probe detection would utilize x-ray film and 
autoradiography. 
Probes labeled with non-isotopic or chemicals such as a high energy 
transfer fluorescent moieties and their use and detection in 
immunofluorescent assays is described in U.S. Pat. Nos. 3,996,345; 
3,998,943; 4,160,016; 4,174,384; and 4,199,559, each of which are 
incorporated in their entirety by reference. These patents pertain to 
assays which utilize fluorescent light emitted from an irradiated sample 
and the use of chemical species(quenchers) to absorb some of the light 
energy. 
European Patent Publication No. 70,685 describes the design, detection and 
use of non-radiative energy transfer probes in a homogeneous nucleic acid 
diagnostic assay. This technique uses two probes which hybridize to 
adjacent sequences on the target DNA. A chemiluminescent moiety and an 
absorber/emitter moiety are attached to the 3' and 5' ends of the probes 
so that when the probes hybridize, the moieties are brought into close 
enough proximity to allow for non-radiative energy transfer. Presence of 
the target DNA allows the probes to hybridize and emit radiation having 
the wavelength specific to the absorber/emitter moiety. 
The recent advances in automated nucleic acid oligonucleotide (ribo- and 
deoxyribo-) synthesis and the polymerase chain reaction (PCR) method of 
DNA amplification have increased the power and sensitivity of nucleic acid 
hybridization assays. The use of automated chemical nucleic acid 
synthesizers for the synthesis of short gene fragments (DNA and RNA) is 
well described by Alvarado-Urbina et. al., Science, 214:270 (1981). 
Automated synthesizers have increased the efficiency of incorporating 
specific moieties into the short gene fragments which can serve as 
detectable labels and quenchers on probes for the detection and isolation 
of a desired natural gene from a living organism or a virion. The short 
gene fragments can also serve as primers in PCR and reverse 
transcription(RT) assays to enable amplification or copying of the genetic 
information carried in natural genes. 
The PCR method of DNA amplification is well described by Mullis and 
Faloona, Methods in Enzymol., vol. 155, pg. 335 (1987). Improvements in 
the PCR technique are disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; 
and 4,800,159, each of which are incorporated in their entirety by 
reference. PCR is an in vitro method for the enzymatic synthesis of 
specific DNA sequences, using two deoxyoligonucleotide primers that 
hybridize to opposite strands and flank the specific target region of DNA 
that is to be amplified. The use of automated thermal cyclers allows a 
repetitive series of reaction steps involving template denaturation, 
primer annealing and the extension of the annealed primers by DNA 
polymerase resulting in the exponential accumulation of the specific 
target region of DNA whose termini are defined by the 5' end of the 
primers. Selective enrichment of a specific target region of DNA sequence 
by a factor of 10.sup.9 was described by Saiki et. al.,Science, 230:1350 
(1985). 
Reverse transcription is a commonly employed molecular biology technique 
for the in vitro synthesis of single-stranded complementary DNA(cDNA) from 
specific RNA sequences for the preparation of cDNA libraries or can be 
used for the synthesis of first strand cDNA for use in subsequent 
amplification reactions; i.e., PCR. The use of reverse transcriptase for 
cDNA synthesis is described by Maniatis, et al., Molecular Cloning, A 
Laboratory Manual, pg. 8.11 (1989). Reverse transcriptase is a protein 
which extends the 3' end of deoxyoligonucleotide primers annealed to a 
specific complementary sequence of single-stranded RNA. Modification of 
the reverse transcriptase enzyme have allowed longer cDNA synthesis and 
higher yields and are described in U.S. Pat. No. 5,244,797, which is 
incorporated in its entirety by reference. 
The PCR technique alone and in combination with the RT reaction is an 
extremely powerful method for amplifying nucleic acid sequences, however 
the detection of the amplified material may require additional 
manipulation and subsequent handling of the PCR products to determine 
whether the target region of DNA is present. For example, removal of 
labeled probe that has not come into contact with the target sequence 
significantly complicates typical hybridization assays. A more useful 
probe technique would minimize the number of additional handling steps 
currently required for the detection of the amplified material. Ideally, 
such a technique would combine the amplification and detection steps into 
a homogeneous system, thereby eliminating the need for a post 
amplification phase separation of target-contacted and 
target-non-contacted probe prior to signal detection. Such a homogeneous 
system permits repeated detection of the signal permitting a kinetic 
analysis of the amplification process. 
A kinetic analysis offers significant advantages over a single, end-point 
analysis. For example, the qualitative assessment of the development of 
signal can greatly increase the accuracy of amplification systems by 
revealing problems such as false positives or other false quantifications. 
However, the design of homogeneous probe systems is constrained by the 
probe's potential interference with the amplification. In PCR, for 
example, the processivity of the polymerase must not be blocked by the 
presence of a down stream probe. 
U.S. Pat. Nos. 5,210,015 and 5,487,972, both of which are incorporated in 
their entirety by reference, describe methods for nucleic acid detection 
which rely on the 5' to 3' nuclease activity of a nucleic acid polymerase 
to cleave annealed labeled DNA probes and thus release labeled 
oligonucleotide fragments for detection. An enhancement on this technique 
is described by Livak et al., PCR Methods and Applications, 4:357 (1995), 
using a reporter fluorescent dye and a quencher fluorescent dye attached 
to the 5' and 3' ends of an oligonucleotide probe. As the polymerase moves 
along the target DNA sequence in a 3' direction, its 5' nuclease activity 
first displaces and then cleaves the oligonucleotide probe, separating the 
reporter from the quencher. Thus, presence of target DNA sequence may be 
measured by detecting fluorescence of the reporter dye. 
These assays depend on the 5' nuclease activity of the polymerase which 
places significant constraints on the design of probes that can be used. 
For example, the label must be attached to DNA and the probe must be 
designed to allow cleavage from the 5' end. Moreover, since one enzyme is 
being required to provide both polymerase and nuclease activity, it is not 
possible to independently select or optimize those events. 
Existing alternatives to PCR based assays rely on amplification of the 
signal produced by the target sequence, instead of amplifying the target 
directly. These methods require significant handling steps and are 
directed to an end point analysis as opposed to a kinetic, real time 
determination of target sequence presence. 
For example, U.S. Pat. Nos. 4,876,187 and 5,011,769, which are incorporated 
in their entirety by reference, Duck et al., BioTechniques, 9:142 (1990) 
and Bekkaoui et al., BioTechniques, 20:240 (1996) disclose a cycling probe 
method that employs probes comprising RNA, preferably DNA:RNA:DNA 
chimeras. The reaction is carried out isothermally, using a temperature at 
which the chimeric probes will anneal to the target DNA. An enzyme such as 
RNase H is used to digest the RNA portion of the probe and generate 
shorter, labeled oligonucleotides which dissociate at the reaction 
temperature. The target DNA sequence is then available for hybridization 
with another probe and, after a number of cycles, sufficient label has 
been generated to collect and detect. In general, these methods rely on 
immobilizing a portion of the label to allow for phase separation and 
signal recovery and measurement. Bekkaoui et al. report a modification of 
this technique, dealing with the formation of a RNase-streptavidin fusion 
enzyme and its use with a biotinylated probe. The streptavidin-biotin 
binding brings the fusion enzyme into proximity with the probe and thus 
increases its RNase activity. However, the enzyme becomes non-functional 
once the attached probe is cleaved, preventing it from participating in 
subsequent cycles. 
The above signal amplification strategies do not generate a real time 
signal since a number of cycles are required before sufficient label is 
released to permit detection. Further, the techniques are designed to be 
used as an alternative to conventional target amplification strategies and 
require isothermal conditions. However, the methods rely on phase 
separation for detection of the label, and thus, are not directed to 
homogenous systems. Also, the choice of probe design is limited because 
the nuclease activity of polymerases could attack the DNA portion of a 
chimeric probes, generating false signal. 
Accordingly, there remains a need for strategies capable of providing real 
time homogenous detection of nucleic acid amplification capable of using 
more versatile probe designs. 
SUMMARY OF THE INVENTION 
The invention comprises a method for the detection of a target DNA sequence 
in a sample which includes: a) contacting and annealing a labeled probe to 
a target single-stranded DNA sequence having a region complementary to the 
probe; b) cleaving with a ribo-nucleic acid nuclease capable of 
hydrolyzing ribonucleotides in a double stranded RNA:DNA duplex to release 
labeled probe fragments; and c) making sequential measurements of the 
released labeled fragments to permit the kinetic characterization of the 
target DNA sequence. This assay is carried out homogeneously in one 
reaction mixture. 
Preferably, the assay is used in conjunction with amplification of the 
target DNA sequence, using PCR for example. In these embodiments, the 
method comprises a) providing primers containing sequences complementary 
to regions in the the target DNA sequence, each capable of priming the 
synthesis of a complementary oligonucleotide, such that the complementary 
oligonucleotide primed can serve as a template for the synthesis of the 
complementary oligonucleotide primed by the other; b) providing labeled 
probe having a sequence complementary to a portion of the target DNA 
sequence; c) amplifying the target DNA sequence employing a polymerizing 
agent with the cycling steps of: 
i) contacting and annealing the labeled probe to the target DNA sequence, 
ii) annealing the first and/or second primer(s), 
iii) cleaving the RNA of the annealed probe with a ribo-nucleic acid 
nuclease to release labeled ribo-oligonucleotide fragments, 
iv) extending the primers with the polymerization agent, and 
v) denaturing the extended primer(s) and the target DNA sequence; and d) 
making sequential measurements of the release of labeled fragments to 
permit the kinetic characterization of target DNA sequence amplificiation. 
In another preferred embodiment, the method comprises the use of a labeled 
probe having a reporter fluorescent dye at one terminus and a quencher 
fluorescent dye at the other terminus and the step of detecting the 
release of labeled ribo-oligonucleotide fragments comprises measuring 
reporter fluorescence. The quencher suppresses reporter fluorescence until 
the annealed probe is cleaved, allowing discrimination between 
target-contacted probe and target non-contacted probe. 
The methods of this invention allow detection of a target DNA sequence in a 
homogeneous, real time system. When combined with target DNA 
amplification, the method offers probe release independent of polymerase 
activity and great flexibility in probe design. Futher, the kinetic 
detection of this invention allows real time analysis unlike the prior art 
endpoint assays.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
The term "sample" or "specimen" refers to nucleic acid isolated from an 
individual(s) or any nucleic acid containing entity, including but not 
limited to; skin, plasma, serum, spinal fluid, lymph fluid, synovial 
fluid, urine, tears, blood cells, organs, tumors, in vitro cell culture 
constituents, bacteria and viruses. 
As used herein, the terms "nucleic acid", "polynucleotide" and 
"oligonucleotide" refer to primers, probes, oligomer fragments to be 
detected, oligomer controls and unlabeled blocking oligomers and shall be 
generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to 
polyribonucleotides (containing D-ribose) as well as chimeric 
polynucleotides (containing 2-deoxy-D-ribose and D-ribose nucleotides), 
and to any other type of polynucleotide which is an N glycoside of a 
purine or pyrimidine base, or modified purine or pyrimidine bases. There 
is no conceived distinction in length between the term "nucleic 
acid","polynucleotide" and "oligonucleotide", and these terms are used 
interchangeably. Thus, these terms include double-and single stranded DNA, 
as well as double- and single stranded RNA. The oligonucleotide is 
composed of a sequence of at least 8 nucleotides, by preference at least 
10-12 nucleotides, and more preferably at least 15-20 nucleotides 
coterminous to a region of the designated nucleotide sequence. 
"Coterminous" means identical to or complementary to the determined 
sequence. 
The oligonucleotide is not necessarily limited to a physically derived 
species isolated from any existing or natural sequence but may be 
generated in any manner, including chemical synthesis, DNA replication, 
reverse transcription or a combination thereof. The terms 
"oligonucleotide" or "nucleic acid" refers to a polynucleotide of genomic 
DNA or RNA, cDNA, semisynthetic, or synthetic origin which, by virtue of 
its derivation or manipulation: (1) is not affiliated with all or a 
portion of the polynucleotide with which it is associated in nature; 
and/or (2) is connected to a polynucleotide other than that to which it is 
connected in nature; and (3) is unnatural(not found in nature). 
Oligonucleotides are composed of reacted mononucleotides to make 
oligonucleotides in a manner such that the 5' phosphate of one 
mononucleotide pentose ring is attached to the 3' oxygen of its neighbor 
in one direction via a phosphodiester linkage, and is referred to as the 
"5' end" end of an oligonucleotide if its 5' phosphate is not linked to 
the 3' oxygen of a mononucleotide pentose ring and subsequently referred 
to as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a 
subsequent mononucleotide pentose ring. A nucleic acid sequence, even if 
internalized to a larger oligonucleotide, also may be said to have 5' and 
3' ends. Two distinct, non-overlapping oligonucleotides annealed to two 
different regions of the same linear complementary nucleic acid sequence, 
so the 3' end of one oligonucleotide points toward the 5' end of the 
other, will be termed the "upstream" oligonucleotide and the latter the 
"downstream" oligonucleotide. In general, "downstream" refers to a 
position located in the 3' direction on a single stranded oligonucleotide, 
or in a double stranded oligonucleotide, refers to a position located in 
the 3' direction of the reference nucleotide strand. 
The term "primer" may refer to more than one oligonucleotide, whether 
isolated naturally, as in a purified restriction digest, or produced 
synthetically. The primer must be capable of acting as a point of 
initiation of synthesis along a complementary strand (DNA or RNA) when 
placed under reaction conditions in which the primer extension product 
synthesized is complementary to the nucleic acid strand. These reaction 
conditions include the presence of the four different deoxyribonucleotide 
triphosphates and a polymerization-inducing agent such as DNA polymerase 
or reverse transcriptase. The reaction conditions incorporate the use of a 
compatible buffer (including components which are cofactors, or which 
affect pH, ionic strength, etc.), at an optimal temperature. The primer is 
preferably single-stranded for maximum efficiency in the amplification 
reaction. 
A complementary nucleic acid sequence refers to an oligonucleotide which, 
when aligned with the nucleic acid sequence such that the 5' end of one 
sequence is paired with the 3' end of the other. This association is 
termed as "antiparallel." Modified base analogues not commonly found in 
natural nucleic acids may be incorporated (enzymatically or synthetically) 
in the nucleic acids including but not limited to primers, probes or 
extension products of the present invention and may include, for example, 
inosine and 7-deazaguanine. Complementarity of two nucleic acid strands 
may not be perfect; some stable duplexes may contain mismatched base pairs 
or unmatched bases and one skilled in the art of nucleic acid technology 
can determine their stability hypothetically by considering a number of 
variables including, the length of the oligonucleotide, the concentration 
of cytosine and guanine bases in the oligonucleotide, ionic strength, pH 
and the number, frequency and location of the mismatched base pairs. The 
stability of a nucleic acid duplex is measured by the melting or 
dissociation temperature, or "Tm." The Tm of a particular nucleic acid 
duplex under specified reaction conditions. It is the temperature at which 
half of the base pairs have disassociated. 
As used herein, the term "target sequence" or "target nucleic acid 
sequence" refers to a region of the oligonucleotide which is to be either 
amplified, detected or both. The target sequence resides between the two 
primer sequences used for amplification or as a reverse transcribed 
single-stranded cDNA product. The target sequence may be either naturally 
derived from a sample or specimen or synthetically produced. 
As used herein, a "probe" comprises a ribo-oligonucleotide which forms a 
duplex structure with a sequence in the target nucleic acid, due to 
complementarity of at least one sequence of the ribo-oligonucleotide to a 
sequence in the target region. The probe, preferably, does not contain a 
sequence complementary to the sequence(s) used to prime the polymerase 
chain reaction (PCR) or the reverse transcription (RT) reaction. The probe 
may be chimeric, that is, composed in part of DNA. Where chimeric probes 
are used, the 3' end of the probe is generally blocked if this end is 
composed of a DNA portion to prevent incorporation of the probe into 
primer extension product. The addition of chemical moieties such as 
biotin, fluorescein, rhodamine and even a phosphate group on the 3' 
hydroxyl of the last deoxyribonucleotide base can serve as 3' end blocking 
groups and under specific defined cases may simultaneously serve as 
detectable labels or as quenchers. Furthermore, the probe may incorporate 
modified bases or modified linkages to permit greater control of 
hybridization, polymerization or hydrolyzation. 
The term "label" refers to any atom or molecule which can be used to 
provide a detectable (preferably quantifiable) real time signal. The 
detectable label can be attached to a nucleic acid probe or protein. 
Labels provide signals detectable by either fluorescence, phosphorescence, 
chemiluminescence, radioactivity, colorimetric (ELISA), X-ray diffraction 
or absorption, magnetism, enzymatic activity, or a combination of these. 
The term "absorber/emitter moiety" refers to a compound that is capable of 
absorbing light energy of one wavelength while simultaneously emitting 
light energy of another wavelength. This includes phosphorescent and 
fluorescent moieties. The requirements for choosing absorber/emitter pairs 
are: (1) they should be easily functionalized and coupled to the probe; 
(2) the absorber/emitter pairs should in no way impede the hybridization 
of the functionalized probe to its complementary nucleic acid target 
sequence; (3) the final emission (fluorescence) should be maximally 
sufficient and last long enough to be detected and measured by one skilled 
in the art; and (4) the use of compatible quenchers should allow 
sufficient nullification of any further emissions. 
As used in this application, "real time" refers to detection of the kinetic 
production of signal, comprising taking a plurality of readings in order 
to characterize the signal over a period of time. For example, a real time 
measurement can comprise the determination of the rate of increase of 
detectable product. Alternatively, a real time measurement may comprise 
the determination of time required before the target sequence has been 
amplified to a detectable level. 
The term "chemiluminescent and bioluminescent" include moieties which 
participate in light emitting reactions. Chemiluminescent moieties 
(catalyst) include peroxidase, bacterial luciferase, firefly luciferase, 
functionlized iron-porphyrin derivatives and others. 
As defined herein, "nuclease activity" refers to that activity of a 
template-specific ribo-nucleic acid nuclease, RNase H. As used herein, the 
term "RNase H" refers to an enzyme which specifically degrades the RNA 
portion of DNA/RNA hybrids. The enzyme does not cleave single or 
double-stranded DNA or RNA and a thermostable hybrid is available which 
remains active at the temperatures typically encountered during PCR. 
Generally, the enzyme will initiate nuclease activity whereby 
ribo-nucleotides are removed or the ribo-oligonucleotide is cleaved in the 
RNA-DNA duplex formed when the probe anneals to the target DNA sequence. 
As used herein, the term "thermostable nucleic acid polymerase" refers to 
an enzyme which is relatively stable to heat when compared, for example, 
to nucleotide polymerases from E. coli and which catalyzes the 
polymerization of nucleosides. Generally, the enzyme will initiate 
synthesis at the 3'-end of the primer annealed to the target sequence, and 
will proceed in the 5'-direction along the template. 
The term "hybridization or reaction conditions" refers to assay buffer 
conditions which allow selective hybridization of the labeled probe to its 
complementary target nucleic acid sequence. These conditions are such that 
specific hybridization of the probe to the target nucleic acid sequence is 
optimized while simultaneously allowing for but not limited to 
amplification of the target nucleic acid in a PCR assay. The reaction 
conditions are optimized for co-factors, ionic strength, pH and 
temperature. 
General Method 
The practice of this invention will engage, unless otherwise indicated, 
standard techniques of molecular biology, microbiology and recombinant DNA 
techniques, which are within the skill of the art. 
The various conditions of the invention exploit a property of RNase H. 
RNase H is an enzyme known to degrade the RNA moiety of RNA-DNA hybrid 
molecules. RNA:DNA duplexes are a substrate for RNase H due to the 
particular secondary structure. Thus, RNase is active along the length of 
the RNA:DNA duplex without positional restriction and thus is not limited 
to either terminus. RNase H will cleave monoribonucleotides or small 
ribo-oligonucleotide fragments from the duplex which are destabilized to 
the point that they dissociate from the larger, complementary 
polynucleotide (DNA). Thus, cleavage does not depend on the 
characteristics of the 5' end. This property allows great flexibility in 
the design of suitable probes. 
The present invention exploits the ribonuclease activity of the RNase H 
when used alone or in conjunction with PCR. This present invention differs 
from previously described PCR amplification wherein the post-PCR amplified 
target nucleic acid sequence(s) are detected, for example, by 
hybridization to a probe which forms a stable duplex with that of the 
target sequence under stringent hybridization and wash conditions. In 
contrast to those known detection methods used in post-PCR amplifications, 
the present invention permits the detection of the target nucleic acid 
sequences during amplification of the target nucleic acid sequence. In the 
present invention, a labeled probe is added simultaneously with the PCR 
primers and RNase H at the start of PCR. The reaction conditions utilized 
allow for the labeled probe to hybridize to the target nucleic acid 
sequence which permits the activity of the RNase H to cleave and 
dissociate the labeled probe fragments prior to the annealing of the PCR 
primers and the extension activity of the DNA polymerase. The signal 
generated from hydrolysis (cleavage) and release of the labeled 
ribo-nucleotide(s) fragments of the probe provides a means for detection 
of the target nucleic acid sequence during its amplification. 
The methods of this invention are also easily adaptable to other nucleic 
acid amplification systems. For example, homogenous assays of 
self-sustained sequence replication (3SR) and ligase chain reaction (LCR) 
systems are within the scope of this invention. 
In the present invention, a label is attached to the probe so that the 
cleaved monoribonucleotides or small ribo-oligonucleotides which are 
generated by the nuclease activity of the RNase H can be detected. Several 
strategies may be employed to distinguish the uncleaved labeled ribo- or 
chimeric oligonucleotide probes from the cleaved labeled probe fragments. 
This feature of the present invention allows identification of those 
nucleic acid containing samples or specimens which contain sequences 
complementary to the ribo- or chimeric oligonucleotide probe. 
In the present invention, a sample or specimen is provided which is 
suspected of containing the particular "target nucleic acid" sequence of 
interest. The target nucleic acid contained in the sample may be first 
reverse transcribed (RT) into cDNA, if isolated as single-stranded RNA or 
it may be isolated as double-stranded genomic DNA. The cDNA or genomic DNA 
is then denatured, using any suitable denaturing method, including 
physical, chemical, or enzymatic means, which are known to those skilled 
in the art. Physical means for strand separation involves heating the 
nucleic acid until it is completely denatured. Typical heat denaturation 
involves the use of temperatures between 80.degree. C. and 100.degree. C., 
for 3 to 10 minutes. The target nucleic acid may exist in a 
single-stranded form in the sample, such as, for example, single stranded 
RNA or DNA viruses and only moderate heating may be necessary to alleviate 
secondary fold back structures. 
The denatured nucleic acid strand(s) are then incubated with preselected 
oligonucleotide primers and a probe under hybridization or reaction 
conditions which enable the binding of the primers and probe(s) to the 
single nucleic acid strands. The primers are selected so that their 
relative positions along a duplex sequence are such that an extension 
product produced from one primer serves as a template for the extension of 
the other primer to yield a replicate chain of defined length, when the 
extension product is separated from its template (complement) under 
subsequent denaturation conditions. 
Because the complementary nucleic acid strands synthesized are longer than 
either the probe or primer, the strands have more points of contact and 
thus a greater chance of finding each other over any given period of time. 
To prevent reannealing of the longer template, a high molar excess of 
probe and primer(s) are employed to help sway the hybridization kinetics 
toward primer and probe annealing rather than template reannealing. 
The primer(s) length must be adequate to prime the synthesis of extension 
products in the presence of the reaction conditions. The length and 
composition of the primer is dependent on many factors, including 
temperature of the reaction, composition of the primer, the position of 
the probe annealing site to the primer annealing site, and the ratio of 
primer to probe concentration. Depending on the complexity of the target 
sequence, the oligonucleotide primer(s) typically contains about 15-30 
nucleotides, although it may contain more or fewer nucleotides. The 
primers must be sufficiently complementary to selectively anneal to their 
respective strands and form stable duplexes. The primers used are selected 
to be completely complementary to the different strands of each specific 
sequence to be amplified. One skilled in the art may select or design 
primers which have non-complement sequences the 5' end, such as 
restriction enzyme digestion sequences, although the 3' end must maintain 
its complementarity to insure proper extension and amplification by the 
DNA polymerase. 
In the practice of this invention, the labeled probe must be first annealed 
to its complementary nucleic acid target before the primers anneal. The 
activity of the RNase H must supersede the DNA polymerase activity, 
allowing the cleaved probe fragments to dissociate from the nucleic acid 
target, as to not interfere with the primer extension and amplification of 
the nucleic acid target region. 
To ensure that the labeled probe will anneal to its complementary nucleic 
acid before primer extension polymerization reaches this duplex region, a 
variety of techniques may be employed. The invention allows for 
significant optimization of this characteristic as opposed to the prior 
art systems limited to DNA oligonucleotide probes. RNA:DNA hybrids are 
known to have a higher melting temperature than DNA:DNA or chimeric:DNA 
hybrids of the same base composition permitting greater specificity. The 
length of complementary nucleic acids is also known to effect the 
hybridization rate and the relative stability of the duplexes. Shorter 
nucleic acid molecules generally require a cooler temperature to form 
sufficiently stable hybrid complexes with the target nucleic acid. 
Therefore, the probe can be designed to be longer than the primer so that 
the labeled probe anneals preferentially to the target at higher 
temperatures relative to primer annealing. Furthermore, the addition of a 
denaturation solution such as formamide allows for an optimal temperature 
for the association of RNA:DNA hybrids as compared to DNA:DNA hybrids. 
One can also vary the base composition of the primers and the probe to 
affect thermal stability. For example, the nucleotide composition of the 
probes can be chosen to have greater G/C content and, consequently, 
greater thermal stability than the primer(s). One skilled in the art can 
then utilize the thermocycling parameters to take advantage of the 
differential thermal stability of the labeled probe(s) and primer(s). 
Following the denaturation step in thermocycling, one could employ an 
intermediate temperature which is permissible for probe annealing and 
RNase H cleavage but not primer binding, and then the temperature can be 
further reduced to permit primer annealing and extension by the DNA 
polymerase. 
In certain embodiments, it may be desirable to provide a second probe 
complementary to a different target sequence. Such a probe should have a 
label that generates an independently detectable signal. The probes may be 
designed to have different but compatible melting temperatures based on 
these techniques. 
To ensure binding of the labeled oligonucleotide before the primer, a high 
molar excess of labeled ribo- or chimeric oligonucleotide probe to primer 
concentration can also be used. Such probe concentrations range from about 
5 to 25 times higher than the respective primer concentration, which is 
generally 0.5-5.times.10.sup.7 M. 
The oligonucleotide primers and labeled probes may be prepared by a number 
of methods. Methods for preparing oligonucleotides (deoxy-, ribo, and 
chimeric) of a specific sequence are known in the art, and include, for 
example, cloning and restriction of appropriate sequences, direct 
automated chemical syntheses and enzymatically. Such techniques include, 
for example, the phosphotriester method, the phosphodiester method, the 
diethylphosphoramidate method, and the solid support method. 
The composition of the probes can be designed to inhibit nuclease activity. 
The incorporation of modified phosphodiester linkages (e.g., methyl 
phosphorylthioate or methylphosphonates) in the labeled probe during 
chemical synthesis may be used to prevent cleavage at a selected site. 
Depending on the length of the probe, the composition of its 5' 
complementary region, and the position of the label, one can design a 
probe to preferentially favor the generation of short or long labeled 
probe fragments for use in the practice of the invention. Great 
flexibility in the modification of the probes of this invention is 
possible so long as a 4-6 base pair RNA:DNA sequence is available as a 
substrate for RNase H. 
The probe is labeled, as described below, by incorporating moieties 
detectable by spectroscopic, photochemical, biochemical, immunochemical, 
enzymatic or chemical means. The method of linking or conjugating the 
label to the probe depends, of course, on the type of label(s) used and 
the position of the label on the probe, but in general comprises any 
suitable means of attachment known in the art. Further, the label may be 
considered attached to a particular nucleotide even though the attachment 
may comprise one or more intervening nucleotides. 
A number of detectable labels which would be suitable for use in this 
invention, as well as methods for their incorporation into the probe, are 
known in the art and include, but are not limited to, enzymes (e.g., 
alkaline phosphatase and horseradish peroxidase) and enzyme substrates, 
radioactive atoms, fluorescent dyes, chromophores, 
chemiluminescent/bioluminescent labels, electrochemiluminescent labels, 
labeled receptor-ligand binding, labeled antibody-antigen coupling, or any 
other labels that may interact with each other to enhance, alter, or 
diminish a detectable signal in real time. Should the PCR be practiced 
using a thermo-cycler instrument, the label must be able to survive the 
high temperature cycling required in this automated process. 
Preferably, two interactive labels may be used on a single probe while 
maintaining an appropriate spacing of the labels on the probe to permit 
the separation of the labels during cleavage with the RNase H. In some 
instances it may be desirable to use a single probe having two different 
label moieties. 
In a preferred embodiment, the interactive labels comprise a reporter (such 
as a fluoroscein) and quencher (such as a rhodamine) fluorescent dye pair. 
Each dye is attached to the probe, separated by at least a 4-6 base 
sequence to provide an adequate substrate for RNase H. In its single 
stranded state, the probe has sufficient flexibility that the rhodamine 
comes into proximity with the fluorescein with enough frequency to quench 
the reporter. However, when the probe anneals to the target nucleic acid 
sequence and is digested by RNase H, the fluoroscein is separated from the 
rhodamine, increasing the detectable reporter fluorescence. The 
fluorescence may be measured in any suitable way, including the Taq-Man 
LS-50B System (Perkin-Elmer). 
A number of modifications may be made to the probe to maximize quenching 
prior to hybridization and release. For example, the reporter and quencher 
may be separated by about 10 nucleotides or less so that quenching occurs 
without depending upon the flexibility of the single stranded probe. In 
general, the dyes may be attached either at the termini or internally, to 
optimize detection characteristics. Alternatively, the probe can be 
designed so that it forms a secondary structure, such as a hairpin, that 
brings the reporter and quencher into proximity when unhybridized. The use 
of ribo-oligonucleotides may be used to great advantage in this 
embodiment. RNA forms inherently more stable secondary structures than DNA 
or chimeric oligonucleotides. Accordingly, probes can be designed which 
very efficiently quench reporter fluorescence prior to hybridization 
leading to assay systems with very low background noise. Additionally, 
this technique may not be possible using conventional homogeneous assay 
systems because the DNA:DNA hairpin could be a substrate for the nuclease, 
leading to false release of label. 
In similar, embodiment, detection of the cleaved labeled probe can be 
achieved using fluorescence polarization. This technique is able to 
distinguish between large and small molecules based on molecular tumbling. 
Large molecules (e.g., intact labeled probe) tumble in solution much more 
slowly than small molecules. Upon linkage of a fluorescent moiety to the 
molecule of interest, this fluorescent moiety can be measured (and 
differentiated) based on molecular tumbling, thus differentiating between 
intact and digested probe. Detection may be measured during PCR using the 
ABI Prism 7700 Sequence Detector (Perkin Elmer). 
In another embodiment, two labeled ribo- or chimeric oligonucleotide probes 
are used, each complementary to separate regions of a double-stranded 
target region, but not to each other. For example, the presence of two 
probes can potentially double the intensity of the signal generated from a 
single label and may further serve to reduce product strand reannealing, 
as often occurs during PCR amplification. 
In yet other embodiments, the use of radioactive atoms, such as .sup.32 P , 
may be suitable for labeling and detection. Enzymatic methods for 
introducing .sup.32 P into nucleic acids are known in the art, and 
include, for example, 5' end labeling with polynucleotide kinase, or 
random insertion by nick translation and the Klenow fragment. Labels at 
the 3' terminus may employ polynucleotide terminal transferase to add the 
desired moiety, such as for example, cordycepin .sup.35 S-dATP, and 
biotinylated dUTP. The labels may be attached to the ribo- or chimeric 
oligonucleotide probe directly or indirectly by a variety of techniques. 
Depending on the precise type of label used, the label might be located at 
the 5' or 3' end of the probe, located internally in the probe's 
nucleotide sequence, or attached to carbon spacer arms of various sizes 
and compositions to facilitate signal interactions. Using commercially 
available phosphoramidite reagents, one can produce oligomers containing 
functional groups (e.g., thiols or primary amines) at either terminus via 
an appropriately protected phosphoramidite. Enzymes can be detected by 
their activity on a secondary substrate. 
Methods for introducing oligonucleotide functionalizing reagents to 
introduce one or more sulfhydryl, amino or hydroxyl moieties into the 
oligonucleotide probe sequence, typically at the 5' terminus are described 
in U.S. Pat. No. 4,914,210. A 5' phosphate group can be introduced as a 
radioisotope by using polynucleotide kinase and .gamma.-.sup.32 P-ATP to 
provide a reporter group. Biotin can be added to the 5' end by reacting an 
aminothymidine residue, introduced during synthesis, with an 
N-hydroxysuccinimide ester of biotin. 
Oligonucleotide (DNA and RNA) derivatives are also available labels. For 
example, etheno-dA and etheno-A are known fluorescent adenine nucleotides 
which can be incorporated into an ribo- or chimeric oligonucleotide probe. 
Similarly, etheno-dC is another analog that could be used in probe 
synthesis. The probes containing such nucleotide derivatives may be 
hydrolyzed to release much more strongly fluorescent mononucleotides 
during PCR. 
Template-dependent extension of the oligonucleotide primer(s) is catalyzed 
by a polymerizing agent in the presence of adequate amounts of the four 
deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP) or analogs 
as discussed above, in a reaction medium which is comprised of the 
appropriate salts, metal cations and pH buffering system. Suitable 
polymerizing agents are enzymes known to catalyze primer and 
template-dependent DNA synthesis and possess the 5' to 3' nuclease 
activity. Known DNA polymerases include, for example, E. coli DNA 
polymerase I, Thermus thermophilus (Tth) DNA polymerase, Bacillus 
stearothermophilus DNA polymerase, Thermococcus littoralis DNA polymerase, 
and Thermus aquaticus (Tag) DNA polymerase. The reaction conditions for 
catalyzing DNA synthesis with these DNA polymerases are well known in the 
art. To be useful in the present invention, the RNase H must efficiently 
cleave the ribo- or chimeric oligonucleotide probe and release labeled 
fragments so that the signal is directly or indirectly generated. 
The products of the synthesis are duplex molecules consisting of the 
template strands and the primer extension strands, which include the 
target sequence. By-products of this synthesis are labeled oligonucleotide 
fragments which consist of a mixture of mono-, di- and larger nucleotide 
fragments. Repeated cycles of denaturation, labeled probe and primer 
annealing, and primer extension and cleavage of the labeled probe result 
in the exponential accumulation of the target region defined by the 
primers and the exponential generation of labeled fragments. Sufficient 
cycles are run to achieve a detectable species of label, which is 
generally several orders of magnitude greater than background signal. 
In a preferred method, the PCR reaction is carried out as an automated 
process which utilizes thermostable enzymes. In this process the reaction 
mixture is cycled through a denaturing step, a probe and primer annealing 
step, and a synthesis step, whereby cleavage and displacement occurs 
simultaneously with primer dependent template extension. A DNA thermal 
cycler, such as the commercially available machine from Perkin-Elmer/ABI 
Instruments, which is specifically designed for use with a thermostable 
enzyme, may be employed. 
Temperature stable polymerases are preferred in this automated process 
because the preferred way of denaturing the double stranded extension 
products is by exposing them to a high temperature (about 95.degree. C.) 
during the PCR cycle for example, U.S. Pat. No 4,889,818 discloses a 
representative thermostable enzyme isolated from Thermus aquaticus. 
Additional representative temperature stable polymerases include, e.g., 
polymerases extracted from the thermostable bacteria Thermus flavus, 
Thermus ruber, Thermus thermophilus, Bacillus stearothermophilus (which 
has a somewhat lower temperature optimum than the others listed), Thermus 
lacteus, Thermus rubens, Thermatoga maritima, Thermococcus littoralis, and 
Methanothermus fervidus. 
Detection or verification of the labeled oligonucleotide fragments may be 
accomplished by a variety of methods and may be dependent on the source of 
the label or labels employed. One convenient embodiment of the invention 
is to subject the reaction products, including the cleaved label fragments 
to size analysis. Methods for determining the size of the labeled nucleic 
acid fragments are known in the art, and include, for example, gel 
electrophoresis, sedimentation in gradients, gel exclusion chromatography 
and homochromatography. 
During or after amplification, separation of the labeled fragments from the 
PCR mixture can be accomplished by, for example, contacting the PCR 
mixture with a solid phase extractant (SPE) for example, materials having 
an ability to bind oligonucleotides on the basis of size, charge or 
interaction with the oligonucleotide bases can be added to the PCR 
mixture, under conditions where labeled, uncleaved oligonucleotides are 
bound and labeled fragments are not. Such SPE materials include ion 
exchange resins or beads, such as the commercially available binding 
particles Nensorb.TM. (DuPont Chemical Co.). Nucleogen.TM. (the Nest 
Group) and hyroxylapatite. In a specific embodiment, if a dual labeled 
probe comprising a 3' biotin label separated from a 5' label by a nuclease 
susceptible cleavage site is employed as the signal means, the PCR 
amplified mixture can be contacted with materials containing a specific 
binding partner such as avidin or streptavidin, or an antibody or 
monoclonal antibody to biotin. Such materials can include beads and 
particles coated with specific binding partner and can also include 
magnetic particles. 
Following the step wherein the PCR mixture has been contacted with a SPE, 
the SPE material can be removed by filtration, sedimentation or magnetic 
attraction leaving the labeled fragments free of uncleaved labeled 
oligonucleotides and available for detection. 
Reagents employed in the methods of the invention can be packaged into 
diagnostic kits. Diagnostic kits include the labeled oligonucleotides and 
the primers in separate containers. If the oligonucleotide is unlabeled, 
the specific labeling reagents may also be included in the kit. The kit 
may also contain other suitably packaged reagents and materials needed for 
amplification, for example, buffers, dNTPs, and/or polymerizing means, and 
for detection analysis, for example, enzymes and solid phase extractants, 
as well as instructions for conducting the assay. 
EXAMPLES 
Table 1 shows the oligonucleotide sequences of the PCR primers and the 
ribo-oligonucleotide probe used in this example. The primers were selected 
for amplification of a segment of the BETA-actin gene. The probe was 
labeled with 6-carboxyfluorescein (6-FAM) at the 5' end and 
6-carboxytetramethylrhodamine (TAMRA) at the 3' end. The primers and probe 
were obtained as a custom synthesis from Perkin-Elmer. 
Table 1. Primer and Probe Sequences 
SEQ ID NO. 1: 
Forward Primer: 5' CAC ACT GTC CCC ATC TA 3' 
SEQ ID NO. 2: 
Reverse Primer: 5' GGA ACC GCT CAT TG 3' 
SEQ ID NO. 3: 
Probe sequence: 5' AUG CCC CCC CCA UGC CAU CCU GCG U 3' 
The PCR amplification was performed using a GeneAmp PCR System 
(Perkin-Elmer) using 50 .mu.l reactions that contained 1X Bicine buffer 
(Perkin-Elmer), 2.5 mM Mn(OAc).sub.2, 200 .mu.M dNTP's (Perkin-Elmer), 
0-16 Units of thermostable RNase H (Epicentre Technologies), 1.25 Units of 
.DELTA. Tth DNA Polymerase (Clonetech), human male DNA (Perkin-Elmer), 400 
nM of each primer and 50 nM of labeled probe. The thermal regimen was 
95.degree. C. for 2 min, and then 40 cycles of 60.degree. C. for 20 sec, 
45.degree. C. for 1 min and 95.degree. for 15 sec. FAM fluorescence was 
measured using a Taq-Man LS-50B System (Perkin-Elmer). 
FIG. 1 shows the FAM fluorescence detected real time during PCR cycling. 
Curve 1 represents the baseline fluorescence obtained with no RNase H 
added to the reaction and thus no release of label. Curves 2-5 represent 
the addition of 1, 2, 4 and 16 Units of RNase H to the reaction, 
respectively. Amplification of the BETA-actin gene segment is reflected by 
the real time increase in fluorescence, directly dependent on the amount 
of RNase H available to cleave the probe and release the label. 
The invention has been described with a particular view to the presently 
preferred embodiments. However, it will be obvious that certain changes 
and modifications may be practiced within the scope of the invention by 
those of skill in the art. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CACACTGTCCCCATCTA17 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GGAACCGCTCATTG14 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: RNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AUGCCCCCCCCAYGCCAUCCUGCGTU26 
__________________________________________________________________________