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This invention relates to the field of bioassays that involve nucleic acid
hybridization probes. These bioassays are useful for the detection of
specific genes, gene segments or RNA molecules. The assays are useful
clinically, for, e.g., tissue, blood and urine samples, as well as in food
technology, agriculture, and biological research.
BACKGROUND OF THE INVENTION
The use of nucleic acid hybridization probes for bioassays is well known.
One of the early papers in the field directed to assays for DNA is
Gillespie, D. and Spiegelman, S., A Quantitative Assay for DNA-RNA Hybrids
with DNA Immobilized on a Membrane, J. Mol. Biol. 12:829-842 (1965). In
general terms such an assay involves separating the nucleic acid polymer
chains in a sample, as by melting, fixing the separated DNA strands to a
nitrocellulose membrane, and then introducing a probe sequence which is
complementary to a unique sequence of the material being sought, the
"target" material, and incubating to hybridize probe segments to
complementary target segments, if targets are present. Non-hybridized
probes are removed by known washing techniques, and then the amount of
probe remaining is determined by one of a variety of techniques outlined
below which provides a measurement of the amount of targets in the sample.
A more recently developed form of bioassay that uses nucleic acid
hybridization probes involves a second probe, often called a "capture
probe." Ranki, M., Palva, A., Virtanen M., Laaksonen, M., and Soderlund,
H., Sandwich Hybridization as a convenient Method for the Detection of
Nucleic Acids in Crude Samples, Gene 21:77-85 (1983); Syvanen, A.-C.,
Laaksonen, M., and Soderlund, H., Fast Quantification of Nucleic Acid
Hybrids by Affinity-based Hybrid Collection, Nucleic Acids Res.
14:5037-5048 (1986). A capture probe contains a nucleic acid sequence
which is complementary to the target, preferably in a region near the
sequence to which the radioactively labeled probe is complementary. The
capture probe is provided with a means to bind it to a solid surface.
Thus, hybridization can be carried out in solution, where it occurs
rapidly, and the hybrids can then be bound to a solid surface. One example
of such a means is biotin. Langer, P. R., Waldrop, A. A. and Ward, D. C.,
Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel Nucleic Acid
Affinity Probes, Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981). Through
biotin the capture probe can be bound to streptavidin covalently linked to
solid beads.
The present invention is directed to the methods and means, including
assays and pharmaceutical kits containing requisite reagents and means,
for detecting in an in vitro or ex vitro setting the presence of nucleic
acid species.
It is a goal in this art to detect various nucleic acid sequences in a
biological sample, in which the said sequences, as so-called target
sequences, are present in small amounts relative to its existence amongst
a wide variety of other nucleic acid species including RNA, DNA or both.
Thus, it is desirable to detect the nucleic acid encoding polypeptides
that may be associated with pathological diseases or conditions, such as,
for example, RNA of the human immunodeficiency virus. In addition to the
detection of nucleic acids encoding the proteins of such viral particles,
it is desirable to detect other nucleic acids characteristic of a
pathological disease or condition such as a defective gene, as in the case
of hemophilia. It is also desirable to detect other nucleic acids whose
presence in the sample indicates that the organism is able to resist the
action of a drug, such as an antibiotic.
Several approaches have been used for detecting the probe. One is to link a
readily detectable reporter group to the probe. Examples of such reporter
groups are fluorescent organic molecules and .sup.32 P-labeled phosphate
groups. These detection techniques have a practical limit of sensitivity
of about a million targets per sample.
A second approach is to link a signal generating system to the probe.
Examples are enzymes such as peroxidase. Probes are then incubated with a
color-forming substrate. Leary, J. J., Brigati, D. J. and Ward, D. C.,
Rapid and Sensitive Colorimetric Method for Visualizing Biotin-Labeled DNA
Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-Blots,
Proc. Natl. Acad. Sci. USA 80:4045-4049 (1983). Such amplification reduces
the minimum number of target molecules which can be detected. As a
practical matter, however, nonspecific binding of probes has limited the
improvement in sensitivity as compared to radioactive tagging to roughly
an order of magnitude, i.e., to a minimum of roughly 100,000 target
molecules.
Yet another approach is to make many copies of the target itself by in vivo
methods. Hartley, J. L., Berninger, M., Jessee, J. A., Bloom, F. R. and
Temple, G. S., Bioassay for Specific DNA Sequences Using a Non-Radioactive
Probe, Gene 49:295-302 (1986). This can also be done in vitro using a
technique called "polymerase chain reaction" (PCR). This technique was
reported in Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G.
T., Erlich, H. A., and Arnheim, N., Enzymatic Amplification of Beta-globin
Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle
Cell Anemia, Science 230:1350-1354 (1985); Saiki, R. K., Gelfand, D. H.
Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and
Erlich, H. A., Primer-directed Enzymatic Amplification of DNA With a
Thermostable DNA Polymerase, Science 239:487-491 (1988); Erlich, H. A.,
Gelfand, D. H., and Saiki, R. K., Specific DNA Amplification, Nature
331:461-462 (1988), and Mullis et al., European Patent Application
Publication Nos. 200362 and 201184 (see also U.S. Pat. Nos. 4,683,195 and
4,683,202). In PCR, the probe is complementary only to the beginning of a
target sequence but, through an enzymatic process, serves as a primer for
replication of an entire target. Each repetition of the process results in
another doubling of the number of target sequences until a large number,
say, a million copies, of the target are generated. At that point
detectable probes, e.g., radioactively labeled probes, can be used to
detect the amplified number of targets. The sensitivity of this method of
target amplification is generally limited by the number of "false positive
signals" generated, that is, generated segments that are not true copies
of the target. Nonetheless, this method is quite sensitive. The procedure
requires at least two nucleic acid probes and has three steps for a single
cycle. This procedure is cumbersome and not always reliable.
Yet another method for amplification is to link to the probe an RNA that is
known to be copied in an exponential fashion by an RNA-directed RNA
polymerase. An example of such a polymerase is bacteriophage Q-beta
replicase. Haruna, I., and Spiegelman, S., Autocatalytic Synthesis of a
Viral RNA In Vitro, Science 150:884-886 (1965). Another example is brome
mosaic virus replicase. March et al., POSITIVE STRAND RNA VIRUSES Alan R.
Liss, New York (1987). In this technique, the RNA serves as a template for
the exponential synthesis of RNA copies by a homologous RNA-directed RNA
polymerase. The amount of RNA synthesized is much greater than the amount
present initially. This amplification technique is disclosed in Chu, B. C.
F., Kramer, F. R., and Orgel, L. E., Synthesis of an Amplifiable Reporter
RNA for Bioassays, Nucleic Acids Res. 14:5591-5603 (1986); Lizardi, P. M.,
Guerra, C. E., Lomeli, H., Tussie-Luna, I. and Kramer, F. R., Exponential
Amplification of Recombinant-RNA Hybridization Probes, Bio/Technology
6:1197-1203 (October, 1988), which is incorporated herein by reference and
is attached hereto in manuscript form [hereinafter referred to as "Lizardi
et al."]; published European Patent Application 266,399 (EP Application
No. 87903131.8). After non-hybridized probes are removed by washing, the
RNA polymerase is used to make copies of the replicatable RNA. According
to the disclosure of published European Patent Application No. 266,399,
replication of the RNA may take place while the RNA is linked to the
probe. Alternatively, the replicatable RNA may be separated from the
remainder of the probe prior to replication. That application also
discloses a variety of chemical links by which a probe sequence can be
joined to a replicatable RNA. In addition, it discloses that the probe
sequence may be part of a replicatable RNA, as described in Miele, E. A.,
Mills, D. R., and Kramer, F. R., Autocatalytic Replication of a
Recombinant RNA, J. Mol. Biol. 171:281-295 (1983). That European
application also discloses that such recombinant RNAs must be able to
hybridize specifically with the target sequence as well as to retain their
ability to serve as a template for exponential replication by an
appropriate RNA-directed RNA polymerase, as is demonstrated in the results
obtained by Lizardi et al., supra.
Replication of RNA, as opposed to target amplification using PCR, can be
done in a single step. In that step one can synthesize as many as a
billion copies of the replicatable RNA that was joined to the probe in as
little as twenty minutes, which theoretically could lead to detection of a
single target molecule. However, in practice the sensitivity of this type
of probe replication is limited by the persistence of nonspecifically
bound probes. Nonspecifically bound probes will lead to replication just
as will probes hybridized to targets.
A major problem in the implementation of bioassays that employ
hybridization technology coupled to signal amplification systems is the
background signal produced by nonspecifically bound probe molecules. These
background signals introduce an artificial limit on the sensitivity of
bioassays. In conventional bioassays this problem is sometimes alleviated
by the utilization of elaborate washing schemes that are designed to
remove nonspecifically bound probes. These washing schemes inevitably add
to the complexity and cost of the assay.
As a means to reduce the background noise level of assays employing probes
linked to replicatable RNA by covalently joined linking moieties, European
Patent Application No. 266,399 discloses what it refers to as "smart
probes," that is, probes whose linked RNA is said not to serve as a
template for replication unless and until the probe has hybridized with a
target sequence. In that application two embodiments are disclosed for
smart probes.
In a first embodiment in that application, the smart probe comprises a
probe portion consisting of about 75-150 deoxynucleotides, made by in
vitro or in vivo methods known in the art. The smart probe also comprises
a recombinant, replicatable RNA containing an inserted heterologous
sequence of about 10-30 nucleotides, made by, e.g., the method of Miele,
E. A., Mills, D. R., and Kramer, F. R., Autocatalytic Replication of a
Recombinant RNA. J. Mol. Biol. 171:281-295 (1983). Joining those two
portions at their 5' ends is a linking moiety of the formula
--O(PO.sub.2)NH(CH.sub.2).sub.a SS(CH.sub.2).sub.b NH(PO.sub.2)O--, where
a and b are each 2 to 20. Furthermore, the sequence at the 3' end of the
DNA portion of the smart probe is capable of being (and very likely to be)
hybridized to the heterologous sequence of the RNA portion of the smart
probe. The enzyme ribonuclease H is said to be capable of cleaving the RNA
portion of smart probes which have not hybridized to targets, but not be
capable of cleaving the RNA portion of smart probes which have hybridized
to targets, because when the probe sequence in the DNA portion of a smart
probe is bound to its target, it is said to be incapable of also being
hybridized to the heterologous sequence in the RNA portion of the smart
probe, thereby providing a way to eliminate nonspecifically bound probes
prior to amplification. Amplification via RNA replication is said to
optionally include the preliminary step of cleaving the disulfide bond in
the linking moiety.
In that embodiment, cleavage of probes not hybridized to targets is said to
be possible for ribonuclease H, because the 3' end of the DNA portion of
the smart probe (which contains the probe sequence) is hybridized to the
recombinant replicatable RNA portion, presumably thereby providing a site
wherein ribonuclease H can cleave the RNA and render it inoperative as a
template for amplification by an RNA-directed RNA polymerase.
In the other embodiment of a smart probe disclosed in published European
Patent Application 266,399, there is a probe portion, a linking moiety,
and a replicatable RNA portion, linked as described above. Here, however,
the probe portion comprises not only a probe segment of 50-150
nucleotides, but also additional segments, called "clamp" segments, on
either side of it, that is, a 5'-clamp segment and a 3'-clamp segment,
each of about 30-60 nucleotides. Each clamp segment is said to hybridize
with a segment of the replicatable RNA portion, rendering the RNA inactive
as a template for replication, unless and until the probe is hybridized
with a target. That hybridization causes the clamps to release, thereby
rendering the RNA replicatable, either directly or after optional cleavage
of the disulfide bond.
The smart probes disclosed in published European Patent Application No.
266,399 comprise a somewhat complicated linking moiety containing a weakly
covalent and rather easily dissociable disulfide linkage. Disulfide bonds
readily dissociate under reducing conditions. The two versions of smart
probes disclosed in that application rely on distant intramolecular
interactions to render the probe smart. This is a disadvantage which makes
such probes difficult to design, particularly since distant interactions
are not well understood. The second version, reported above, has a further
complication that it utilizes two distant clamps which must displace a set
of relatively strong neighboring compliments. And, the design depends on
both distant clamps hybridizing or none, which makes design very
difficult.
An object of the present invention is a simple molecular allosteric switch
that renders a nucleic acid hybridization probe smart, that is, capable,
in an appropriate assay, of generating a signal only if the probe is
hybridized to a target sequence.
It is a further object of this invention to couple the activity of a signal
generating system to the state of such a switch.
It is yet another object of this invention to develop probes containing
such an allosteric switch that are linked to any of a number of different
signal generating systems whose activity is dependent on the state of the
switch.
It is another object of this invention to develop assays of improved
sensitivity that utilize the above constructs, as well as kits for
performing such assays.
SUMMARY OF THE INVENTION
The present invention is predicated on a simple molecular allosteric switch
that works on the principle that when a nucleic acid double helix is
formed between a relatively short probe sequence and a target sequence,
the ends of the double helix are necessarily located at a distance from
each other due to the rigidity of the double helix. That rigidity is
discussed in detail in Shore, D., Langowski, J. and Baldwin, R. L., DNA
Flexibility Studied by Covalent Closure of Short Fragments into Circles,
Proc. Natl. Sci. USA 78:4833-4837 (1981); and Ulanovsky, L., Bodner, M.,
Trifonov, E. N., and Choder, M., Curved DNA: Design, Synthesis, and
Circularization, Proc. Natl. Acad. Sci. USA 83:862-866 (1986).
This invention involves the use of a nucleic acid hybridization probe
comprising at least the following essentials: a probe sequence of
approximately 15-115 nucleotides in length surrounded on both sides by
complementary nucleic acid sequences which are considerably shorter than
the probe sequence, preferably not greatly in excess of one-half the
length of the probe sequence. This combination of three sequences forms a
simple molecular allosteric switch. When not hybridized to a target
sequence, the switch secluences are hybridized to each other, which we
refer to as a closed switch. When the probe sequence hybridizes to a
predetermined complementary target sequence for which the probe is
designed, the strong interaction between the probe and target sequences to
form a rigid double helix necessarily results in the dissociation of the
switch sequences, which we refer to as an open switch. In the open
configuration, the switch sequences are unable to interact with each
other.
The invention comprises probe molecules containing the above switch wherein
one of the switch sequences, or both switch sequences in combination,
comprise a biologically functional nucleic acid moiety useful for
selectively generating a detectable signal indicative of the hybridization
of the probe with its predetermined target sequence.
The invention further comprises bioassay methods which take advantage of
the allosteric change in the switch sequences in the above probe molecules
to generate a detectable signal indicative of the hybridization of the
probe with its predetermined target sequence. The assay may be qualitative
(a qualitative demonstration) or quantitative (a quantitative
determination). It may include amplification, which may be linear or
exponential in nature.
The invention also includes kits of reagents and macromolecules for
carrying out the above bioassays.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of a closed switch according to the
invention.
FIG. 2 is a schematic representation of the switch of FIG. 1, but in an
open state.
FIG. 3 is a schematic representation of the probe of Example I, containing
a switch in an open state.
FIG. 4 is a schematic representation of the probe of Example II, containing
a switch in an closed state.
FIG. 5 is a schematic representation of the probe of Example II, containing
a switch in an open state.
FIG. 6 is a schematic representation of the probe of Example III,
containing a switch in an closed state.
FIG. 7 is a schematic representation of the probe of Example III,
containing a switch in an open state.
FIG. 8 is a schematic representation of the probe of Example IV, containing
a switch in an closed state.
FIG. 9 is a schematic representation of the probe of Example IV, containing
a switch in an open state and additionally showing a ribozyme.
FIG. 10 is a detailed schematic showing the nucleotide sequences of the
ribozyme shown in FIG. 9.
FIG. 11 is a schematic representation of the probe of Example IV,
containing a switch in an open state and additionally showing an
additional strand.
FIG. 12 is a schematic representation of the probe of Example V, containing
a switch in an closed state.
FIG. 13 is a schematic representation of the probe of Example V, containing
a switch in an open state.
DETAILED DESCRIPTION OF THE INVENTION
Shown in FIG. 1 is a probe, or probe portion, comprising the three
essential ingredients of a probe according to this invention, namely, a
probe sequence and complementary switch sequences on both sides of the
probe. As depicted in FIG. 1, the switch is closed. FIG. 2 is the same
probe or probe portion in its open state.
Referring to FIG. 1, probe sequence 1 is a nucleic acid probe sequence
extending from its 5' side 2 to its 3' side 3. Immediately adjacent to the
5' side of the probe sequence is a nucleic acid first switch sequence 4.
Immediately adjacent to the 3' side of the probe sequence is a nucleic
acid second switch sequence 5. Switch sequences 4 and 5 are complementary
and hybridize to each other via hydrogen bonds 7, forming the stem 6 of a
"hairpin" secondary structure. Referring to FIG. 2, probe sequence 1 is
hybridized via hydrogen bonds 9 to its predetermined target sequence 8.
Switch sequences 4 and 5 are apart and not interacting with one another.
The probe may be RNA or DNA. The probe sequence 1 must be of sufficient
length to ensure a very specific interaction with its predetermined target
sequence 8. It should be at least about 15 nucleotides in length, although
we prefer that it be at least about 20 nucleotides in length.
The probe sequence 1 should be short enough to ensure that the sides 2, 3
of probe sequence 1, when hybridized to the target sequence 8 (FIG. 2) are
physically prevented by the rigidity of the hybridized region between
sides 2 and 3 from approaching each other within a distance that would
permit switch sequences 4, 5 from interacting with each other. In other
words, when the probe sequence is hybridized, the switch sequences
necessarily are not hybridized to each other. An additional force helps to
drive the transition to an open state, namely, torsional forces tending to
unwind stem 6 when the hybridized region shown in FIG. 2 forms a double
helix. In practice, the probe sequence is no longer than about 100
nucleotides. We prefer that the probe sequence be 20-60 nucleotides in
length, and most preferably, about 30 nucleotides in length.
The switch sequences are related to the length of the probe sequence. Most
preferably, we prefer that the length of the switch sequences be no more
than half the length of the probe sequence. The switch sequences should be
at least about 10 nucleotides in length to permit formation of a stable
stem 6. Turner, D. H., Sugimoto, N., Jaeger, J. A., Longfellow, C. E.,
Freier, S. M. and Kierzek, R., Improved Parameters for Prediction of RNA
Structure, Cold Spring Harbor Symp. Quant. Biol. 52:123-133 (1987). The
length of switch sequences for certain embodiments described below must
also be sufficiently long to contain necessary functional sequences. We
prefer switch sequences of about 10-40 nucleotides or preferably about
10-30 nucleotides.
In designing a probe according to the invention, attention should be paid
to the relative strengths of the open switch hybrid (FIG. 2) as compared
to the closed switch hybrid (FIG. 1) under the assay conditions to be
used: the former should be greater. There are assay conditions, however,
in which the strengths of hybrids is only length-dependent. Wood, W. I.,
Gitschier, J., Lasky, L. A., and Lawn, R. M., Base Composition-independent
Hybridization in Tetramethylammonium Chloride: A Method for
oligonucleotide Screening of Highly Complex Gene Libraries, Proc. Natl.
Acad. Sci. USA 82:1585-1588 (1985).
Switch design can be readily tested by digesting probes or probe portions
(FIGS. 1, 2) with appropriate nucleases before and after hybridization to
model nucleic acids containing target sequences and then analyzing the
digestion products by polyacrylamide gel electrophoresis. This will be
apparent to those skilled in the art and will not be described further.
To help drive the transition from closed to open, one may take advantage of
the principle of strand displacement to provide an additional force.
Green, C., and Tibbetts, C., Reassociation Rate Limited Displacement of
DNA Strands by Branch Migration, Nucleic Acids Res. 9:1905-1918 (1981).
This may be accomplished by overlapping a switch sequence with a probe
sequence, which means that at least one nucleotide of the switch sequence
is also a nucleotide of the probe sequence.
While the switch sequences must be adjacent to the probe sequence, they
need not be immediately adjacent to it. A few nucleotides may separate the
switch sequences from the probe sequences, but not so many that the
functioning of the switch is materially affected, as those skilled in the
art will readily appreciate.
Probe molecules of this invention, containing the switch described above,
can be of diverse design and still take advantage of the allosteric change
that accompanies probe sequence hybridization (FIG. 2) in signal
generation.
For example, a switch sequence may, by virtue of the conformation it
assumes in the open state, enable an interaction with another
macromolecule, or even a different portion of the same molecule, which is
required for the generation of a detectable signal. In Example I below,
the second switch sequence, in the open state, is able to hybridize with a
complementary nucleic acid strand. In Example III, the first switch
sequence, in the open state, forms a hairpin structure that enables it to
bind specifically to a viral protein. In Example IV, the second switch
sequence, in the open state, is able to interact with an
oligoribonucleotide or with an oligodeoxyribonucleotide. In Example V, the
first switch sequence, in the open state, assumes a structured
conformation that enables it to interact with a relatively distant region
of the same probe molecule.
It is also possible to do the reverse. In Example II, the switch sequences
can bind to a specific enzyme only when they are in the closed state.
Signal generation using probe molecules and methods of this invention may
vary widely. The state of the simple allosteric switch governs signal
generation, which means that there is no signal generated unless the probe
sequence hybridizes with its target sequence. We prefer signal generating
systems that involve amplification, particularly exponential
amplification, to increase sensitivity.
The Examples which follow illustrate a few of the myriad variations
involving amplification. They all utilize the exponential replication of a
replicatable RNA by an RNA-directed RNA polymerase to generate a readily
detectable signal. The Examples utilize MDV-1 RNA, which is described in
Kacian, D. L., Mills, P. R., Kramer, F. R., and Spiegelman, S., A
Replicating RNA Molecule Suitable for a Detailed Analysis of Extracellular
Evolution and Replication, Proc. Nat. Acad. Sci. USA 69:3038-3042 (1972).
The Examples also use Q-beta replicase, which is the specific polymerase
for replicating MDV-1 RNA. Q-beta replicase is described in Haruna, I. and
Spiegelman, S., Specific Template Requirements of RNA Replicases, Proc.
Nat. Acad. Sci. USA 54:579-587 (1965). Any replicatable RNA and its
homologous replicase could, of course, be used. Other useful signal
generating systems could employ enzymes, enzyme cofactors, ribozymes, DNA
and RNA sequences required for biological activity (e.g., promoters,
primers, or linkers required for the ligation of plasmids used to
transform bacteria). Detectable signals are diverse and include, for
example, radiation, light absorption, fluorescence, mass increase, and the
presence of biologically active compounds.
Assay techniques which can be used to detect hybridized probes of this
invention are also diverse. In the following Examples, synthesis of a
replicatable RNA is used to signal that hybridization of the probe
sequence has occurred. The signal generating systems illustrated in the
Examples fall into three broad classes: in Examples II-III, the switch is
incorporated within a replicatable RNA; in Examples IV-V, a replicatable
RNA sequence is joined with a probe portion but can only be replicated
after cleavage, which is dependent upon the presence of an open switch;
and in Example I, the transcription of a replicatable RNA from a template
added after hybridization, can only occur when an open switch sequence
forms a part of a functional promoter of transcription.
Each of the specific embodiments set forth in the accompanying Examples
satisfies the objective of generating a signal only if the probe is
hybridized to a target sequence. Either the biological activity of the
signal generating systems illustrated depends strictly on the state of the
switch, or the state of the switch provides a means for rendering
nonspecifically bound probes unable to generate signals, or the state of
the switch provides a means for separating hybridized probes from
nonspecifically bound probes. Thus, each of the specific embodiments
markedly reduces the background caused by nonspecifically bound probes,
thereby significantly improving the sensitivity of the assays, including
assays which include amplification.
EXAMPLE I
In this example, the probe is a single DNA strand designed to contain three
sequences: a probe sequence approximately 34 nucleotides in length; a
first switch sequence of about 17 nucleotides immediately adjacent to the
5' side of the probe sequence; and a second switch sequence of about 17
nucleotides immediately adjacent to the 3' side of the probe sequence. The
switch sequences are designed to be complementary to one another. When
hybridized to each other, the hybridized switch sequences comprise a
promoter for the DNA-directed RNA polymerase, bacteriophage T7 RNA
polymerase. In this application, we refer to the first switch sequence as
a "promoter sequence" and the second switch sequence as a
"promoter-complement" sequence. In this example, the switch sequences
comprise the ends of the probe molecule. The design of promoter and
promoter-complement sequences is according to Osterman, H. L. and Coleman,
J. E., "T7 Ribonucleic Acid Polymerase-Promoter Interactions,"
Biochemistry 20:4885-4892 (1981). The particular promoter-complement
sequence we have chosen to work with is TAATACGACTCACTATA.
The probe molecule, including a probe sequence complementary to a
predetermined target sequence, can be made by chemical synthesis of
oligodeoxyribonucleotides using methods well known in the art, e.g., Gait,
M. J., OLIGONUCLEOTIDE SYNTHESIS, IRL Press, Oxford, United Kingdom
(1984).
The probe of this example can be used to detect a DNA or RNA target
sequence which is complementary to the probe sequence. The target sequence
may be in a sample containing other, unrelated nucleic acids and other
materials, for example, proteins. The probe may be used to detect a gene
segment of an infectious agent (virus, bacterium, protozoan, etc.) in a
clinical sample of, for example, human blood or urine.
The target sequence must be exposed to the probe. This is done by
techniques well known to the art. Commonly, but not necessarily, nucleic
acid is isolated from a sample before the probe is added.
The probe and the sample, which may contain nucleic acid target sequences,
are next incubated under conditions, including time and temperature,
appropriate to cause hybridization of probe sequences with target
sequences. Appropriate conditions are well known in the art. For
quantitative determination of the number of target sequences present, an
amount of probe in excess, preferably in substantial excess, of the
highest anticipated target amount should be used. If only a qualitative
demonstration of the presence of target sequences is desired, a lesser
amount of probe can be used.
Probes hybridized to targets are separated from unbound probes by methods
well known to the art, for example, through the use of capture probes.
After separation, the treated sample will contain probes hybridized to
targets (FIG. 2) and also nonspecifically bound probes. The two are not in
the same form, however. In the hybridized probes the allosteric switches
are open; that is, the switch sequences are not hybridized to each other.
In the nonspecifically bound probes, however, the switch sequences remain
hybridized to each other.
Detecting those probes with open switches will now be described. This
example includes amplification prior to detection.
Referring to FIG. 3, the sample is incubated with a single-stranded DNA
molecule 10 comprising a promoter sequence 11 and a template sequence 12
for the transcription of a replicatable RNA. The promoter sequence 11
allows hybridization via hydrogen bonds 13, under conditions known to the
art, to the promoter-complement of the second switch sequence 5 of probes
having open switches. Specifically, this DNA molecule consists of the 17
deoxyribonucleotides of the promoter sequence (complementary to the
promoter-complement set forth above) followed by the 244
deoxyribonucleotides complementary to MDV-poly (+) RNA described in
Lizardi et al., supra. This DNA molecule can be prepared by isolating one
of the complementary strands of a suitable restriction fragment of a
plasmid containing that sequence by methods known in the art. Maniatis,
T., Fritsch, E. F., and Sanbrook, J., MOLECULAR CLONING: A LABORATORY
MANUAL Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). | | |
