JP2003505107A - Nucleic acid amplification method - Google Patents

Abstract

(57) Abstract: A probe molecule having a single-stranded nucleic acid, wherein the probe is adjacent to or substantially adjacent to a single-stranded sequence complementary to a target nucleic acid sequence, a single-stranded RNA polymerase promoter sequence, and a promoter sequence. The present invention provides a probe molecule having a blocking portion, a method for detecting a target nucleic acid sequence using a probe, and a kit including the probe.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to nucleic acid probe molecules, methods involving the use of probe molecules, and
The present invention relates to a kit having a probe molecule.

BACKGROUND OF THE INVENTION Many nucleic acid amplification methods have been previously disclosed. One such method is the polymerase chain reaction (PCR), disclosed in US4683195 and 4683202. PCR
Although the method is well known and widely used, it involves alternating reactions between intermediate temperatures (e.g. 50 ° C to 55 ° C) and elevated temperatures (e.g. 90 ° C to 95 ° C) involving repeated thermal cycling. It has some drawbacks such as the need to control the temperature. The width of time required for multiple cycles of large-scale temperature transitions to achieve nucleic acid amplification, and
The occurrence of sequence errors in amplified copies of nucleic acid sequences is also a major disadvantage due to the errors that occur during multiple copies of long sequence bands. Furthermore, the detection of amplified nucleic acid sequences generally requires additional steps such as agarose gel electrophoresis.

Another method for amplifying nucleic acid using RNA polymerase is WP88 / 10315 (Siska Diagnost
ics), EP329822 (Cangene), EP373960 (Siska Diagnostics), US5,554,516 (Gen-Pr
obe Inc.), WO89 / 01050 (Burg et al), WO88 / 10315 (Gingeras et al) and EP32.
9822 (Organon Teknika), the latter document relating to a method known as NASBA. Such an amplification method describes a cyclic reaction in which the synthesis of DNA and RNA is alternately repeated. This alternating RNA / DNA synthesis is accomplished primarily by annealing oligonucleotides that flank specific DNA sequences, which causes these oligonucleotides to have transcriptional promoters. The RNA copy of the specific sequence thus generated, or the input sample (US5,554,526) having the specific RNA sequence is then copied as a DNA strand using nucleic acid primers, and the formed DNA: RNA RNA derived from the hybrid is removed by denaturation (W
O88 / 10315) or using RNase H (EP329822, EP373960 and US5,
554,516).

WO93 / 06240 also discloses two further amplification methods, one for the thermal process and one for the isothermal process. Both the thermal process type and the isothermal process type respond to the hybridization of two nucleic acid probes whose regions are complementary to the target nucleic acid. The portion of the probe is such that the probes are adjacent or substantially adjacent to each other, the first and second
Of the probe can hybridize to the sequence of interest so that the complementary arm-specific sequences of the probe can anneal to each other. After annealing, strand extension of one of the probes is performed by using a portion of the other probe as the template. Amplification of the elongated probe is performed by either of two methods, and in the case of the thermal process cycle type, the thermal process separation of the elongated first probe is performed to obtain a new probe of the elongated first probe. Hybridization of the earlier probe, which is substantially complementary to a portion of the synthesized sequence, is performed. Extension of this further probe is accomplished by using the appropriate polymerase with the extended first probe as the template. Thermal process separation of the products of the extended first and earlier probes can act as a template for the extension of the earlier first probe molecule, and the extended first probe can It can act as a template for the extension of the previous probe molecule.

In the isothermal process type, primer extension of the first probe creates a functional RNA polymerase promoter that transcribes multiple copies of RNA in the presence of the associated RNA polymerase. The RNA formed is further amplified as a result of the interaction of complementary DNA oligonucleotides containing the earlier RNA polymerase promoter sequence.
A large amount of RNA is generated by this cyclic process, and its detection is possible by various means.

DISCLOSURE OF THE INVENTION In the first feature, a probe molecule having a single-stranded nucleic acid, wherein the probe is a single-stranded sequence complementary to a target nucleic acid sequence, a single-stranded chain of an RNA polymerase promoter sequence, and a promoter Provided is a probe molecule as above having a blocking moiety flanking or substantially flanking the sequence.

RNA polymerase promoter sequences are well known in the art. Preferred promoter sequences are those known as bacteriophage polymerases, especially T3, T7 or SP6 polymerases. The T3, T7 and SP6 promoter sequences are typically 17 bases long. The promoter sequence must be present in double-stranded form in order to be fully functional.

An example of a double-stranded promoter sequence recognized as T7 polymerase is shown below.

[0009]

[Chemical 1]

The promoter sequence is underlined (the meanings of -5 and +12 sequences are described separately).
The promoter sequence recognized as T7 polymerase is called T7 promoter sequence. In fact, several small variants of the sequence (typically differing by only one base) are recognized as T7 polymerases and are known to act as functional promoters.

It has been conventionally known that a single-stranded probe molecule having one strand of RNA polymerase promoter can be obtained. When combined with a complementary strand, a fully functional double heavy chain promoter is formed, which in turn allows RNA synthesis (transcription) using flanking sequences downstream of the promoter's template strand as template. Typically, in the prior art, the formation of double-stranded functional promoters only occurs as a result (directly or indirectly) of the presence of a particular target nucleic acid sequence of interest, thereby causing RNA synthesis. It indicated the presence (directly or indirectly) of the sequence of interest.

However, the present invention is such that such a system involves a relatively high level of "background" signal (ie RNA synthesis can occur in the absence of the sequence of interest), which is the sensitivity of the test. It was also discovered that reproducibility may be reduced. Surprisingly, we have found that the signal: noise ratio (SN ratio) can be dramatically improved by including a "blocking moiety" in the single-stranded probe molecule that is not recognized by RNA polymerase. It was discovered that the unique blocking moiety is located adjacent or substantially adjacent to the RNA polymerase promoter sequence.

The term “adjacent” as used herein refers to the absence of nucleobases between the blocking moiety and the promoter sequence. In practice, it is usually preferred that the blocking moiety not be directly adjacent to (ie, "substantially adjacent to") the promoter. As used herein, the term "substantially contiguous" means between 1 and 50 nucleobases, preferably between 1 and 40, more preferably between 1 and 30, most preferably between 1 and 50 between the blocking portion and the promoter sequence. It shall mean that there are 20 bases. In particular, the inventors have found that a spacing of about 12-15 bases between the promoter and the blocking moiety gives optimal results, especially when the blocking moiety has an alkylene moiety.

As a recognized numbering convention, for the template strand, the terminal 3'base of the promoter (assuming the promoter sequence consists of 17 bases) is designated as position -17. Thus, the flanking blocking moiety located 3'of the promoter on the template strand is located at position -18, and the "substantially contiguous" blocking moiety is located at any of positions -19 to -68, the preferred position being Is located at positions -19 to -38, more preferably at positions -22 to -35.

When the nucleic acid probe molecule comprises the template strand of an RNA promoter, it is generally preferred that the blocking moiety be located 3'to the promoter. Conversely, if the probe molecule has the non-template strand of the promoter, it is preferable to position the blocking moiety 5'of the promoter.

It is usually preferred, but not essential, for the probe molecule to have a template strand of RNA promoter sequence.

As mentioned above, it is usually preferred that there are several nucleobases between the promoter sequence and the blocking moiety. Preferably, at least some sequences of intervening nucleobases (especially sequences immediately adjacent to the promoter) are arranged to optimize the promoter activity of the functional double stranded promoter. It is particularly preferable that the "-5" sequence is present. The -5 sequence is a 5 base sequence immediately 5'to the template strand of the promoter sequence which is expected to enhance the promoter activity. The optimal sequence of the bases forming the -5 sequence differs depending on each polymerase. For T7 polymerase, the optimal -5 sequence is shown in bold on page 3.

[0018]   As a general rule, the blocking moiety contained in the probe is
Any actual condition may not be recognized as a suitable template. In fact, the block
The king moiety is a suitable precursor (eg,
It can be inserted into a synthetic oligonucleotide by introducing
Wear. Desirably, the blocking moiety is unable to base pair with the nucleic acid.
Yes, and conveniently located outside the extreme 5'or 3'end. Preferred block
As the king moiety, alkylene, alkanol and alkyl residues, especially C₂-C ₂₀ Alkyl, alkanol or alkylene, more preferably C₃-C_TenAlkyl
, Alkanols or alkylenes. To give a specific example, Octangio
Alcohol, propanediol, hexaethylene glycol, propyl residue, etc.
It

In certain preferred embodiments, the nucleic acid probe molecule has multiple blocking moieties, which may be located adjacent to each other within the probe or may be interrupted by intervening nucleobases. If there are multiple blocking moieties, they may be of the same chemical entity or different.

The probe molecule of the present invention may be DNA, RNA, PNA (peptide nucleic acid) or LNA (locked nucleic acid) or any combination thereof. Generally, the single-stranded promoter sequence is usually substantially composed of DNA, but several (for example, 1 to 3) bases may be those of RNA, PNA or LAN, and the promoter sequence is complementary. DNA
The formation of a functional promoter is still possible if it hybridizes to the sequence.

PNA is a peptide bond chain of sugar / phosphate skeleton (typically of repeating N- (2-aminoethyl) -glycine unit in which bases are linked by a methylene carbonyl bond.
) Is a synthetic nucleic acid analog. PNA / DNA hybrid is a double-stranded DN
It has a higher Tm value compared to the A molecule, because in DNA the highly negatively charged phosphate backbone induces electrostatic repulsion between the corresponding strands.
This is because the skeleton of PNA is uncharged. Another feature of PNA is that the single base mismatch is relatively more destabilized than the single base mismatch of the heteroduplex DNA. Therefore, PNAs are useful for inclusion in probes for use in the present invention, as the resulting probe has a higher specificity than probes consisting entirely of DNA. The synthesis and use of PNAs is described, for example, by Orum et al., (19
93, Nucl. Acid Res. 21, 5332); Egholm et al., (1992 J. Am. Chem. Soc., 114, 18
95); and Egholm et al., (1993, Nature 365, 566).

LNA is a synthetic nucleic acid analog that incorporates an “internally crosslinked” nucleoside analog. For the synthesis of LNA and its properties, many investigators, such as Nielson et al.
97, J. Chem. Soc. Perkin Trans, 1, 3423); Koshkin et al. (1998, Tetrahedron L
etters 39, 4381); Singh & Wengel (1998 Chem. Commun. 1247); and Singh et al.
(1998 Chem. Commun. 455). As with PNA, LNA is DNA
When paired with, it shows greater thermal stability than conventional DNA / DNA heteroduplex. However, LNA can be synthesized by a conventional nucleic acid synthesizer, but PNA cannot be synthesized.

In addition to non-conventional nucleic acids such as LNAs and PNAs, the probe molecules of the present invention may also optionally contain modified nucleobases such as inosine. Especially DNA
In order to prevent chain extension of the probe by the polymerase, it is preferable to obtain a probe with a modified 3'end such as dideoxynucleotides or other modified bases known in the art.

At least such portion of the probe molecule that hybridizes to the nucleic acid target is PN
It is preferred to have A and / or LNA. The target to which the probe molecule hybridizes typically comprises RNA and / or DNA.

The target to which the probe hybridizes is the actual sequence of interest present in the sample under consideration (eg a human or other gene which may be a marker for a genetic or infectious disease). Good. Alternatively, the presence of the sequence of interest in the sample may lead (directly or indirectly) to the presence of nucleic acid sequences ahead of which the nucleic acid probe hybridizes.

For example, the presence of a sequence of interest in a sample (eg, a bodily fluid sample derived from a human or animal subject or an environmental sample) may result in the synthesis of RNA or DNA target sequences ahead of which the nucleic acid probe of the invention hybridizes. And the target nucleic acid sequence is eg WO93
/ 06240, WO99 / 37805 or WO99 / 37806.

Thus, in a particular embodiment, the nucleic acid probe of the invention is (in the 5 ′ to 3 ′ direction)
Template part to be transcribed into RNA; (optional) +12 "part (12 base sequence immediately adjacent to the 5'end of the promoter that enhances promoter activity in the same manner as the -5 sequence: T7
Specific examples of +12 sequences for polymerases are in italics on page 3; promoter sequences; (optional) -5 sequences; and a portion complementary to the target; generally the target complement of the probe. Within the target moiety with one or more blocking moieties that are typically located 3'of the -5 sequence.

Desirably, the sequence of the probe molecule is selected to hybridize near the 3'end of the target such that the 3'end of the target is in the presence of deoxyribonucleotide triphosphate using the probe as a template. It is extended by a suitable DNA polymerase. This extension of the target creates a complementary strand of the single-stranded promoter present in the probe, which in turn forms a double-stranded functional RNA polymerase promoter, which in turn (selects the appropriate RNA polymerase and ribonucleotide triphosphates). (In the presence) can induce the formation of RNA. This RNA species is either directly detected or hybridized to a probe of the earlier side (having a single-stranded RNA promoter) and extended, thereby creating an earlier double-stranded RNA polymerase promoter, This leads to the synthesis of older RNA species. If desired, the sequence of the probe is such that the RNA species that precedes it (at least in part)
Selected to match that of the original target, which allows the RNA species ahead of it to hybridize to the original probe molecule, thereby restarting the process and leading to an amplification cycle. Such a cycle forms the basis of a very sensitive detection test.

Alternatively, the RNA species may be subjected to "stepped" amplification, in which case
Each RNA species produced hybridizes to the corresponding probe and is extended to create a double-stranded promoter, which leads to the synthesis of the earlier RNA species. The number of amplification steps may be increased as desired, resulting in significant amplification without (finally) cycling the original signal.

Although amplification cycles and steps of this kind have been disclosed in the prior art, the prior art does not disclose the use of probes with blocking moieties which are also expected to interfere with the method. Conversely, and surprisingly, we use the probes according to the invention to reduce the background signal (i.e., the amount of RNA produced in the absence of target), which improves the test system. I have found

As is known to those skilled in the art, the probe molecule of the present invention can be conveniently used in a test method for detecting a nucleic acid sequence of interest in a sample.

That is, in the second aspect, the present invention provides a method for detecting a nucleic acid sequence of interest in a sample, the method comprising the following steps, namely: adding a nucleic acid target molecule to the first aspect of the present invention. Contacting the nucleic acid probe molecule according to claim 1, wherein the target is the sequence of interest or is formed as a result of the presence of the sequence of interest in the sample; Extending the 3'end, thereby creating a functional double-stranded RNA polymerase promoter; creating RNA molecules by performing RNA synthesis from the RNA polymerase promoter; and RNA molecules thus produced Is directly or indirectly detected.

The step of causing extension of the 3'end of the target is conveniently carried out by a DNA polymerase (eg Klenow).
Fragment or Bst polymerase) and by adding deoxyribonucleotide triphosphate in a suitable reaction buffer. Appropriate reaction conditions are known to those skilled in the art.

[0034] Typically, the blocking moiety, in addition to blocking RNA polymerase,
It also tends to reduce non-specific interactions involving NA polymerase. Therefore,
The DNA polymerase goes beyond the blocking moiety (i.e. it is present in that portion of the probe that hybridizes to the target, but of course the blocking moiety itself cannot base pair and thus does not base pair with the target). It is usually preferred that the probe hybridize to the target so that the target need not be extended.

The step of causing RNA synthesis is typically performed by the presence of an RNA polymerase that recognizes the RNA promoter along with the appropriate ribonucleotide triphosphate. Preferred polymerases are T3, T7 and SP6 polymerases.

The RNA molecule produced is either directly detected (ie, “direct detection”) or is used as described above to detect one or more RNA molecules by any desired method (“indirect detection”). Formation of the destination RNA molecule (conveniently by a cycle or step amplification method)
May be done. Many detection methods are known in the art. In preferred detection, a label binding partner (e.g., original or earlier RNA molecule) typically labeled with an enzyme label (preferably horseradish peroxidase or alkaline phosphatase) or a fluorophore, radiolabel or biochemical label. Hybridize to
PNA, LNA, DNA or RNA sequences) are used.

For example, newly synthesized RNA can be detected by conventional methods (eg, by gel electrophoresis) with or without the introduction of labeled bases during synthesis.

Alternatively, for example, the newly synthesized RNA is captured on a solid phase surface (eg on beads or in a microplate) and the captured molecule is a labeled nucleic acid probe (eg radiolabel, or more preferably an enzyme, Detection number R by hybridization with chromophore, fluorescent group, etc.). Preferred enzyme labels are horseradish peroxidase and alkaline phosphatase.

One preferred detection method utilizes molecular beacons or fluorescence resonance energy transfer (FRET), delayed fluorescence energy transfer (DEFRET) or homogeneous time-resolved fluorescence (HTRD) techniques. A molecular beacon is a molecule that produces or does not produce a fluorescent signal depending on the shape of the molecule. Typically, one portion of the molecule has a fluorophore and another portion of the molecule has a "quencher" that quenches the fluorescence from the fluorophore. That is, when the shape of the molecule is such that the fluorophore and the quencher are in close proximity, the molecular beacon does not fluoresce, but when the fluorophore and the quencher are relatively widely separated, the molecule fluoresces.
The molecular beacon conveniently comprises a nucleic acid molecule and a quencher labeled with a suitable fluorophore.

A case where the shape of the molecular beacon can be changed is, for example, by hybridization of a portion of the molecular beacon to a nucleic acid that induces loop-out. Alternatively, the molecular beacon may initially have a hairpin-type structure (stabilized by self-complementary base pairing), which is altered by hybridization or by enzymatic or ribozyme degradation. .

FRET (Fluorescence Resonance Energy Transfer) occurs when a fluorescence donor molecule transfers energy to an acceptor molecule via a non-radiative dipole-dipole interaction. Energy transfer depending on the R ^-6 distance between the donor and acceptor reduces the lifetime and quantum yield of the donor and increases or sensitizes the fluorescence of the acceptor.

Another method (DEFRET, delayed fluorescence energy transfer) is a unique property of certain metal ions (lanthanides such as europium) that can show highly efficient and long-lived emission when excited to the excited state. Used (excitation = 337 nm, emission = 620 nm
). The advantage of such long-lived luminescence is that time-resolved (TR) techniques can be used, in which case luminescence measurements are initiated after an initial resting period, so that all background fluorescence and light Scattering can be eliminated. Cy5 (Amersham Pharm
acia) (excitation = 620 nm, emission = 665 nm) can be used as DEFRET partner.

HTRF (see WO92 / 01224; US5,534,622) occurs when a donor (eg europium) is encapsulated in a protective cage (cryptate) and ligated to the 5'end of the oligomer. The acceptor molecule developed for this system is a protein fluorophore called XL665. This molecule is linked to the 3'end of the second probe. This system was developed by Packard.

In another embodiment, the newly synthesized RNA forms ribozymes before and after amplification, which is due to degradation of specific nucleic acid substrate sequences (eg, degradation of fluorophore / quencher dual-labeled oligonucleotides). Can be detected.

In a third aspect, the invention provides a kit for use in the method of the second aspect, the kit comprising one or more probe molecules of the first aspect of the invention and packaging means.

The kit may optionally further comprise one or more of instructions for carrying out the method of the invention; buffer; DNA polymerase; RNA polymerase; dNTP; rNTP; and label binding partner.

The invention will be described in more detail by way of illustrative examples and with reference to the following figures:

Example 1 This example demonstrates the use of a hexaethylene glycol (Hex) linker as a blocker for RNA polymerase single-stranded read-through and other non-specific side reactions induced by DNA polymerase. Hex linkers are incorporated between the -52 / -53, -41 / -42 and -19 / -30 bases. Moreover, the Hex linker is incorporated at all three positions.

1.1 Preparation of Oligonucleotides All oligonucleotides were applied according to the manufacturer's instructions Applied Biosys
Synthesized by phosphoramidite chemistry using tems 380A synthesizer. Hex incorporation is 18-dimethoxytritylhexaethylene glycol, 1-[(2-cyanoethyl)-(N,
It was carried out by a chain extension reaction using N-diisopropyl)]-phosphoramidite.
Biotinylation of the oligonucleotide probe was performed by incorporation of biotin phosphoramidite. Oligonucleotides functionalized with alkaline phosphatase were prepared by the manufacturer's proprietary method (Oswel). All oligonucleotides were purified by HPLC according to a standard method.

1.2 Preparation of RNA RNA is 1 pmol of probes 1 and 2, T7 RNA polymerase buffer (Promega) (40 mM Tris-HCl (pH 7.9), 6 mM MgCl ₂ , 2 mM spermidine, 10 mM NaCl); T7 RNA polymerase (Promega) (25 μM). Units) and 2 μl rNTP mixture (Amersham Pharmacia Biotech) (each
NTP 20 mM; adenosine 5'-triphosphate (ATP), guanosine 5'-triphosphate (GTP), cytidine 5'-triphosphate (CTP) uridine 5'-triphosphate (U
TP)). The reaction volume was 20 μl with RNase-free distilled water. The mixture
Incubated for 3 hours at 37 ° C. RNase-free DNase (A
(3 units of DNase were added to 20 μl of test mixture and incubated for 10 minutes at 37 ° C., 3 minutes at 90 ° C.).

1.3 Synthesis of RNA Low Hybridization Oligonucleotide 10 fmol of linear DNA template (probe 3,4,5,6 or 7) and 10 fmol of RNA complementary to 3'end of linear template (prepared as above); Bst DNA Polymerase (New England Biolabs) (
3 '->5' exo-, 4 units); 1 μl dNTP mixture (Amersham Pharmacia Biotech) (each dNTP2
.5 mM: 2'-deoxyadenosine 5'-triphosphate (dATP), 2'-deoxythymidine
5'-triphosphate (dTTP), 2'-deoxyguanosine 5'-triphosphate (dGTP)
) And 2'-deoxycytidine 5'-triphosphate (dCTP)); T7 RNA polymerase buffer (Promega); T7 RNA polymerase (25 units) and 2 μl rNTP mixture (Amers
Hybridization was performed in a test mixture containing Ham Pharmacia Biotech). The reaction volume was 20 μl using RNase-free distilled water. Control reaction is complementary RN
A linear template was used in the absence of A. The mixture was incubated for 3 hours at 41 ° C.

1.4 Capture and detection of synthetic RNA Hybridization buffer containing 0.9 pmol of biotinylated capture oligonucleotide (specific for the RNA to be detected) and 6 pmol of specific alkaline phosphatase oligonucleotide in streptavidin-coated wells. Liquid (50m
Test sample 5 in 145 μl of M Tris-HCl, pH 8.0, 1 M NaCl, 20 mM EDTA and 0.1% BSA)
μl was added. RNA was immobilized in the wells via a biotinylated capture probe by incubation (60 min at room temperature, shaking at 300 rpm) and annealed to the detection probe. Unbound material was removed from the wells by washing 4 times with TBS / 0.1% TweeN-20 and then once with alkaline phosphatase substrate buffer (Boehringer Mannheim). Finally, alkaline phosphatase substrate buffer containing 4-nitrophenyl phosphate (5 mg / mL) was added to each well. The plates were incubated at 37 ° C in a Labsystems EIA plate reader and readings were recorded every 2 minutes at 405 nm. The results are shown in a bar graph in FIG. In FIG. 8, the pair of graphs is the amount of RNA produced in various reactions (left side shows the amount of RNA produced as a result of complete reaction, the right side shows the amount of RNA produced, ie background signal). From left to right, a) Hex unused in the probe; b) Hex between bases -53 / -54 (3'-most end of promoter sequence-
Assumed to be in position 17; c) Hex between bases -41 / -42; d) Hex between bases -29 / -20; and e) 3 Hex in the probe are bases -53 / -54, -41 / -41 /. Results are shown between -42 and -29 / -30. The numerical values at the top of the graph are the amplification factor (top) and S / N
(Bottom) is shown. The results show that background RNA is generated when only the linear DNA template is reacted with T7 RNA polymerase and Bst DNA polymerase. This is inconvenient in the amplification test because the background is amplified and the SN ratio becomes poor. By introducing the hexaethylene glycol linker into the region of the DNA template hybridized with the DNA or RNA primer, the background value was significantly reduced. The position of this linker with respect to the distance from the T7 promoter was important in controlling the degree of background reduction. Furthermore, the single hexaethylene glycol linker between bases -20 / -30 is similar to the three hexaethylene glycol linkers between bases -53 / -54, -41 / -42 and -29 / -30. Gave a background drop.

[0053]     1.5 List of oligonucleotides (H = Hex linker) Probe 1 SEQ ID NO: 2

[0054]

[Chemical 2]

[0055] Probe 2 SEQ ID NO: 3

[0056]

[Chemical 3]

[0057] Sequence of transcribed RNA SEQ ID NO: 4

[0058]

[Chemical 4]

[0059] Probe 3 (template, no Hex) SEQ ID NO: 5

[0060]

[Chemical 5]

[0061] Probe 4 (template, similar to SEQ ID NO: 5 except Hex is between bases -53 / -54)

[0062]

[Chemical 6]

[0063] Probe 5 (template, similar to SEQ ID NO: 5 except Hex is between bases 41 / -42)

[0064]

[Chemical 7]

[0065] Probe 6 (template, similar to SEQ ID NO: 5 except Hex is between bases 29 / -30)

[0066]

[Chemical 8]

[0067] Probe 7 (similar to SEQ ID NO: 5 except for template and Hex at all 3 locations)

[0068]

[Chemical 9]

[0069] Capture probe SEQ ID NO: 6

[0070]

[Chemical 10]

[0071] Detection probe SEQ ID NO: 7

[0072]

[Chemical 11]

According to the results of this experiment (FIG. 8), the signal-to-noise ratio was significantly affected by the position and number of blocking Hex linkers contained within the DNA probe. To investigate further, we used essentially identical probe molecules, but changed the Hex residue to bases -24 / -25, -27 / -28, -31 /-
The experiment was repeated with another probe at 32 or -33 / -34. The results are shown in FIG. 92, and the signs and the like are the same as in FIG.

According to these results, a single hexaethylene glycol linker between bases 31 / -32 resulted in high yields of RNA transcription and low background. Three hexaethylene glycol linkers are bases -53 / -54, -41 / -42 and -29 /-
Background was undetectable when between 30, but the signal was low as well.

Example 2 This example demonstrates the use of Hex linkers in RNA cyclic amplification.
Two DNA templates were used. The first DNA template was annealed to RNA1, extended with DNA polymerase and transcribed RNA2. The second template was annealed to RNA2 and extended with DNA polymerase to transcribe RNA1. Reaction is DNA without Hex linker
This was done using 2 templates and 2 DNA templates with 3 Hex linkers.

2.1 Preparation of Oligonucleotides All oligonucleotide probes were synthesized and purified as described in Example 1.

2.2 Preparation of RNA RNA1 is 1 pmol of probes 1 and 2, T7 RNA polymerase buffer (Promega); T7 RNA
Polymerase (Promega) (25 units) and 2 μl rNTP mixture (Amersham Pharmacia B
iotech). The reaction volume was 20 μl with RNase-free distilled water. The mixture was incubated for 3 hours at 37 ° C. DNA (probes 1 and 2) is RNase-free DNa
It was removed from the test mixture using se (Ambion) (3 units of DNase was added to 20 μl of the test mixture and incubated for 10 minutes at 37 ° C, 3 minutes at 90 ° C).

2.3 Synthesis of RNA Low Hybridizing Oligonucleotide The reaction mixture contained 1 fmol of probe 3 (no Hex) or probe 4 (3 Hex), and 2.2 above. 1 fmol of RNA prepared in 1., but this RNA is complementary to the 3'ends of probes 3 and 4; Bst DNA polymerase (New England Biolabs) (3 '->5' exo-
minus, 4 units); 1 μl dNTP mixture (Amersham Pharmacia Biotech); T7 RNA polymerase buffer; T7 RNA polymerase (25 units) and rNTP mixture 2 μl. The reaction mixture was adjusted to 20 μl with RNase-free distilled water. Mix for 1 or 2 hours
Incubated at 41 ° C. 100 fmol of probe 5 (no Hex) or probe 6 (3 Hex) were added to the reaction mixture containing probe 3 or probe 4 in 3 portions. The mixture was reincubated at 41 ° C. for a total reaction time of 3 hours.
The control reaction used the same probe used in the complete reaction, but no complementary RNA.

2.4 Capture and detection of synthetic RNA 5 μl of test sample was processed as described in Example 1.4. The results are shown in Fig. 10. Figure 10 shows H
It shows the RNA yield of the test reaction and the control reaction (paired) of the DNA template without ex-blocking portion (2 on the left side of the graph) or the probe with the Hex portion (2 on the right side of the graph). . For each set of probes, experiments were performed with a second DNA template (for cycle amplification reactions) added after an incubation time of 1 or 2 hours as described. The importance of the hexaethylene glycol linker was revealed when performing cycle amplification with two DNA templates. If the DNA template is used without a chemical linker, the RNA yield will be low without amplification and SN
The ratio will also be low. However, when a chemical linker is used the signal is increased and the signal to noise ratio is also improved. This improves the amplification process and increases the sensitivity of nucleic acid amplification tests.

[0080]     2.5 List of oligonucleotides (H = Hex linker) Probe 1 SEQ ID NO: 8

[0081]

[Chemical 12]

[0082] Probe 2 Same as SEQ ID NO: 3 in Example 1 Sequence of transcribed RNA SEQ ID NO: 9

[0083]

[Chemical 13]

[0084] Probe 3 (without Hex) SEQ ID NO: 10

[0085]

[Chemical 14]

Probe 4 (SEQ ID NO: except Hex is between bases -29 / -30, -41 / -42 and -53 / -54)
(Same as 10)

[0087]

[Chemical 15]

[0088] Probe 5 SEQ ID NO: 11

[0089]

[Chemical 16]

Probe 6 (SEQ ID NO: except Hex is between bases -28 / -29, -40 / -41 and -53 / -54)
(Same as 11)

[0091]

[Chemical 17]

Capture Probe Similar to SEQ ID NO: 6 in Example 1 Detection Probe Similar to SEQ ID NO: 7 in Example 1 Example 3 This example is a hexaethylene glycol (Hex) linker located between bases 31 / -32. Indicates that the result is a decrease in background. When extended with Bst, Klenow or Phi29 DNA polymerase, the Hex linker reduced background when using three different DNA templates.

[0093]     3.1 Preparation of oligonucleotides   All oligonucleotide probes were synthesized and purified as described above.

3.2 Preparation of RNA RNA was prepared using T7 RNA polymerase buffer (Promega); T7 RNA polymerase (Promega) (25 units) and 2 μl rNTP mixture (Amersham Pharmacia Biotech). The reaction volume was 20 μl with RNase-free distilled water. The mixture was incubated for 3 hours at 37 ° C. DNA was removed from the test mixture using RNase-free Dnase (Ambion) (3 units of DNase was added to 20 μl of test mixture and incubated for 10 minutes at 37 ° C, 3 minutes at 90 ° C). RNA1A is transcribed with 1 pmol of probe 1A and 2A and RNA2A is 1 pmo
was prepared using 1 probe 2A and 3A, and RNA 3A was prepared using 1 pmol probe 2A and 4A.

3.3 Synthesis of RNA Low Hybridization Oligonucleotide 10 fmol of linear DNA template and 10 fmol of RNA complementary to the 3'end of the linear template; Bst (New En
gland Biolabs) (3 '->5' exo-, 4 units); Klenow (New England Biolabs) (3 '->
5'exo-, 4 units) or Phi29 (Amersham Pharmacia Biotech) DNA polymerase
(3 '->5' exo-, 1 unit); 1 μl dNTP mixture (Amersham Pharmacia Biotech); T7 R
NA polymerase buffer; T7 RNA polymerase (25 units) and 2 μl rNTP mixture (Am
Hybridization was performed in a test mixture containing ersham Pharmacia Biotech). The reaction volume was 20 μl using RNase-free distilled water. Control reactions used linear template in the absence of complementary RNA. The mixture was incubated for 3 hours at 41 ° C.

3.4 Capture and Detection of Synthetic RNA 5 μl of test sample was treated with biotinylated capture oligonucleotide (strand from probe 9A and 10A using capture probe 1 to detect transcripts from probe 9A and 10A; Transcripts from probes 5A, 6A, 7A and 8A are detected) 0.9 pmol and alkaline phosphatase oligonucleotide (detection probe 1
Is used to detect transcripts from probes 9A and 10A; detection probe 2 is used to detect transcripts from probes 5A, 6A, 7A and 8A) 145 μl of hybridization buffer containing 6 pmol (50 mM Tris-HCl) , PH8.0, 1M NaCl, 20mM EDTA and
0.1% BSA). The microplate was then processed as described in the previous example. The results are shown in 11a, 11b and 11c. 11a to 11c use the same reference numerals as those in FIGS. 8 to 10. In Figures 11a-11c, three graphs (i), (ii) and (iii) show the results obtained with Bst, Klenow or Phi29 polymerase, respectively.
For each polymerase, the left pair of graphs are the results obtained with the probe without the Hex moiety and the right pair of graphs are the results obtained with the Hex containing probe. FIG. 11a shows the results of the reaction with RNA 1A and probe 5A (without hex) or 6A (with hex). Figure 11b shows RNA 2A and probe 7A (without hex) or 8A (with hex)
The result of the reaction using is shown. Figure 11c shows RNA3A and probe 9A (hex free) or 1
The result of the reaction using 0A (containing hex) is shown. This experiment shows that a similar reduction in background was observed when DNA templates containing hexaethylene glycol linkers were incubated with DNA polymerases other than Bst DNA polymerase. Furthermore, the hexaethylene glycol linker located between bases -3 / -32 also reduces the background of three different DNA template sequences. However, the amount of transcribed RNA was dependent on the sequence of the DNA template used.

[0097]     3.5 List of oligonucleotides (H = Hex linker) Probe 1A Same as SEQ ID NO: 2 in Example 1 Probe 2A Same as SEQ ID NO: 3 in Example 1 RNA 1A Same as SEQ ID NO: 4 in Example 1 Probe 3A SEQ ID NO: 12

[0098]

[Chemical 18]

[0099] RNA 2A SEQ ID NO: 13

[0100]

[Chemical 19]

[0101] Probe 4A SEQ ID NO: 14

[0102]

[Chemical 20]

[0103] RNA 3A SEQ ID NO: 15

[0104]

[Chemical 21]

[0105] Probe 5A Same as SEQ ID NO: 5 in Example 1 Probe 6A (same as SEQ ID NO: 5 except Hex is between bases 31 / -32)

[0106]

[Chemical formula 22]

[0107] Probe 7A Hex-free, SEQ ID NO: 16

[0108]

[Chemical formula 23]

[0109] Probe 8A (similar to SEQ ID NO: 16 except Hex is between bases 31 / -32)

[0110]

[Chemical formula 24]

[0111] Probe 9A Hex-free, (similar to SEQ ID NO: 12)

[0112]

[Chemical 25]

[0113] Probe 10A (similar to SEQ ID NO: 12 except Hex is between bases 31 / -32)

[0114]

[Chemical formula 26]

[0115] Capture probe 1 Similar to SEQ ID NO: 6 in Example 1 Detection probe 1 Same as SEQ ID NO: 7 of Example 1 Capture probe 2 SEQ ID NO: 17

[0116]

[Chemical 27]

[0117] Detection probe 2 SEQ ID NO: 18

[0118]

[Chemical 28]

Example 4 This example shows that various chemical linkers could be used as blockers for RNA polymerase single-stranded readthroughs and other non-specific side reactions by nucleic acid polymerases. In all cases, the chemical linker tested was a base
It was located between -31 / -32.

4.1 Preparation of Oligonucleotides All oligonucleotide probes were synthesized as described above. Octanediol
(Oct) was incorporated by a chain extension reaction with 8-dimethoxytrityloctanediol and 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite. Propanediol (Pro) was incorporated by chain extension reaction using 3-dimethoxytritylpropanediol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite. All oligonucleotides were purified by HPLC according to a standard method.

4.2 RNA Preparation 100 fmol of probes 1 and 2, T7 RNA polymerase buffer (Promega); T7 RNA polymerase (Promega) (25 units) and 2 μl rNTP mixture (Amersham Pharmacia Bi
otech) was used to prepare RNA1. The reaction volume was 20 μl with RNase-free distilled water.
The mixture was incubated for 3 hours at 37 ° C.

4.3 Synthesis of RNA Low Hybridization Oligonucleotides The reaction mixture contained 10 fmol of probe 3, probe 4 or probe 5 and 10 fmol of RNA1 transcribed with probes 1 and 2, provided that this RNA was probe 3, 4 or Complementary to the 3'end of 5; Bst DNA polymerase (New England Biolabs
) (3 '->5' exo-minus, 4 units); 1 μl dNTP mixture (Amersham Pharmacia Biotech);
T7 RNA polymerase buffer; 2 μl of T7 RNA polymerase (25 units) and rNTP mixture (A
mersham Pharmacia Biotech). The reaction mixture was adjusted to 20 μl with RNase-free distilled water. The mixture was incubated at 37 ° C for 3 hours.

4.4 Capture and Detection of Synthetic RNA 10 μl of test sample was processed as described in Example 1.4. The results are shown in Figure 12. Figure 12 shows the results obtained using the probe for the reaction, the probe being similar except for the type of blocking moiety present (hex, octanediol or propanediol). The result of the experiment performed in duplicate is shown. All three chemical linkers tested between bases 31 / -32 resulted in the same degree of background inhibition. Therefore, the same effect can be obtained by using any of these three types of linkers.

[0124]     4.5 List of oligonucleotides Probe 1 SEQ ID NO: 19

[0125]

[Chemical 29]

[0126] Probe 2 Same as SEQ ID NO: 3 in Example 1 Probe 3 (H = hexaethylene glycol) SEQ ID NO: 20

[0127]

[Chemical 30]

[0128] Probe 4 (same as SEQ ID NO: 20 except having O = octanediol)

[0129]

[Chemical 31]

[0130] Probe 5 (same as SEQ ID NO: 20 except having P = propanediol)

[0131]

[Chemical 32]

Sequence of Transcribed RNA-Capture probe similar to SEQ ID NO: 13 of Example 3 Detection probe similar to SEQ ID NO: 6 of Example 1 Similar to SEQ ID NO: 7 of Example 1 Example 5 How This Example Hex with T7 RNA polymerase and Bst DNA polymerase
It is intended to clarify whether it interacts with the linker. These experiments are shown in Figure 13a.
And 13b.

These experiments show that no RNA signal could be detected when performing the reaction with the Hex linker located in the granules of the T7 promoter (FIG. 13a). This occurs in both transcription (Fig. 14a) and elongation plus transcription (Fig. 14b) reactions. Hex
RNA signals were detected in the control reaction without the linker (Probe 3). Also,
No RNA signal was detected (Figure 15) when using DNA primers that did not overlap the Hex linker located in the distillation of the T7 promoter (Probe 7; Figure 13b). A control reaction (Probe 5) with a DNA primer that overlaps the Hex linker gives an RNA signal. This result is that (1) transcription by T7 RNA polymerase is terminated when the Hex linker is located downstream of the T7 promoter in the template to be transcribed; (2) extension and transcription reactions should be transcribed. It is shown to be unsuccessful if the 3'end of the DNA is located upstream of the Hex linker in the template.

[0134]     5.1 Preparation of oligonucleotides   All oligonucleotide probes were synthesized and purified as described above.

5.2 RNA Preparation 100 fmol of probes 1 and 2, T7 RNA polymerase buffer; T7 RNA polymerase (2
RNA1 using 5 units) and 2 μl rNTP mixture (Amersham Pharmacia Biotech)
Was prepared. The reaction volume was 20 μl with RNase-free distilled water. Mix for 3 hours at 37 ° C
Incubated at.

5.3 Synthesis of RNA by transcription 10 fmol of probe 2 and probe 3 or 10 fmol of probe 2 and probe 4
Was used. Probe 2 was complementary to the -5 and T7 promoter sequences of probes 3 and 4, thus creating a double stranded T7 promoter. T7
RNA polymerase buffer (Promega); T7 RNA polymerase (Promega) (25 units) and 2
A reaction mixture containing μl of rNTP mixture (Amersham Pharmacia Biotech) was used. The reaction volume was 20 μl with RNase-free distilled water. The mixture was incubated for 3 hours at 37 ° C.

5.4 RNA Low Hybridization RNA or DNA Primer Synthesis Reaction mixture is 10 fmol RNA1 and probe 3 or 10 fmol RNA1 and probe 4; 10 fmol probe 5 and probe 6 or 10 fmol probe 7 and probe 6; 10 fmol probe Those containing 5 and probe 8 or 10 fmol of probe 7 and probe 8 were used. RNA or DNA primers (RNA1, probe 5 or probe 7) were complementary to the 3'ends of probes 3,4,5 and 8. For each reaction; Bst
DNA polymerase (New England Biolabs) (3 '->5' exo-minus, 4 units); 1 μl dN
TP mix (Amersham Pharmacia Biotech); T7 RNA polymerase buffer; T7 RNA polymerase (25 units) and 2 liters of rNTP mix. The reaction mixture is RNa
It was made up to 20 l with se-free distilled water. The mixture was incubated at 37 ° C for 3 hours.

5.5 Capture and Detection of Synthetic RNA Some of the test samples were processed as described in the previous examples. Probes 3 and 4
RNA transcripts from S. alba were quantified using capture probe 1 and detection probe 1; RNA transcripts from probes 6 and 8 were quantified using capture probe 2 and detection probe 2. The results are shown in Figures 14 and 15. FIG. 14 (a) shows the amount of RNA produced with probes 2 and 3 (left pair of graphs) or probes 2 and 4 (right pair of graphs) in a transcription-only reaction. Figure 14 (b) shows elongation, followed by transcription of probe 3 and RNA.
The amount of RNA produced in the reactions required to be performed with 1 (left pair of graphs) or probe 4 and RNA1 (right pair of graphs) is shown. For each pair of graphs, the graph on the left shows the yield of complete reaction and the graph on the right shows the yield of control reaction.
In both 14 (a) and 14 (b), the hex part was located downstream of the T7 promoter as shown in Figure 13 (a).

FIG. 15 shows RNA produced using a template without hex (a) or a template with hex (b).
Indicates the amount of a (i) shows the results with probes 5 and 6, a (ii) shows the results with probes 6 and 7, b (I) shows the results with probes 5 and 8. The results are shown, and b (ii) shows the results when probe 7 and probe 8 were used.

[0140]     5.6 List of oligonucleotides Probe 1 SEQ ID NO: 21

[0141]

[Chemical 33]

[0142] Probe 2 Same as SEQ ID NO: 3 in Example 1 Probe 3 Same as SEQ ID NO: 5 in Example 1 Probe 4 (same as probe 3 above, except that H = hexaethylene glycol)

[0143]

[Chemical 34]

[0144] Probe 5 SEQ ID NO: 22

[0145]

[Chemical 35]

[0146] Probe 6 (same as SEQ ID NO: 20 except without Hex) Probe 7 SEQ ID NO: 23

[0147]

[Chemical 36]

[0148] Probe 8 (H = hexaethylene glycol), similar to SEQ ID NO: 20 Capture probe 1 Similar to SEQ ID NO: 17 of Example 3 Detection probe 1 Similar to SEQ ID NO: 18 of Example 3 Capture probe 2 Same as SEQ ID NO: 6 in Example 1 Detection probe 2 Same as SEQ ID NO: 7 in Example 1

[0149]

[Chemical 37]

Example 6 This example compares the step amplification of 50 amol of DNA primer with two linear DNA templates with or without chemical linkers.

[0151]     6.1 Preparation of oligonucleotides   All oligonucleotide probes were prepared and purified as described above.

6.2 Synthesis of RNA Low Hybridizing Oligonucleotide The following method was used for the reaction using the probe having the chemical linker. 1 fmol of probe 1 and 50 amol of a DNA primer (probe 2) complementary to the 3'end of the linear template (probe 1); Bst DNA polymerase (New England Biolabs) (3 '->5' exo-,
Hybridization was performed in a test mixture containing 1 μl dNTP mixture (Amersham Pharmacia Biotech). Reaction volume is 18 μl with RNase-free distilled water
And The mixture was incubated at 37 ° C for 1 hour. Then 20 fmol of probe 3
Was added in a volume of 2 l to bring the reaction volume to 20 l. Probes 4 and 5 were used in reactions involving probes without chemical linkers. Only probes 1 and 3 or probes 4 and 5 were used in control reactions.

6.3 Capture and Detection of Synthetic RNA 20 μl of test sample (or a dilution thereof) was processed as described in the previous example. The results are shown in Figure 16. FIG. 16 shows the RNA yield (femtomolar units) of a `` step '' (i.e., non-cycle) amplification reaction in which the template has a blocking moiety of the invention (A) or when a conventional template without a blocking moiety is used (B). Is shown. In all reactions, 1 fmol of the first template and 20 or 40 fmol of the second template were used as shown in the lower part of the graph. The numbers at the top of the graph show the amplification factor (top) and the signal-to-noise ratio (bottom). As in the previous figure, for each pair of graphs, the left graph shows the results of the complete reaction and the right graph shows the results of the control reaction. These show that a chemical linker is required in the DNA template to improve the step amplification process.
In this experiment, the octanediol linker was positioned between bases 31 / -32 of the first DNA template and hexaethylene glycol between bases 31 / -32 of the second DNA template. According to the result of the reaction, the S / N ratio and the RNA transcription amount were lower than those in the case where no chemical linker was contained.

[0154]     6.4 List of oligonucleotides Probe 1 (O = Oct), similar to probe 4 in Example 4

[0155]

[Chemical 38]

[0156] Probe 2 Same as SEQ ID NO: 22 of Example 5 Probe 3 (H = Hex) SEQ ID NO: 24

[0157]

[Chemical Formula 39]

[0158] Similar to SEQ ID NO: 20 except without probe 4, Hex Similar to SEQ ID NO: 24 except without probe 5 and Hex Transcription RNA sequence, SEQ ID NO: 25

[0159]

[Chemical 40]

[0160] Capture probe SEQ ID NO: 26

[0161]

[Chemical 41]

[0162] Detection probe SEQ ID NO: 27

[0163]

[Chemical 42]

[0164]

[Sequence list]

[Brief description of drawings]

FIG. 1 is a schematic diagram showing hexaethylene glycol which is a blocking portion incorporated into a DNA probe according to the present invention.

FIG. 2 is a schematic diagram of a nucleic acid probe of the present invention, showing a preferred embodiment.

FIG. 3 is a schematic diagram of a nucleic acid probe of the present invention.

FIG. 4 is a schematic diagram of a pair of nucleic acid probes of the present invention, which may be used in an amplification cycle.

FIG. 5 is a schematic diagram of a pair of nucleic acid probes of the present invention, which may be used in an amplification cycle.

FIG. 6 is a schematic diagram of the nucleic acid probe of the present invention, showing a preferred embodiment.

FIG. 7 is a schematic diagram showing amplification using the probes shown in FIGS. 4 and 5.

FIG. 8 is a bar graph comparing the yield of various reactions (femtomolar units of RNA) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 9 is a bar graph comparing the yields of various reactions (femtomolar RNA) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 10 is a bar graph comparing the yield of various reactions (femtomolar RNA) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 11a is a bar graph comparing the yield of various reactions (RNA in femtomolar units) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 11b is a bar graph comparing the yield of various reactions (femtomolar RNA) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 11c is a bar graph comparing the yields of various reactions (RNA in femtomolar units) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 12 is a bar graph comparing the yields of various reactions (RNA in femtomolar units) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 13a is a schematic diagram of a reaction scheme.

FIG. 13b is a schematic diagram of a reaction scheme.

Figure 14a is a bar graph comparing the yield of various reactions (RNA in femtomolar units) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

Figure 14b is a bar graph comparing the yield of various reactions (RNA in femtomolar units) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 15 is a bar graph comparing the yield of various reactions (femtomolar RNA) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

FIG. 16 is a bar graph comparing the yields of various reactions (RNA in femtomolar units) with a probe of the invention having a blocking moiety compared to a conventional probe without a blocking moiety.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] September 6, 2000 (200.9.6)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0137

[Correction method] Change

[Contents of correction]

5.4 RNA Low Hybridization RNA or DNA Primer Synthesis Reaction mixture is 10 fmol RNA1 and probe 3 or 10 fmol RNA1 and probe 4; 10 fmol probe 5 and probe 6 or 10 fmol probe 7 and probe 6; 10 fmol probe Those containing 5 and probe 8 or 10 fmol of probe 7 and probe 8 were used. RNA or DNA primers (RNA1, probe 5 or probe 7) were complementary to the 3'ends of probes 3,4,5 and 8. For each reaction; Bst
DNA polymerase (New England Biolabs) (3 '->5' exo-minus, 4 units); 1 μl dN
TP mix (Amersham Pharmacia Biotech); T7 RNA polymerase buffer; T7 RNA polymerase (25 units) and 2 liters of rNTP mix. The reaction mixture is RNa
20 μl of se-free distilled water was prepared . The mixture was incubated at 37 ° C for 3 hours.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0152

[Correction method] Change

[Contents of correction]

6.2 Synthesis of RNA Low Hybridizing Oligonucleotide The following method was used for the reaction using the probe having the chemical linker. 1 fmol of probe 1 and 50 amol of a DNA primer (probe 2) complementary to the 3'end of the linear template (probe 1); Bst DNA polymerase (New England Biolabs) (3 '->5' exo-,
Hybridization was performed in a test mixture containing 1 μl dNTP mixture (Amersham Pharmacia Biotech). Reaction volume is 18 μl with RNase-free distilled water
And The mixture was incubated at 37 ° C for 1 hour. Then 20 fmol of probe 3
Was added in a volume of 2 μl to give a reaction volume of 20 μl . Probes 4 and 5 were used in reactions involving probes without chemical linkers. Only probes 1 and 3 or probes 4 and 5 were used in control reactions.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] July 17, 2001 (2001.17)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Weston, Anthony             You Be 5 54 UK             Middle sex, no salt, dorch             Wester Close, New Court, 8 (72) Inventor Cardi, Donald Leonard Nicola             Su             United Kingdom, N.N. 11 6 YOU             Nunortamptonshire, Aston-Le             -Walls, Blacksmiths Ray             N, Trinlan (No address) (72) Inventor Marsh, Peter             UK, CV 31 2 A             Warwickshire, Leamington             Spa, Eagle Street, 4 F term (reference) 4B024 AA11 AA20 CA09 CA11 HA14                 4B063 QA18 QQ42 QQ52 QR08 QR56                       QS26 QS34 QX02 QX07

Claims (26)

[Claims] 1. A probe molecule having a single-stranded nucleic acid, wherein the probe is adjacent to or substantially adjacent to a single-stranded sequence complementary to a target nucleic acid sequence, a single strand of an RNA polymerase promoter sequence, and a promoter sequence. The above probe molecule having a blocking portion. 2. The probe according to claim 1, which has a template strand of an RNA polymerase promoter. 3. A -5 sequence flanking the 3'end of a promoter sequence.
The probe according to 1 or 2. 4. The method according to claim 1, which has a +12 sequence adjacent to the 5'end of the promoter.
The probe according to any one of 2 or 3. 5. The probe according to claim 1, wherein the 3 ′ end of the target is extendable by a DNA polymerase when hybridized to the target. 6. The probe according to any one of the preceding claims, wherein the target complementary portion is located at the 3'end of the promoter sequence. 7. The probe according to claim 1, wherein the blocking portion is located between the -19th position and the -68th position. 8. The probe according to claim 1, wherein the blocking portion is located between the -19th position and the -38th position. 9. A probe according to any one of the preceding claims, wherein the blocking moiety is located between the -22 and -35 positions. 10. A probe according to any one of the preceding claims wherein the blocking moiety has C ₂ -C ₂₀ alkyl, alkanol or alkylene residue. 11. The probe according to any one of the preceding claims, wherein the probe has C ₃ -C ₁₀ alkyl, alkanol or alkylene residues. 12. The probe according to claim 1, which has an octanediol, propanediol or hexaethylene glycol residue. 13. The probe according to claim 1, which has a PNA and / or an LNA. 14. A probe according to any one of the preceding claims, wherein the target complementary portion of the probe has a PNA and / or LNA. 15. A method for detecting a nucleic acid sequence of interest in a sample, the method comprising the steps of: contacting a nucleic acid probe molecule according to any one of the preceding claims with a nucleic acid target molecule. , Provided that the target is the sequence of interest or is formed as a result of the presence of the sequence of interest in the sample; extending the 3'end of the target using the probe as a template, This allows the functional duplex
Creating an RNA polymerase promoter; creating RNA molecules by synthesizing RNA from the RNA polymerase promoter; and RNA thus produced
Detecting the molecule directly or indirectly. 16. An RNA molecule is hybridized to a probe molecule of a further side and extends, whereby a RNA polymerase promoter of a further side is created, which causes synthesis of the RNA molecule of a further side, and RNA of the RNA produced thereby is generated. 16. The method of claim 15, wherein the amount is amplified. 17. The method of claim 16, wherein the earlier RNA molecule is hybridized to the second earlier probe molecule and extended. 18. The sequence of the earlier RNA molecule is substantially the same as that of the original target molecule so that it hybridizes to the original nucleic acid probe molecule under the test conditions used by the earlier RNA molecule. 18. The method according to claim 16 or 17, which is capable. 19. The method according to any one of claims 16, 17 or 18, wherein the target sequence comprises DNA or RNA. 20. The method according to any one of claims 16 to 19, wherein the target sequence is DNA or RNA formed as a result of the presence of the nucleic acid sequence of interest in the sample. 21. The method of any one of claims 16-20, wherein the RNA molecule is detected directly or indirectly using a labeled binding partner. 22. The method of claim 21, wherein the label binding partner is an enzyme, fluorophore, radiolabel or biochemical label. 23. The method of claim 21 or 22, wherein the labeled binding partner comprises DNA, RNA, LNA, PNA or any combination thereof. 24. A kit for use in carrying out the method according to any one of claims 16 to 23, which comprises the probe molecule according to any one of claims 1 to 14 and a packaging means. 25. The following further elements: instructions for carrying out the method according to any one of claims 16 to 23; buffer; DNA polymerase; RNA polymerase;
25. The kit of claim 24, further comprising one or more of deoxyribonucleotide triphosphates; ribonucleotide triphosphates; and label binding partners. 26. A method for detecting a nucleic acid sequence of interest in a sample, the method comprising the steps of: a further probe and a nucleic acid target molecule. Contacting the nucleic acid probe molecule, provided that the target is the sequence of interest or is formed as a result of the presence of the sequence of interest in the sample; Hybridize to the probe molecule adjacent to each other to create a functional double-stranded RNA polymerase promoter; perform RNA synthesis from the RNA polymerase promoter
Creating the RNA molecule; and detecting the RNA molecule thus produced, either directly or indirectly.