TWI754652B - Improvements in or relating to nucleic acid amplification processes - Google Patents

Abstract

Disclosed is a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a) mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation event in which the primers hybridise to the target, which hybridisation event, directly or indirectly, leads to the formation of a duplex structure comprising two nicking sites disposed at or near opposite ends of the duplex; and performing an amplification process by; (b) causing a nick at each of said nicking sites in the strands of the duplex; (c) using a polymerase to extend the nicked strands so as to form newly synthesised nucleic acid, which extension with the polymerase recreates nicking sites; (d) repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesised nucleic acid; characterised in that the temperature at which the method is performed is non-isothermal, and subject to a reduction of at least 2℃ during the amplification process of steps (b)-(d).

Description

Improvements in or related to nucleic acid amplification methods

The present invention relates to a method of amplifying nucleic acid and an apparatus for carrying out the method.

The polymerase chain reaction (PCR) is the most widely used in vitro method for DNA amplification. Although extremely effective, this technique requires the use of a thermal cycler to subject the reaction mixture to periodic temperature changes to achieve amplification. Therefore, PCR is not particularly suitable for use outside of a laboratory environment, such as in the context of a point-of-care ("PoC") diagnostic device.

To partially overcome this shortcoming, various isothermal amplification techniques have been devised that avoid the need for thermal cycling. Such techniques include, for example: signal-mediated RNA amplification techniques ("SMART"; WO 99/037805); nucleic acid sequence-based amplification ("NASBA" Compton 1991 Nature 350, 91-92); rolling circle amplification ("NASBA" Compton 1991 Nature 350, 91-92) "RCA", eg, see Lizardi et al., 1998 Nature Genetics 19, 225-232); loop-mediated amplification ("LAMP", see Notomi et al., 2000 Nucl. Acids Res. 28, (12)e63); recombinase polymerase Amplification ("RPA" see Piepenberg et al, 2006 PLoS Biology 4(7)e204); Strand Displacement Amplification ("SDA"); Helicase Dependent Amplification ("HDA" Vincent et al, 2004 EMBO Rep. 5,795-800); Transcription-Mediated Amplification ("TMA"); Single Primer Isothermal Amplification ("SPIA" see Kurn et al., 2005 Clinical Chemistry 51, 1973-81); Sequence sustaining amplification ("3SR"); and nickase amplification reaction ("NEAR").

SDA is a technique (disclosed by Walker et al., 1992 Nucl. Acids Res. 20, 1691-1696) that involves the use of a pair of short "buffer" primers upstream of a pair of primers comprising one of the complementary portions of the target And the 5' of the complementary part of the target (the recognition and cleavage site of the endonuclease). "Buffer" primers help initiate SDA reactions by generating complementary single-stranded targets for primer amplification. The primers hybridize to respective complementary single-stranded target molecules. Using a reaction mixture comprising DNA polymerase and at least one modified nucleotide triphosphate, the 3' end of the target strand is extended using the primer as a template (and again, the 3' end of the primer is extended using the target as a template. )

Extension of the target strand creates a double-stranded recognition site for the endonuclease. However, because the target is extended using a modified triphosphate, the endonuclease does not cleave the two strands, but instead creates a single-stranded nick in the primer. The 3' end at the nick is then extended by a DNA polymerase (typically a Klenow fragment of DNA polymerase I lacking exonuclease activity). As the nick primer is extended, it displaces the extension product originally generated. Because the substitution product essentially replicates the target sequence of the opposite primer, it is then free to hybridize to the opposite primer. In this way, exponential amplification of both strands of the target sequence is achieved.

The amplification stage of the SDA method is substantially isothermal - typically carried out at 37°C, which is the optimum temperature for endonucleases and polymerases. However, before reaching the amplification stage, the double-stranded target needs to be completely dissociated into its constituent single strands to allow the primer pair to hybridize to its complementary target strand.

This dissociation or "melting" is typically accomplished by heating the double-stranded target to an elevated temperature, typically about 90°C, to break the hydrogen bonds between the two strands of the target. The reaction mixture is then cooled to allow the addition of enzymes required for the amplification reaction. Due to the use of high temperature to generate single-stranded targets, SDA The technology is not ideally suited for a PoC environment.

US 6,191,267 discloses the selection and performance of the N.BstNBI nickase and its use in SDA as an alternative to restriction endonucleases and modified triphosphates.

Another amplification technique similar to SDA is the nickase amplification reaction (or "NEAR").

In "NEAR" (eg, as disclosed in US 2009/0017453 and EP 2,181,196), forward and reverse primers (referred to as "templates" in US 2009/0017453 and EP 2,181,196) hybridize to the double-stranded target Separate chains and extend. Further copies of the forward and reverse primers (present in excess) hybridize to the extension product of the opposite primer and extend themselves, creating an "amplified duplex". Each amplified duplex thus formed contains a nicking site towards the 5' end of each strand which is nicked by a nickase, allowing synthesis of further extension products. The previously synthesized extension product can simultaneously hybridize to a further copy of the complementary primer, extending the primer and thereby producing a further copy of the "amplified duplex". In this way, exponential amplification can be achieved.

NEAR differs from SDA in particular in that no "buffer" primer and initial thermal dissociation steps are required. Although the target is still substantially double-stranded, the initial primer/target hybridization event required to trigger the amplification method still occurs: the initial primer/target hybridization is believed to utilize partial dissociation of the target strand - a phenomenon known as "respiration" (See Alexandrov et al., 2012 Nucl. Acids Res. and reviewed by Von Hippel et al., 2013 Biopolymers 99(12), 923-954). Respiration is the local and temporary loosening of base pairing between DNA strands. The melting temperature (Tm) of the initial primer/target heteroduplex is typically much lower than the reaction temperature, so the primer tends to dissociate, but for the polymerase, the brief hybridization lasts long enough to extend the primer, which increases the heteroduplex. Tm of the helix and make it stable.

The amplification phase in NEAR is performed isothermally at a constant temperature. In fact, Xizhi is communicating The initial target/primer hybridization and subsequent rounds of amplification are often performed at the same constant temperature ranging from 54°C to 56°C.

Avoiding the need for thermal cycling means that isothermal techniques may be more useful than PCR in a PoC environment. In addition, the synthesis of a large number of amplification products can be achieved even with a very low number of target molecules (eg, as few as 10 double-stranded target molecules) as starting materials.

WO 2011/030145 (Enigma Diagnostics Ltd.) discloses an "isothermal" nucleic acid amplification reaction carried out under temperature shaking conditions, wherein the reaction is initially carried out at a predetermined temperature, the temperature is allowed to deviate upward or downward from the predetermined temperature, and The temperature is then returned to the predetermined temperature at least once during the amplification reaction. More typically, the temperature is allowed to "wiggle" a small amount (about 5°C) above and below the predetermined temperature.

The present invention aims to provide, inter alia, an improved nucleic acid amplification technique having one or more advantages over the prior art, including, for example, reduced reaction time, and/or increased yield, and/or reduced non-specific amplification products .

In a first aspect, the present invention provides a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a) combining a target sequence with one or more complementary single-stranded primers in a manner allowing the primers to hybridize to the target sequence Mixing under conditions of a hybridization event of the target that results, directly or indirectly, in the formation of a duplex structure comprising two nick sites disposed at or near opposite ends of the duplex; and amplifying by (b) creating a nick at each of the nick sites in the strands of the duplex; (c) using a polymerase to extend the nicked strand to form a newly synthesized nucleic acid using a polymerase (d) repeating steps (b) and (c) as necessary to generate multiple copies of the newly synthesized nucleic acid; characterized in that the temperature at which the method is performed is non-isothermal, and The temperature is lowered by at least 2°C, preferably at least 5°C during the amplification process of step (b)-step (d).

In a second aspect, the present invention provides an apparatus for carrying out the method of the first aspect of the present invention, the apparatus comprising a temperature regulating member and a programmable control member, in a reaction for carrying out the method of the first aspect During the amplification method of the mixture, the programmable control means are programmed to operate the temperature regulation means to reduce the temperature by at least 2°C, preferably at least 5°C.

The amplification methods of the methods of the present invention can be applied to commonly known and conventional amplification techniques including SDA and NEAR.

Thus, for example, the amplification method can be based on the amplification method employed in strand displacement amplification, or based on NEAR or indeed any other nucleic acid amplification that relies on the creation of a single-stranded nick and subsequent extension from the 3' end of the nicked strand The amplification method used in the amplification method. Thus, prior art teachings related to the amplification stage of SDA or NEAR will generally be equally applicable to the amplification method of the method of the present invention (except for prior art teachings related to maintaining a constant temperature during amplification).

Preferably, step (a) comprises mixing the sample containing the double-stranded target with two single-stranded primers, one of which is complementary to the first strand of the target, and the other of which is complementary to the first strand of the target. Complementary to the second strand of the target such that two primers hybridize to the target with the free 3' ends of the primers facing each other.

The two primers can be conveniently described as "forward" and "reverse" primers.

Desirably, both the forward and reverse primers will contain a nickase recognition site sequence. Typically, the nicks made by the nickase will only be external and typically 3' of the nickase recognition site.

In a preferred embodiment, the forward primer will comprise at or near its 3' end a portion complementary to and hybridizable to the 3' end of the antisense strand of the target sequence, and the reverse primer at its 3' end At or near the portion of the reverse primer that is complementary to and hybridizable to the 3' end of the sense strand of the target sequence.

In this way, a nickase recognition site is introduced at the opposite end of the target sequence, and amplification of the target sequence (along with the intervening sequence of the primer downstream of the nick site) is accomplished by performing forward and reverse primers Multiple cycles of extension of the polymerase to form a double-stranded nicking site, and nicking the nicking site with a nickase, such that the nicked primer is further extended by a polymerase or the like, substantially as disclosed, for example, in US 2009/0017453, which The contents are incorporated herein by reference.

Detailed description

In particular, the present invention relates to a method for the amplification of selected target nucleic acids.

The target can be single-stranded, double-stranded, or a mixture comprising both. The target may comprise RNA, DNA, or a mixture of both. In particular, the target may incorporate one or more modified nucleotide triphosphates (ie, nucleotide triphosphates not normally found in naturally-occurring nucleic acids), although this is not necessary and in practice is not preferred .

The target can be selected from the following non-exhaustive list: genomic nucleic acid (this term encompasses any animal, plant, fungal, bacterial or viral genomic nucleic acid), plastid DNA, mitochondrial DNA, cDNA, mRNA, rRNA, tRNA or synthetic Oligonucleotides or other nucleic acid molecules.

In particular, the method may additionally comprise an initial reverse transcription step. For example, RNA (eg, viral genomic RNA, or cellular mRNA, or RNA from some other source) can be For the synthesis of DNA or cDNA using reverse transcriptase according to methods well known to those skilled in the art. DNA can then be used as a target sequence in the methods of the present invention. The original RNA will typically be degraded by the ribonuclease activity of reverse transcriptase, but additional RNase H may be added after completion of reverse transcription if necessary. RNA molecules are often present in samples in greater numbers of copies than corresponding (eg, genomic) DNA sequences, therefore, DNA transcripts are preferably prepared from RNA molecules to effectively increase the number of copies of DNA sequences.

A "target sequence" is a sequence of bases in a target nucleic acid, and can refer to the sense and/or antisense strands of a double-stranded target, and, unless the context dictates otherwise, also encompasses amplification from the original target nucleic acid The nucleotide sequence identical to the nucleotide sequence reproduced or replicated in the amplification product, extension product or amplification product.

The target sequence can be present in any kind of sample, such as biological or environmental samples (water, air, etc.). The biological sample can be, for example, a food sample or a clinical sample. Clinical samples may include the following: urine, saliva, blood, serum, plasma, mucus, sputum, tears, or feces.

The sample may or may not be subjected to treatment prior to contact with the primer. Such processing may include one or more of: filtration, concentration, partial purification, sonication, dissolution, and the like. Such methods are well known to those skilled in the art.

The methods of the present invention involve the use of nick sites and means for creating nicks at the nick sites. A "nick" is the cleavage of the phosphodiester backbone of only one strand of a complete or at least partially double-stranded nucleic acid molecule. A nick site is a location in a molecule where a nick is made.

In preferred embodiments, a "nick recognition site" will exist at, within, or near a nick site. ("Nearby" in this context means that the nearest base of the nick recognition site is within 10 bases of the nick site, preferably within 5 bases of the nick site).

The nick recognition site may comprise at least one of the recognition sites of the restriction endonuclease one strand, and the nicking site may comprise at least one strand of the nucleic acid base sequence cleaved by restriction endonucleases when present as a double-stranded molecule. Typically, restriction endonucleases will cleave both strands of a double-stranded nucleic acid molecule. In the present invention, double-strand breaks can be avoided by incorporating at or near the nick site one or more modified bases that render the strand of nucleic acid less susceptible to cleavage by restriction endonucleases. In this way, restriction endonucleases, which typically cleave both strands of a double-stranded nucleic acid molecule, can be used to introduce single-stranded nicks into the double-stranded molecule. Modified bases and analogs suitable for accomplishing this are well known to those skilled in the art and include, for example, all alpha phosphate-modified nucleoside triphosphates and alpha borane-modified nucleoside triphosphates; Oxyadenosine 5'-O-(thiotriphosphate), 5-methyldeoxycytidine 5'-triphosphate, 2'-deoxyuridine 5'-triphosphate, 7-deaza -2'deoxyguanosine 5'-triphosphate, 2'deoxyguanosine-5'-O-(1-borane triphosphate), and the like. Triphosphates comprising modified bases can be present in the reaction mixture used to perform the amplification method so that modified bases are incorporated at relevant positions during subsequent rounds of amplification to prevent the formation of sites that are cleaved by endonucleases point.

However, in a preferred embodiment, the nick is made at the nick site with the aid of a nickase. These are enzymes that break only single strands in double-stranded nucleic acid molecules under normal circumstances. The nickase has a nick recognition site and the nick site can be within the nick recognition site or can be 5' or 3' of the recognition site. Many nicking enzymes are known to those skilled in the art and are commercially available. A non-exhaustive list of examples of nickases includes: Nb.Bsml, Nb.Bts, Nt.Alwl, Nt.BbvC, Nt.BstNBI, and Nt.Bpu101. The latter enzyme is available from ThermoFisher Scientific; other enzymes are available from eg New England Biolabs.

In a preferred embodiment, the nickase is introduced into the reaction mixture at the beginning of the method (eg, within 1 minute of contacting the sample with the primers and DNA polymerase). In some cases, however, it may be desirable to introduce the nicking enzyme into the reaction mixture after a longer delay (eg, lowering the temperature closer to the optimal temperature for the nicking enzyme).

The methods of the present invention involve the use of DNA polymerases. Preferably, the method of the present invention comprises the use of at least one thermophilic DNA polymerase (ie having an optimum temperature in excess of 60°C). Preferably, the DNA polymerase is a strand displacement polymerase. Preferably, the DNA polymerase has no exonuclease activity. Preferably, the DNA polymerase is a strand displacement polymerase without exonuclease activity and is also preferably thermophilic.

Examples of preferred DNA polymerases include Bst polymerase, Vent® DNA polymerase, ^90N polymerase, Manta 1.0 polymerase (Qiagen), ^BstX polymerase (Qiagen) and large fragment of Bsm DNA polymerase (ThermoFisher Scientific) .

In some embodiments, the methods of the invention desirably comprise a pre-amplification or enrichment step. This is the step in which the target sequence is contacted with the forward and reverse primers and DNA polymerase but not with the nickase. This step typically lasts about 2-5 minutes and results in an initial (linear) amplification of the target sequence of about 1,000-fold, which may be particularly useful when the target sequence is present in the sample at a low replica number.

In some embodiments, pre-amplification is performed at a temperature below 50°C using a mesophilic DNA polymerase such as Exo-Minus Klenow DNA polymerase or Exo-Minus psychrophilic DNA polymerase from Cenarchaeum symbiosum or An enrichment step and the mixture is then heated to over 50°C to denature or inactivate the thermolabile DNA polymerase, and then the thermophilic DNA polymerase is added for downstream amplification.

Typically, the methods of the invention comprise a detection step wherein one or more direct or indirect products of the amplification method are detected and optionally quantified, which is indicative of the presence and/or amount of the target in the sample. There are many known suitable detection and/or quantification techniques, including: gel electrophoresis, mass spectrometry, lateral flow capture, incorporation of labeled nucleotides, intercalating dyes, molecular beacons, and other probes, especially hybridization oligos. Nucleotides or other nucleic acid-containing molecules.

The products or products detected in the detection step may be referred to herein as "detection targets." The "target" with respect to the detection step is not necessarily the same as the "target" in the amplification method and in fact the two molecules will usually differ at least to some extent, although they may have some sequence in common (typically 10- 20 bases), wherein the detection target comprises a nucleic acid molecule or oligonucleotide.

Nucleic acid detection methods may employ the use of dyes that allow specific detection of double-stranded DNA. Intercalating dyes that exhibit enhanced fluorescence upon binding to DNA or RNA are well known. Dyes can be, for example, DNA or RNA intercalating fluorophores and can include among others the following: acridine orange, ethidium bromide, Pico Green, propidium iodide, SYBR I, SYBR II, SYBR Gold, TOTO-3 (thiazole orange di polymer) Oli Green and YOYO (oxazole yellow dimer).

Nucleic acid detection methods may also employ the use of labeled nucleotides that are incorporated directly into the detection target sequence or in probes containing sequences complementary or substantially complementary to the relevant detection target. Suitable labels can be radioactive and/or fluorescent and can be resolved in any manner known in the art. Labeled nucleotides that can be detected but otherwise function as natural nucleotides (eg, are recognized by natural enzymes and can serve as their substrates) are distinguished from modified nucleotides that do not function as natural nucleotides.

Molecular beacons can be used to detect and monitor the presence and/or amount of target nucleic acids and nucleic acid sequences. Molecular beacons are hairpin-shaped oligonucleotides containing a fluorophore at one end and a quencher dye (quencher) at the opposite end. The hairpin loop contains a probe sequence that is complementary or substantially complementary to the detection target sequence and the stem is formed by the gluing of self-complementary or substantially self-complementary sequences on either side of the probe sequence.

The fluorophore and quencher are bound at opposite ends of the beacon. The fluorophore and quencher interact with each other under conditions that prevent the molecular beacon from hybridizing to its target or when the molecular beacon is free in solution Proximity to prevent fluorescent light. Hybridization occurs when the molecular beacon encounters the detection target molecule; the loop structure switches to a stable, more rigid configuration, causing the fluorophore to separate from the quencher, allowing fluorescence (Tyagi et al. 1996, Nature Biotechnology 14:303-308). Because of the specificity of the probe, the generation of fluorescence is substantially unique due to the presence of the expected amplification product/detection target.

Molecular beacons are highly specific and can distinguish nucleic acid sequence differences (eg, single nucleotide polymorphisms) by a single base. Molecular beacons can be synthesized using different colored fluorophores and complementary sequences of different detection targets, enabling simultaneous detection and/or quantification of several different detection targets in the same reaction, allowing a single PoC assay to "multiplex" detection Measure multiple different pathogen or biochemical markers. For quantitative amplification methods, molecular beacons can specifically bind to the amplification detection target after amplification, and since unhybridized molecular beacons do not fluoresce, there is no need for quantitative determination of the amount of amplification product Probe-target hybrids are isolated. The signal generated is proportional to the amount of amplified product. This can be done in real time. As with other real-time formats, specific reaction conditions must be optimized for each primer/probe set to ensure accuracy and precision.

The production or presence of target nucleic acids and nucleic acid sequences can also be detected by fluorescence resonance energy transfer (FRET) detection and monitoring. FRET is an energy transfer mechanism between two fluorophores of donor and acceptor molecules. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. Subsequent emission from the donor molecule as it returns to its ground state can transfer excitation energy to the acceptor molecule (through long-range dipole-dipole interactions). FRET is a useful tool for quantifying molecular dynamics, eg, in DNA-DNA interactions, as seen under Molecular Beacons. To monitor the production of specific products, the probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Probe-detection target hybridization causes a change in the distance or orientation of the donor and acceptor and FRET is observed Changes in characteristics. (Joseph R. Lakowicz. "Principles of Fluorescent Spectroscopy", Plenum Publishing Corporation, 2nd edition (July 1, 1999)).

The production or presence of target nucleic acids can also be detected and monitored by lateral flow devices. Lateral flow devices are well known. These devices typically include a fluid-permeable solid-phase flow path through which the fluid flows by capillary forces. Examples include, but are not limited to, dipstick assays and thin layer chromatography plates with various suitable coatings. Various binding reagents for the sample, binding partners for the sample or conjugates involving the binding partners, and signal generating systems are immobilized in or on the flow path. The detection of analytes can be achieved by a number of different methods including: enzymatic detection, nanoparticle detection, colorimetric detection, and fluorescence detection. Enzymatic detection can involve enzyme-labeled probes that hybridize to complementary or substantially complementary nucleic acid detection targets on the surface of the lateral flow device. The resulting complex can be treated with a suitable label to generate a readable signal. Nanoparticle detection involves bead technology, which can use colloidal gold, latex, and paramagnetic nanoparticles. In one example, the beads can bind to anti-biotin antibodies. The target sequence can be directly biotinylated, or the target sequence can hybridize to a sequence-specific biotinylated probe. Gold and latex produce colorimetric signals that are visible to the naked eye and paramagnetic particles when excited in a magnetic field produce invisible signals that can be interpreted by a dedicated reader.

Fluorescence-based lateral flow detection methods are also known, such as dual-luciferin and biotin-labeled oligonucleotide probe methods or the use of quantum dots.

Nucleic acids can also be captured on lateral flow devices. Capture means can include antibody-dependent and antibody-independent methods. Antibody-independent capture typically uses a non-covalent interaction between two binding partners, such as a high affinity and irreversible bond between a biotin-labeled probe and a streptavidin capture molecule. Capture probes can be directly immobilized on lateral flow membranes.

The entire method of the invention, or at least the amplification method portion of the method, can be carried out in reaction vessels such as, for example, conventional laboratory plastic reagent tubes from ^Eppendorf® or can be carried out in and/or on solid supports. The solid support can be porous or non-porous. In a specific embodiment, the solid support may comprise a porous membrane material (eg, nitrocellulose or the like). More particularly, the solid support may comprise or form part of a porous lateral flow analytical device, as described above. Alternatively, the solid support may comprise or form part of a microfluidic-based assay in which one or more solid narrow-bore capillaries are used to transport liquid along the assay device.

In preferred embodiments, all or at least a portion of the methods of the present invention may be performed using a point-of-care (PoC) assay device. PoC devices are typically characterized as being inexpensive to manufacture, disposable after a single use, generally self-contained, do not require any other device or equipment to perform or interpret analysis and ideally require no clinical knowledge or training to use.

Examples of primers suitable for use in the present invention are disclosed herein. Other examples of methods applicable to the present invention are disclosed inter alia in US 2009/0017453 and EP 2,181,196, the contents of which are incorporated herein by reference. Those skilled in the art will readily be able to design other primers suitable for amplification of other target sequences without undue experimentation.

As explained elsewhere, primers used in the present invention will preferably include not only target complementary portions, but also nicking endonuclease binding and nicking sites, as well as stabilizing portions. Preferred primers may contain self-complementary sequences that can form a stem-loop structure in the primer molecule.

Primers used in the methods of the invention may comprise modified nucleotides (ie, nucleotides not found in naturally occurring nucleic acid molecules). Such modified nucleotides are preferably present in the target-complementary portion of the primer, and/or elsewhere in the primer. Preferred examples of modified nucleotides are 2'-modified nucleotides, especially 2'O-methyl modified nucleotides, although many other modified nucleotides are known to those skilled in the art.

temperature characteristic curve

Although not isothermal, the method of the present invention does not require thermal cycling. Therefore, the method of the present invention does not require the use of the relatively complex thermal cycling apparatus used in PCR, and thus makes it easier to apply itself in a PoC environment.

"Thermal cycling" or temperature cycling means: in particular, the temperature of the reaction mixture is maintained at a specified temperature (such as t ₁ ) for a specified period of time (typically at least 30 seconds). The temperature is then adjusted (up or down) before returning to the previously maintained temperature.

Typically, non-isothermal nucleic acid amplification reactions such as PCR require performing multiple thermal steps/cycles (ie, at least two or more than two) and multiple thermal cycles/reactions.

The temperature reduction in the method of the invention is intentional and controlled in the sense that the magnitude of the temperature reduction is above a predetermined minimum level and below a predetermined maximum level. Furthermore, the temperature reduction rate is preferably within a predetermined range.

The initial step (a) in the methods of the invention involves contacting the target sequence with a primer having at least a portion complementary to the target sequence under conditions that allow the primer to hybridize at least temporarily to the target. This can be described as the "start" phase.

This step is typically carried out at a temperature in the range of 50°C to 65°C, preferably in the range of 52°C to 62°C, more preferably in the range of 54°C to 62°C, most preferably in the range of 58°C to 62°C. The reaction mixture comprising target and primer can be maintained at this temperature for a suitable period of time. The optimal temperature and the optimal period for which the reaction mixture is maintained at this temperature can be determined by those skilled in the art with the benefit of the present invention, and these conditions can be influenced by parameters such as the length of the target sequence, the size of the primers, Length and especially the length of the primer portion complementary to the target, G:C content of the target:primer hybrid, pH and salt concentration of the reaction mixture. Typical initial temperature hold time can be within 5 seconds In the range of 5 minutes, preferably in the range of 10 seconds to 3 minutes. Typical conditions that permit the initial hybridization event in step (a) will be known to those skilled in the art and are described in the accompanying examples.

A temperature range of 58°C to 62°C is preferred for the initial stage. This temperature range is believed to be high enough to minimize primer dimer formation (and thus reduce the amount of non-specific amplification), and to increase the chance of creating a potential "start site" in the target duplex, while being low enough to allow at least some of the primer molecules to hybridize to the target.

The subsequent reduction in temperature helps stabilize the hybridization of the relatively short primer to the extension product, rather than the hybridization of the primer to the original target molecule. It is further assumed that additional cooling of the reaction mixture promotes hybridization of the detection probe to the detection target.

During the amplification method set forth in steps (b) to (d), the temperature of the reaction mixture is lowered. This can be done in a regulated manner, for example using temperature regulating means to lower the temperature of the reaction mixture according to a predetermined temperature profile. The temperature reduction can begin immediately after the initial phase (step a). The volume of the reaction mixture is preferably small so that the heat capacity of the reaction mixture (and the reaction vessel or substrate in or on it) is reduced.

The volume of the reaction mixture is preferably less than 100 μl, preferably less than 50 μl, more preferably less than 25 μl and most preferably less than 20 μl. In this way, the temperature of the reaction mixture can be adjusted more accurately and more rapidly by means of the temperature adjustment means. In suitable embodiments, the temperature regulating means may be extremely simple (eg, a fan), or may be omitted entirely, wherein sufficient cooling is substantially or completely achieved by passive components (eg, by thermal radiation from the reaction mixture). Typically, the reaction mixture volume may range from 1 μl to 50 μl, preferably 1 μl to 20 μl and more preferably 1 μl to 10 μl.

The volume of the reaction mixture can be less than 10 μl. In particular, the method of the present invention can employ a "digital PCR" type method (see also published by Morley 2014 Biomolecular Detection and Quantification 1, 1-2), where the sample is diluted and divided into many (usually hundreds, thousands or even millions) of simultaneously processed aliquots: some aliquots will contain the target sequence and Some will not contain: if the target sequence is not present, no signal will be generated. The ratio of negative aliquots can be used to infer the number and/or concentration of target sequences in the original sample. In such embodiments, the volume of reaction mixture in each aliquot can be extremely small, however, typically the minimum volume will be 2500 nl, preferably at least 50 μl.

The temperature can be reduced with any desired characteristic curve. For example, the temperature may be decreased according to a substantially linear characteristic curve (eg, having a substantially constant rate of temperature decrease), or may be decreased in any non-linear manner, including a curvilinear or stepwise manner, or a linear and non-linear characteristic curve Any combination (eg, one or more periods of constant rate temperature reduction, which rate may be zero or relatively low, alternating with periods of relatively higher temperature reduction rates).

The temperature profile of the reaction according to the invention is such that the reaction temperature cannot be returned to the temperature at which the "starting" phase is carried out. Thus, in the method of the present invention, there is no oscillation of the predetermined temperature and no "return" to the predetermined temperature, contrary to eg the disclosure of WO 2011/030145.

The temperature of the reaction mixture at the start of the amplification process will typically be the same as in step (a), eg preferably in the range of 54°C to 62°C and optimally in the range of 58°C to 62°C. During the amplification process, the temperature is lowered by at least 2°C, preferably at least 3°C, 4°C or 5°C, more preferably at least 8°C, 9°C or 10°C, and most preferably at least 13°C, 14°C or 15°C, but It will be appreciated that, in absolute terms, the preferred amount of temperature drop may depend, at least in part, on the initial temperature chosen at the start of the amplification method, with lower initial temperatures (eg, in the range of 45°C to 55°C). ) can predict lower temperature drop and/or lower temperature drop rate. Typically, the maximum amount of temperature reduction during the amplification method is about 20°C, although it is understood that the maximum temperature reduction may be below this temperature (eg, 16°C, 17°C, 18°C or 19°C) or higher (eg 25°C or 30°C).

In preferred embodiments, the amount of temperature reduction of the reaction mixture during the amplification process may be in the range of 5°C to 40°C, preferably in the range of 8°C to 35°C, more preferably in the range of 8°C to 30°C or Even in the range of 8°C to 25°C, and optimally in the range of 8°C to 20°C. A typical initial temperature of the reaction mixture at the start of the amplification process is in the range of 50°C to 62°C, preferably in the range of 54°C to 62°C, more preferably in the range of 56°C to 60°C, and most preferably in the range of 58°C to 60°C Inside.

In a preferred embodiment, the temperature reduction during the amplification method (step (b) to step (d) of the method of the invention) comprises encompassing 54°C to 50°C, 56°C to 50°C or 58°C to 50°C, More preferably a reduction in the range of 58°C to 45°C, 58°C to 40°C or even 60°C to 40°C. It will be appreciated that the temperature reduction during the amplification method may exceed the stated ranges as defined above. That is, the maximum temperature may exceed the upper temperature of the stated range and/or the minimum temperature may be lower than the lower temperature of the stated range.

The endpoint temperature of the amplification reaction is preferably chosen to be compatible with the chosen detection method. For example, if the detection method involves the use of an enzyme label, the end temperature of the amplification reaction needs to be arranged to be compatible with the enzyme and, for example, within ±5°C of the optimal temperature of the enzyme. Alternatively, where the detection method involves the use of hybridized detection probes, such as molecular beacons or the like, it is desirable to arrange for the end point temperature of the amplification reaction to be selected to be consistent with the detection probe/detection target duplex. compatible with Tm.

For example, the endpoint temperature is preferably lower than the Tm of the detection probe/detection target duplex, preferably at least 2°C lower to facilitate hybridization of the probe to the detection target.

Typical average rates of temperature reduction during the amplification process are in the range of -0.10°C min ^-1 to -6.0°C min ^-1 , preferably -0.20°C min ^-1 to -3.5°C min ^-1 , more preferably in the range of -0.10°C min -1 to -6.0°C min -1 -0.30°C min ^-1 to -3.5°C min ^-1 range, and optimally -0.40°C min ^-1 to -3.5°C min ^-1 range. As evident from the foregoing, at any point during the amplification process, the actual rate of temperature reduction may deviate from the preferred average rate, depending on the nature of the temperature reduction gradient.

In some embodiments, the temperature reduction gradient lasts at least 3 minutes, more preferably at least 4 minutes and is optimally substantially linear over most of the duration of the amplification method. Typically, the temperature reduction gradient is substantially linear over a period in the range of 3 minutes to 12 minutes, preferably in the range of 4 minutes to 10 minutes, more preferably in the range of 4 minutes to 8 minutes. For the purposes of the present invention, "substantially linear" means that for any second order polynomial describing the temperature gradient, the magnitude of the coefficient of X is 5% smaller than the magnitude of the coefficient of Y.

A number of different techniques can be envisaged for achieving the desired temperature reduction during the amplification process. Such techniques may include one or both of the following: (a) ceasing the application of heat to the reaction mixture and/or removing the reaction mixture from a heated and/or adiabatic environment and allowing the reaction mixture to substantially cool by passive heat loss to Ambient environment; (b) applying active cooling to the reaction mixture. Active cooling may involve exposing the reaction mixture to a cooling environment, such as placing the reaction mixture in heat transfer (conduction, radiation or convection) contact with a cooling medium, especially a fluid. This can include, for example: contacting the reaction mixture with a chilled water bath; using a fan to blow cold air or other gas through or through the reaction mixture; contacting the reaction mixture with a reaction mixture compatible coolant, which can be a gas, liquid, or solid; or use a Peltier-type cooling device. For example, the reaction mixture compatible coolant can be a reaction mixture compatible buffer in the form of a frozen or refrigerated liquid. The addition of cold buffer will tend to dilute the reaction mixture, so if this method is adopted, it is preferable to use a smaller volume (eg, below the temperature of the reaction mixture by more than 20° C.) 1 μl to 2 μl) of coolant.

Any active cooling step can be applied discontinuously during the amplification method to achieve The desired temperature reduction level and/or the desired temperature reduction characteristic curve is now available. In particular, active cooling can be performed at two or more than two interspersed time intervals during the amplification process, and optionally combined with passive cooling simultaneously or alternately.

In general, preferably, the desired amount and rate of temperature reduction can be achieved substantially entirely by passive components because of this simplification of the method and any equipment or kit required to carry out the method. To achieve the desired amount and rate of cooling with substantially completely passive components, the volume of the reaction mixture needs to be small to reduce its heat capacity, as previously mentioned.

A preferred feature of the method of the present invention is that the amplification method can utilize a first polymerase having an optimum temperature and a second polymerase having an optimum temperature, wherein the optimum temperature of the second polymerase is lower than that of the first polymerase the optimum temperature. Thus, since the temperature of the reaction mixture can be at or near the optimal temperature of the first polymerase, the first polymerase can be particularly active near the start of the amplification process.

Thus, for example, the first polymerase may advantageously be a "thermophilic" enzyme (ie, having an optimum temperature in excess of 60°C).

Conversely, the second polymerase has a lower optimal temperature than the first polymerase. As the amplification process continues, the temperature of the reaction mixture decreases and gets closer to the optimum temperature for the second polymerase. Consequently, the second polymerase becomes increasingly active, which at least partially compensates for the decreased reaction rate because (i) the reaction is generally thermodynamically slowed due to lower temperatures and (ii) the temperature of the reaction mixture may drop to Below the optimum temperature for the first polymerase, resulting in reduced catalysis.

Advantageously, the optimum temperature of the second polymerase is in the range of 30°C to 55°C, more preferably in the range of 30°C to 45°C.

In a specific embodiment, the second polymerase can be DNA polymerase I or Bsu Klenow fragment of polymerase.

It is envisaged that a third or other polymerase may even be used, preferably with another lower third optimum temperature.

The second polymerase is preferably in the reaction mixture from the start of the amplification process, but if desired, the second polymerase can be added after a delay such that the temperature of the reaction mixture is lowered from a higher initial temperature. This may be advantageous when, for example, the second polymerase is not particularly thermolabile and may be substantially denatured if present at the relatively high temperatures typically employed at the start of the amplification process.

In a manner completely similar to the previous one, the amplification method can utilize a first nickase and a second nickase with higher and lower optimum temperatures, respectively. The first nickase and the second nickase can be used in combination with a single polymerase or multiple polymerases as the case may be.

As above, the use of a second nickase with a lower optimal temperature can at least partially offset the expected reduction in reaction rate that accompanies the reduction in temperature during the amplification process.

Thus, in some embodiments, the temperature of the reaction mixture may start at a temperature below the optimal temperature of the first polymerase and/or the first nickase or may decrease to below the first temperature during the course of the amplification method The temperature of the optimum temperature for the polymerase and/or the first nicking enzyme will tend to approach and may even reach the optimum temperature for the second polymerase and/or the second nicking enzyme.

In some embodiments, the methods of the invention desirably comprise the step of contacting the reaction mixture with a degrading enzyme that degrades nucleic acids. Desirably, this step is performed after the user obtains the desired results of the amplification reaction (eg, detection of pathogens). Typically, the degradative enzyme is thus added to the reaction mixture after the amplification process has reached the desired endpoint. Preferably, the degrading enzyme is thermolabile, such that if it is unintentionally introduced into or contacted with the reaction mixture before the amplification method reaches the desired endpoint, the temperature is sufficient to substantially denature or otherwise render the enzyme. It is not activated. Suitable examples include cod uracil-DNA glycosidase ("UDG") available from ^{ArcticZymes®} and Antarctic thermolabile UDG (available from New England BioLabs). These enzymes are rapidly and irreversibly not activated upon exposure to temperatures of 55°C or 50°C, respectively, and the term "thermolabile" in relation to degrading enzymes should therefore be interpreted. Alternatively, thermosensitive degrading enzymes (ie degrading enzymes that are at least partially active below 50°C, but reversibly, substantially inactive above 55°C) can be used.

The invention will now be further described with the aid of illustrative examples and with reference to the accompanying drawings, wherein:

1A-1C are schematic diagrams of typical embodiments of primers that can be used to carry out the methods of the present invention.

Figures 2A and 2B are schematic diagrams of the initial stage and the exponential amplification stage, respectively, of a nucleic acid amplification reaction suitable for carrying out the method of the present invention; Figures 3 to 11 are (subtracted background) fluorescence (in arbitrary units) and Curves of temperature (°C) versus time (minutes); Figures 12A to 12C are curves of (subtracted background) fluorescence (arbitrary units) versus time of individual replication reactions of the amplification reaction; Figure 13 is a comparison in the present invention Scatter plot of the time taken to achieve amplification from 10 replicates of the template sequence under "STAR" conditions or under isothermal conditions; Figures 14A and 14B are using isothermal amplification conditions (63°C or 49°C, respectively). ) (background-subtracted) fluorescence (arbitrary units) and temperature (°C) versus time.

Figures 15A, 15B, and 15C are (background-subtracted) fluorescence of individual replicate reactions (10 replicates or 100 replicates; dashed and solid lines, respectively) of amplification reactions of the invention under various temperature profiles (arbitrary units) and temperature (°C) versus time (minutes); Figures 16A and 16B are (arbitrary units) fluorescence (arbitrary units) of amplification reactions according to the present invention under various temperature profiles and Curves of temperature versus time (min); Figures 17A and 17B are ( Figure 17B ) of amplification reactions using primers containing 6 ( Figure 17A ) or 7 ( Figure 17B ) O-methylated bases according to the present invention (subtracted background) fluorescence ( arbitrary units) and temperature (°C) versus time (min); Fluorescence (arbitrary units) versus time (minutes) for the amplification reaction (subtracted background) of the transcriptionally generated DNA target.

Example Example 1: Protocol for testing temperature reduction

The effect of temperature reduction on amplification reactions was tested by comparing amplification using temperature reduction over time with standard isothermal conditions. Amplification at reduced temperature is referred to herein as "STAR" (selective temperature amplification reaction). Unless indicated, these comparisons were performed using the protocol described below.

Enzymes, oligonucleotides and targets

Chlamydia trachomatis (Ct) was used as an initial target for developing the STAR mechanism. Chlamydia trachomatis serovar J (ATCC VR-886) genomic DNA was obtained from the American Type Culture Collection (Manassas, VA). The open reading frame 6 region of the cryptic plastid was amplified with primers STARctF61a (SEQ ID NO: 1, 5'-CGACTCCATATGGAGTCGATTTCCCCGAATTA-3') and STARctR61c (SEQ ID NO: 2, 5'-GGACTCCACACGGAGTCTTTTTCCTTGTTTAC-3'). The resulting DNA template was detected using the molecular beacon STARctMB1 (SEQ ID NO: 3, 5'-FAM/ccattCCTTGTTTACTCGTATTTTTAGGaatgg/BHQ1-3') as described in EP No. 0728218. Manta 1.0 DNA polymerase was purchased from Enzymatics (Beverly, MA). Nt.BstNBI nicking endonuclease was purchased from New England BioLabs (Ipswich, MA) as described in US Pat. No. 6,191,267.

Oligonucleotides and molecular beacons were synthesized by Integrated DNA Technologies (Coralville, IA) and Bio-Synthesis (Lewisville, TX). The general characteristics of primers for STAR reactions are as follows:

Primer sets were constructed with the stabilization region 5' of the nick site and the target-specific binding region 3' of the nick site (Fig. 1A). Primers are constructed in such a way that stem and loop structures can be formed at the 5' end of the oligonucleotide by creating a self-complementary structure that forms at least part of the stem. The _Tm of this construct is chosen to direct linear or exponential amplification depending on the reaction temperature at a given time. The stem further encompasses at least a portion of the nickase recognition sequence. The nickase recognition sequence in the primer is part of the double-stranded stem structure, but at least one nucleotide is single-stranded to avoid nicking. Optionally, the sequence complementary to the target sequence may or may not contain secondary structure. In addition, this sequence may contain modified nucleotides, such as 2' modifications or phosphorothioate linkages.

Referring to Figure 1A, a "primer region" is a sequence that is complementary to and bonded to the target sequence. The "NEB region" is the nicking endonuclease binding region, ie the nickase recognition region of the nicking primer in this case at a site four nucleotides downstream of the end of the NEB region. The "amplification loop" provides primer stabilization and hybridization for the amplification method and surrounds itself by means of self-complementation during the initial phase to reduce background non-specific amplification. 1B and 1C show a brief overview of primers that can be used in the methods of the present invention slightly different specific examples. The primer structure is basically the same as the specific example shown in Figure 1A, but the changed primer includes modified bases in the "primer region". Specifically, at the 3' end of the primer region, there is a continuous chain of 2' O-methylated bases. In Figure IB, this strand is 7 bases long and in Figure 1C, the strand is 6 bases long. Primers of the class shown in Figures IB and 1C were used in Example 8 below.

An overview of the oligonucleotides and amplification mechanisms found in the reaction include: (1) a target nucleic acid molecule; (2) two or more primers comprising some number of oligonucleotides complementary to the target nucleic acid molecule an oligonucleotide molecule; and (3) a site within the primer that can be cut by a nickase. The method involves contacting a target nucleic acid molecule with a polymerase, two or more primer oligonucleotides, and a nickase, each primer oligonucleotide specifically binding to a complementary sequence on the target nucleotide molecule; and in A detectable amplicon comprising at least a portion of the primer oligonucleotide that binds the target sequence is generated under non-isothermal conditions. The entire STAR reaction can be understood as going through two distinct phases: initiation and exponential amplification. The initial stage is the initial formation of an exponential-template duplex in which exponential expansion can occur. These two stages are schematically illustrated in Figures 2A and 2B. In those figures, the triangular symbols represent nickases and the hexagonal symbols represent DNA polymerases. The initial primer is contacted with the target nucleic acid, followed by extension and generation toward the starting template. The opposite strand primer then binds to the newly generated forward starting template, extending in the direction towards and through the nicking site of the starting template. This initial process can be understood as involving the polymerase used for extension and is highly prone to the formation of primer dimers and false amplification of truncated or background products. Once nicking begins on either strand, the polymerase will infiltrate the nick site and extend towards the opposite primer and through the nick site.

Once this nicking occurs on both the forward and reverse starting strands, followed by a cycle of polymerase extension, a duplex called an exponential duplex is formed. The second phase of the reaction begins; since each new template created from nicking and extension is now the target of another primer, This exponential amplification process self-supplies.

It is now understood that the second stage requires an active nicking endonuclease for rapid template generation. The requirement for temperature cycling for this nicked strand displacement replication block is previously known, so the reaction can and always proceed at a constant temperature. The present novel discovery enables unique and distinct amplification methods with significantly higher performance than existing methods, including the production of products with high specificity in high yields in a short period of time.

Amplification conditions

The Basic Selective Temperature Amplification (STAR) mix contains two primers, a polymerase and a nickase (reference above). Reactions were performed in a final volume of 20 μl , including 0.41 μM forward primer, 0.2 μM reverse primer, 0.18 μM molecular beacon, 10 μl STAR master mix, and 5 μl DNA sample. The STAR master mix contains the following reagents; 15 mM MgSO ₄ , 90 mM Tris-HCl (pH 8.5), 300 μM each dNTP, 15 mM (NH ₄ ) ₂ SO ₄ , 15 mM Na ₂ SO ₄ , 1 mM DTT, 0.01% Triton X-100, 7U Nicking endonuclease, 48U polymerase. The reaction temperature is isothermal or varies based on the amount of temperature reduction. If the temperature during each reaction starts at 60°C and is decreased by a specified amount every 15 seconds or 1 minute, for example a negative 0.5°C rate (ie, a temperature decrease of 0.5°C every 15 seconds) for 10 minutes will cause the temperature to run through the reaction The process was reduced from 60°C to 40°C. Amplification and STAR product detection were performed using an Agilent Mx3005 P QPCR device (Agilent). Unless otherwise stated, the following table lists the temperature characteristic curves tested:

Pre-reaction incubations will allow reagents to reach temperature to test the effect of lowering temperature on amplification kinetics, enzyme efficiency, and signal fluorescence. Conducting the reaction in this manner removes incremental temperature variables and allows for direct comparisons between existing isothermal amplification techniques and the novel STAR method.

Amplification step

The exact steps to perform an amplification reaction are as follows: 1) prepare a master mix; 2) prepare primers with or without target; 3) add primer mix to 96 wells depending on the number of reactions to be performed per plate Row A to Row G of plates; 4) Add master mix to Row H of the same 96-well plate; 5) Seal plate and incubate pre-reaction for 2 min; 6) Transfer master mix from Row H to each primer mix row, waiting 15 seconds between transfers; 7) Seal and initiate pre-selected temperature profile and data collection.

During the course of the reaction, amplification products were measured every 15 seconds by using molecular beacons as described above. The fluorescence of the molecular beacon in the reaction mixture is monitored to measure the amount of specific product produced during the reaction. The specific product produced during the reaction binds to the molecular beacon, separating the fluorophore from the quencher, resulting in fluorescence. Fluorescence measurements were subtracted from background, which was based on the average of 3 previous readings in each well before amplification began. Further characterization was performed based on the rise from the baseline threshold level (TL). Choose a TL that is close to the baseline of background-subtracted fluorescence but larger than the range of random fluctuations. Decreasing temperature results in a decrease in molecular beacon baseline fluorescence due to increased stem intensity, resulting in a constant linear baseline decrease with stronger interaction of quencher and fluorophore. A TL of 2000 was chosen for all reactions. For comparison, the exact number was determined based on the time of amplification to TL, referred to as the _AT value. Using the _AT value allows the disks to be compared to each other.

Example 2: Results using unmodified primers

To demonstrate the improvement provided by STAR over current isothermal techniques, amplification was performed using 18 replicates corresponding to the target and 6 replicates corresponding to no target. The STAR reaction demonstrated significant improvements in speed, sensitivity, and total fluorescence compared to isothermal conditions. In particular, the range of -0.8°C min to -3.2°C min was significantly better than all isothermal conditions (Figures 3 to 11). Surprisingly and unexpectedly, such a significant drop in temperature still yields excellent results. Without limiting applicants to any particular theory, it is believed that the amplification improvement can be attributed to at least three characteristics, discussed further below.

The results of experiments using unmodified primers are shown in Figures 3-11. In these figures, the temperature profile is indicated by background shading. The amount of signal (fluorescence) for the "no target" negative control is indicated by the dark curve. The amount of signal generated in the presence of 10 or 100 copies of the target (the genomic DNA of C. trachomatis) is indicated by the lighter curve.

Figures 3, 4 and 5 show the results of isothermal amplification (ie not according to the method of the invention) at 50°C, 56°C and 60°C, respectively. As can be seen in Figure 3, there is essentially no specific amplification at 50°C, there is stronger amplification at 56°C for at least 100 target replicate numbers (Figure 4), and at 60°C Lower amplification (Figure 5).

Figures 6-10 show the results for the non-isothermal (STAR) amplification reaction of the present invention. Results were obtained in which the temperature was decreased during the amplification. The rate of temperature reduction is linear, ranging from -0.1°C/15s (ie -0.4°C/min) in Figure 6 to -1.0°C/15s (ie -4.0°C/min) in Figure 10 . It can be seen that in all cases, the reaction with 100 replicates of the target produced more fluorescent signal than the reaction with 10 replicates of the target, as expected. More remarkably, the reaction produced much more signal than equivalent isothermal amplification, especially for the 10 target replicate number reaction. Furthermore, the generation of a detectable signal is faster than in an equivalent isothermal reaction.

Similar results were obtained when the method of the present invention was performed using a non-linear, stepwise temperature reduction (as shown in Figure 11).

The inventors have also found (for brevity, the information is omitted) that the signal amount change between different replicates in the STAR reaction is much lower than that in the isothermal reaction, demonstrating that the method of the present invention produces more consistent results. The following comments provide a possible mechanism by which the methods of the present invention confer the above-mentioned advantages.

In most nucleic acid amplification reactions, primer-dimers can eventually form, competing for limited reagents and at low target concentrations, primer-dimers can become the primary amplification pathway of the reaction. Limiting or delaying the formation of primer dimers, even in small amounts, provides significant benefits to the reaction. Due to the rapidity of the amplification reaction, delaying primer-dimer formation allows a better amplification pathway (ie, template generation) to be favored, improving all aspects of amplification. By initiating the reaction at high temperature, these templated approaches become advantageous and even preferred. This is reflected in the increased sensitivity, increased fluorescence signal, tighter clustering of replicas (ie, higher reproducibility) and increased speed in the STAR method.

After the initial phase of the reaction, the exponential phase begins. Because the templated pathway favors the error pathway, as many products as possible need to be produced as quickly as possible, and STAR facilitates this. One of the most likely limiting steps for this generation is the nicking of the site by a nicking endonuclease. follow As the temperature of the reaction mixture decreases, it approaches the optimum temperature for the nicking endonuclease, and the efficiency of the reaction increases, producing as much template as possible for detection.

Further molecular beacons facilitate template detection and reduce fluorescent background as temperature decreases. The melting temperature of the template to the molecular beacon becomes significantly higher than the detection temperature, resulting in an improved signal as less template melts from the molecular beacon. Furthermore, as the temperature decreases, the stem melting temperature becomes higher than the reaction temperature. Molecular beacons thus favor the closing phase when no template is present, resulting in less background signal.

The novel non-isothermal reaction method of the present invention is significantly improved over existing isothermal and thermal cycling conditions. By promoting enzymatic activity and optimal reaction kinetics, the method improves changes in _AT , increases the total amount of fluorescence produced, improves amplification consistency, and increases detection sensitivity.

Example 3: Results using SYBR Green II

Because molecular beacons only measure an increase in the total amount of specific single-stranded DNA products, non-specific amplification products that are not related to the expected amplification products are not measured. To measure the production of non-specific amplification products (eg, by primer dimer formation), separate reactions are performed in the presence of SYBR Green II. SYBR Green II is one of the most sensitive dyes known to detect single-stranded DNA, RNA and double-stranded DNA. Because SYBR Green II has low endogenous fluorescence, it is a natural choice for full amplification in detection reactions or non-specific amplification if amplification is performed in the absence of target. Reactions were performed directly under two conditions - isothermal and non-isothermal (STAR), as shown in Table 2 below.

In addition, the reaction compares 50 replicates of genomic DNA to no target. A 10,000x concentration of SYBR Green II was obtained using 0.5x (Life Technologies, Carlsbad) per reaction. The higher TL 9000 was used to calculate _AT due to the inherent nature of intercalating dyes. The fluorescence of SYBR Green II is inversely related to temperature. The lower the temperature, the higher the fluorescent signal, such as "Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature" (Gudnason et al., 2007 Nucl. Acids Res. 35 ( 19) e 127). The results are shown in Table 3 below.

The STAR method presents several improvements: First, the method reduces background generation, as evident from the fact that "no target" takes longer to display SYBR Green II amplification relative to the target signal. Second, the method improves product amplification, reflecting faster amplification times in the presence of the target. Altogether, these improvements make the difference between _AT relative to the isothermal method more than four-fold.

It should be noted that _AT values from isothermal reactions have more variation than those from STAR _. This demonstrates the benefit that the new method has in controlling the amplification method and reflects the unpredictability of non-specific amplification pathways using traditional methods.

Example 4: Results using primers modified with 2'O -methyl groups

The use of 2'O-methyl modified primers is known to reduce primer dimer formation during amplification, as described in US Pat. Nos. 6,794,142 and 6,130,038. US 2005-0059003 describes the use of a 2' O-methyl modification located 3' to the SDA primer, so Bst DNA polymerase I and derivatives can effectively use 2' modified ribonucleotides as primers for DNA synthesis. Contains one or more 2' modified nucleotides (eg 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[ 2(methylamino)-2-oxyethyl], 2'-hydroxy(RNA), 4'-thio, 4'-CH3-O-2'-bridge, 4'-(CH3)3- The target-specific primer regions of the O-2'-bridge, 2'-LN A and 2'-O-(N-methyl carbamate 2'-Suc-OH)) should enhance the isothermal reaction. It would be surprising that the STAR method could further improve amplification if the 2' modified nucleotides completely eliminated primer dimer formation. The reaction was performed directly between two conditions - isothermal and non-isothermal (STAR), as shown below.

The results of amplification using 2' modified nucleotides on the 3' end of the primers are shown in Table 5 below. Reactions were performed with a minimum of 6 replicates in no-target reactions and with a minimum of 12 replicates in target reactions.

The data indicate that the use of at least one primer incorporating a 2'O-methyl nucleotide delays the formation of primer dimers, improving the reaction although slowing it down. Furthermore, using the STAR method not only improved the use of 2'O-methyl amplification, restoring some of the lost speed, but also improved the difference between target and no-target amplification by a factor of three. This indicates that although 2'O-methyl modifications did reduce the generation of nonspecific mis-amplifications, these modifications did not eliminate it. The data further demonstrate that the STAR method utilizes the improvements resulting from the 2'O-methyl modification better than previously disclosed prior art.

Without being bound by any particular theory, the present invention is hypothesized that the potential improvement obtained by using one or more 2' modified nucleotides in the primer region is primarily due to the enhancement of the initial phase of amplification.

During the initial extension of the primer region on the target, the incorporation of one or more 2' modified nucleotides in the primer region of the STAR renders these nucleotides unsuitable to act as nonspecific complexes formed by the interaction of the primers template for polymerase extension, thereby reducing background signal. The polymerase most likely stops when the nucleotide enters the binding pocket. In non-productive reactions (ie, off-target or primer dimer formation), the stopping effect is sufficient to minimize aberrant extension as template binding approaches its melting temperature. Therefore, 2' modifications can limit undesirable amplification pathways because the reaction is already bogged down. However, during favorable amplification, the 2' modification lowers the melting temperature, thus adversely affecting amplification, thereby slowing down the amplification time. STAR can minimize the disadvantage of negative target amplification at the same time When using 2' modification.

This polymerase stop further explains why STAR binds 2'O-methyl modifications to improve each other. In addition to naturally reducing primer dimers, the initial increase in temperature found in the STAR method exacerbates 2' modification stop and primer melting before erroneous amplification can occur, thus the two methods are complementary to each other. Furthermore, because STAR involves lowering the temperature, the reduction in melting temperature caused by the 2' modification in the primer can be minimized as the reaction proceeds.

Example 5: Results using multiple polymerases

Existing amplification techniques are thermally cycled or performed at constant temperature. The method of the present invention is neither, but is performed by reducing the temperature in the absence of cycling. A particular novel feature of the present invention is the ability to use enzymes of similar function but with different temperature optima. For example, this technique would allow the use of multiple primers designed for nicking endonucleases that function at different temperature optima, and different strand displacement polymerases with different optima. Without being bound by any particular theory, this method exploits rapid amplification methods allowing new combinations of enzymes and primers not found in the prior art. The following reactions (Table 6) were performed directly between three conditions - isothermal, non-isothermal (STAR) and non-isothermal (STAR) with BSU polymerase (in addition to the original Manta 1.0 polymerase), as shown below. BSU polymerase was purchased from New England BioLabs (Ipswich, MA) and performed at 0.5 U/reaction. All conditions were performed using 18 on-target replicates and 6 off-target replicates.

Amplification reactions were performed using samples containing 10 replicates of C. trachomatis genomic DNA and the results are shown in Figures 12A-12C.

Figure 12A shows the results of an isothermal reaction (not according to the invention). Figure 12B shows the results of the STAR reaction in the presence of Manta polymerase alone, and Figure 12C shows the results of the STAR reaction in the presence of other BSU polymerases.

The first significant difference was that 10 copies of genomic DNA were not detected by the isothermal method, and only 9 of the 18 copies were beyond the threshold of fluorescence and could be called amplified. Both STAR methods detected 17 out of 18 replicates. (It should be noted that missing replicates in each STAR method were due to faulty multi-shot pipettes).

Although the difference between the STAR methods was not significant, the addition of a second polymerase with a lower optimum temperature of 37°C increased total fluorescence after 10 minutes. In addition, the second polymerase also tightens the replica, thereby reducing _AT changes. This difference will be further demonstrated if commercial strand displacement polymerases with an optimum temperature of 45°C to 50°C are obtained from the market. The results demonstrate that the STAR method is superior to isothermal conditions and further demonstrate that this technology allows for novel novel mechanisms, enzyme combinations and primer amplification protocols.

Example 6: Reproducibility

To verify the consistency of the STAR technology, a large replicate study was performed comparing STAR to the published isothermal conditions as described in US Patent No. 9,562,263. STAR and isothermal amplification were performed using 100+ replicates for reactions containing target and 16 replicates for control reaction mixtures without target. Both conditions used the same buffer, polymerase, nickase and target. As shown in the scatter plot in Figure 13, the STAR technique demonstrated significantly improved mean time ( _AT ) to achieve amplification to the threshold level (TL) of fluorescence, improved sensitivity, and reduced standard deviation between replicates . The _AT time for the reaction according to the present invention was 3.35 minutes, while the _AT value for the reaction according to the conventional isothermal protocol was 4.88 minutes, the difference being statistically significant as judged by a two-tailed t-test of. Without limiting applicants to any particular theory, it is believed that the significant reduction in amplification time is due to improved initiation of the reaction, allowing for more efficient low-duplication amplification, minimized primer dimerization events, and increased specific product Extension produces templates faster than previously disclosed methods.

Example 7: Amplification reaction beyond conventional isothermal temperature range

A further benefit of the STAR technology is the ability to perform amplification outside the most common isothermal amplification temperature ranges. As described in US Pat. Nos. 5,712,124, 9,562,263, and 5,399,391, most isothermal amplification techniques have a precise temperature range in which amplification can occur. Outside these typical temperature ranges, conventional isothermal techniques are difficult to scale up. To demonstrate the versatility of STAR, amplification was performed as described in Table 7 below.

The isothermal reaction is carried out as described in US Patent No. 9,562,263. Figures 14A and 14B are graphs showing the amount of fluorescent signal (minus background; arbitrary units) versus time (minutes) for isothermal amplification reactions performed at 63°C (Figure 14A) or 49°C (Figure 14B). picture. In both figures, the dotted curve represents the results obtained from the negative control reaction without template; the solid curve is the result from the test reaction containing template.

As apparent from Figure 14A, substantially no template-specific amplification occurred when the reaction temperature was kept at 63°C. In Figure 14B, the results appear to indicate that amplification occurs from about 9 minutes at 49[deg.]C, but in fact this may be a false signal generated by the interaction of the molecular beacon with the primer (data not shown).

In contrast to isothermal reactions, "STAR" reactions performed according to the present invention can be initiated at high temperatures and still achieve good amplification. The results from these reactions are shown in Figures 15A, 15B and 15C. These are plots of fluorescence (subtracted background, arbitrary units) versus time (minutes). Solid shading indicates the temperature (°C) during the reaction. Dotted curves represent results obtained using 10 target replicates, solid curves represent results obtained using 100 target replicates. In Figure 15A, the initial temperature is 62°C, and the temperature decrease rate is -0.8°C/15 seconds (ie -3.2°C/min). In Figure 15B, the initial temperature was 63°C, and the temperature decrease rate was -0.8°C/15 seconds. In Figure 15C, the initial temperature was 64°C, and the temperature decrease rate was -0.9°C/15 seconds (ie -3.6°C/min). It is evident from the figure that an initial temperature of 62°C or even 63°C provides good results for the STAR reaction, and there is some amplification even with an initial temperature of 64°C, although this is clearly suboptimal.

In addition, experiments were performed using large temperature drops. The results are shown in Figures 16A and 16B. The figures show the results of a no-target negative control (no fluorescent signal above a threshold level) and STAR reactions performed in the presence of 10 or 100 copies of the target C. trachomatis genomic DNA.

Figure 16A shows the use of an initial temperature of 63°C, followed by a temperature reduction rate of -0.8°C/15 seconds for 1 minute, followed by a sharp decrease to 49°C, and then a rate of -0.2°C/15 seconds (ie -0.8°C/minute) Gradual temperature reduction results obtained for the duration of the continuous reaction. The figures show that amplification was achieved for both the 10-replicate and 100-replicate reactions, although there was approximately twice as much fluorescence signal for the 100-replicate target reaction compared to the 10-replicate target reaction, and the group Significant internal variability.

Figure 16B shows the results obtained using the same initial temperature of 63°C for 1 minute followed by a sharp decrease to 49°C. Thereafter, the reaction temperature was maintained at 49°C for the duration of the experiment. As can be seen from the figure, there is good specific amplification and much less within-group variability (reactions with 100 replicate target or no target only)

The ability of STAR to amplify over the entire 40°C temperature range clearly demonstrates that STAR is compatible with The conventional amplification responses were significantly different. Atypical reaction temperatures with a wide range are not common and would be expected to be ineffective. Without limiting Applicants to any particular theory, surprisingly, these larger temperature ranges appear to be less restrictive for the amplification of STARs than conventional amplification methods. The ability of STAR to achieve superior amplification over a wider temperature range may be due to improved primer specificity and binding and strategic use of enzyme temperature optima. By utilizing the higher temperature of the initial stage, amplification of the correct product is facilitated and thus the efficiency of all subsequent stages, exponential amplification and detection is improved. This selection and subsequent temperature drop opens up an amplification toolbox as new models of enzymes, primers and temperatures can be realized.

Example 8: Results using 6 2'-O-methyl and 7 2'-O-methyl

As previously described, 2'-O-methyl modified primers are known to reduce primer dimer formation during amplification. Further illustrating the cooperative nature of these modifications with the STAR technology is the ability to incorporate larger 2'-O-methyl modified strands and still achieve amplification. Typically, the 2'-O-methyl modification stops the polymerase, permanently delaying amplification; six or more are thought to cause the polymerase to "fall off" from the complex rather than just stop. Figures 17A and 17B demonstrate that STAR is able to tolerate these modifications and achieve significant amplification with longer 2'-O-methyl chains than previously identified 2'-O-methyl chains. The structures of primers containing 2'-O-methylated bases are shown in Figures IB and 1C.

Figures 17A and 17B are graphs of fluorescence (in arbitrary units) versus time (minutes) (subtracted background). Shading indicates the temperature profile (°C) over time during the course of the amplification reaction. Figure 17A shows the results of a reaction performed with bases containing 6 2'-O-methyl modifications, and Figure 17B shows the results of reactions performed with bases containing 7 2'-O-methyl modifications. In both cases, the no-target negative control reaction did not produce any fluorescent signal, while amplification was good with either of the modified primers, although the average fluorescent signal for the 6 modified base primers was slightly higher, And the within-group variability is much less than the results from the 7 modified base primers.

As can be seen in the figure, primers containing strands of 6 and 7 2'O-methyl groups amplified well with STAR. This may be due to STAR's ability to start amplification around 65°C, a highly favorable temperature region for strand displacement polymerase. This favorable region may allow the polymerase to extend longer 2' modified strands, allowing initiation that other techniques lack. For brevity, the data is not shown, but it can also be described that the full-length primer region has been modified with 2'-O-methyl groups and shows amplification, albeit slower and with lower fluorescent signal.

Example 9: Results of using ribonucleic acid

STAR can use any composition of DNA (cDNA and gDNA), RNA (mRNA, tRNA, rRNA, siRNA, microRNA), RNA/DNA analogs, sugar analogs, hybrids, polyamide nucleic acids and other known analogs , amplified from any nucleic acid. Amplification of ribosomal ribonucleic acid was performed as described below.

Enzymes, oligonucleotides and targets:

Listeria monocytogenes was used as a target for the development of STAR RNA assays. Listeria monocytogenes (ATCC VR-886) genomic DNA was obtained from the American Type Culture Collection (Manassas, VA). The gDNA was initially screened and the 23S region of the ribosomal ribonucleic acid was found to be used with primers LMONF72 (SEQ ID NO: 4, 5'-GGACTCGATATCGAGTCCAGTTACGATTTGTTG-3') and LMONR86 (SEQ ID NO: 5, 5'-gGACTCCATATGGAGTCCTACGGCTCCGCTTTT-3' ) to expand. The resulting DNA template was detected using the molecular beacon LMONMB1 (SEQ ID NO: 6, 5'-FAM/gctgcGTTCCAATTCGCCTTTTTCGCagc/BHQ1-3') as described in EP No. 0728218. Total RNA was isolated using the RNeasy Plus Mini Kit Qiagen (Hilden, Germany) combined with rapid mechanical lysis on a Mini Bead Mill 4 (VWR). Listeria monocytogenes (ATCC BAA-2660) was obtained from the American Culture Collection Center (Manassas, VA). were obtained and regenerated by plating on brain heart infusion agar plates (BHI). A single colony was used to inoculate 25 mL of BHI medium, which was grown at 37°C for 18 hours to reach growth arrest. Cultures were then back-diluted and grown for an additional four hours before harvesting. Bacterial pellets were resuspended in RLT lysis buffer and homogenized on a Mini Bead Mill (VWR). Total RNA was purified according to the manufacturer's instructions (Qiagen). Genomic DNA was removed by passing the lysate through a DNA binding column set in the RNeasy Plus purification kit. Genomic DNA contamination was further minimized by on-column DNase I digestion of samples on RNeasy RNA binding columns. Bst X DNA polymerase was purchased from Beverly Qiagen (Beverly, MA). Reverse transcriptase Omniscript was purchased from Qiagen (Hilden, Germany). Nt.BstNBI nicking endonuclease was purchased from New England BioLabs (Ipswich, MA) as described in US Pat. No. 6,191,267. Oligonucleotides and molecular beacons were synthesized by Integrated DNA Technologies (Coralville, IA).

Amplification conditions:

The basic STAR mix contained everything as described in Example 1 above, additionally including the following: 4U reverse transcriptase (referenced above) and the surrogate Manta 1.0 for Bst.X.

The results are shown in Figure 18, which is a graph of fluorescence (arbitrary units) versus time (minutes). Shading indicates the temperature profile during the reaction process. Negative control reactions did not produce any fluorescent signal, while 10 replicates, 100 replicates, or 1000 replicates target reactions produced over-threshold at decreasing amounts of time (approximately 3.5 minutes, 3.0 minutes, and 2.75 minutes, respectively) value of the fluorescent signal. The results demonstrate that STAR can efficiently amplify from reverse transcribed RNA targets.

<110> LumiraDx UK Ltd

<120> Improvements in or related to nucleic acid amplification methods

<140> TW 106123056

<141> 2017-07-10

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Claims (28)

A method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a) combining a target sequence with one or more complementary single-stranded primers under conditions that permit a hybridization event in which the primers hybridize to the target mixing, the hybridization event leads directly or indirectly to the formation of a duplex structure comprising two nicking sites disposed at or near opposite ends of the duplex; and is amplified by (b) creating a nick at each of the nick sites in the strands of the duplex; (c) using a polymerase to extend the nicked strands to form newly synthesized nucleic acids, the polymerase (d) repeating steps (b) and (c) as necessary to generate multiple copies of the newly synthesized nucleic acid; characterized in that the temperature for performing the method is non-isothermal, and in The temperature is subjected to a decrease of at least 2°C during the amplification procedure of steps (b)-(d), and wherein the temperature of the reaction does not return to a predetermined temperature. The method of claim 1, wherein in step (a), the target comprises two nucleic acid complementary strands, and the primers comprise forward and reverse primers, each of which is associated with the The respective strands of the target are complementary such that the 3' ends of the forward and reverse primers are oriented towards each other. The method of claim 1 or claim 2, wherein the temperature is subjected to a controlled decrease of at least 5°C during the amplification reaction. The method of claim 1 or claim 2, wherein the average rate of temperature reduction during the amplification reaction is in the range of -0.10°C min ^-1 to -6.0°C min ^-1 . The method of claim 1 or claim 2, wherein step (b)-step (d) is performed substantially immediately after step (a), and wherein step (a)-step (d) is performed in the same reaction in a vessel or on the same solid support. The method of claim 1 or claim 2, wherein step (a) is carried out at a temperature in the range of 55°C to 62°C. According to the method of item 1 or item 2 of the claimed scope, it further comprises the step of directly or indirectly detecting the newly synthesized nucleic acid. The method of claim 7 of the claimed scope, wherein the detecting step comprises the use of molecular beacons or fluorescent dyes, lateral flow labeled probes, or enzymes that catalyze electrochemical reactions. The method of claim 1 or claim 2, wherein step (b) comprises using a nicking enzyme. The method of claim 1 or claim 2, comprising using a first polymerase and/or a first nickase having an optimum temperature, and a second polymerase and/or having an optimum temperature A second nickase, wherein the optimum temperature of the second polymerase and/or second nickase is lower than the optimum temperature of the respective first polymerase and/or first nickase. The method of claim 10, wherein the second polymerase is Bsu polymerase or Klenow fragment of DNA polymerase. The method of claim 10 of the claimed scope, wherein the initial temperature of the amplification reaction is or higher than the optimal temperature of the first nickase, and the temperature decreases to be lower than the first nickase during the course of the amplification reaction The temperature of the optimum temperature for cutting enzymes. The method of claim 10 of the claimed scope, wherein the temperature of the amplification reaction is lowered to the optimum temperature of the second polymerase and/or the second nickase or lower than the optimum temperature. The method of claim 1 or claim 2, further comprising the step of: contacting the mixture obtained by carrying out the method with a nucleic acid-degrading thermolabile enzyme, the mixture and the thermolabile enzyme in the The thermolabile enzymes are contacted at a temperature at which they are substantially active. The method of claim 14 of the claimed scope, wherein the enzyme is cod uracil-DNA glycosidase (UDG), or Antarctic thermolabile UDG. The method of claim 1 or claim 2, wherein a reverse transcription step is performed before step (a), the reverse transcription step comprising contacting the target RNA analyte with reverse transcriptase to form DNA of the target RNA analyte transcript. The method of claim 16 of the claimed scope, further comprising the step of preparing double-stranded DNA from the DNA transcript. The method of claim 1 or claim 2 further comprises a pre-amplification or enrichment step. The method of claim 1 or claim 2, wherein one or more of the primers comprise modified nucleotides. The method of claim 19, wherein the modified nucleotide is in the target complementary portion of the primer. The method of claim 19, wherein the one or more primers comprise 2' modified nucleotides. The method of claim 21, wherein the one or more primers comprise 2'O-methyl modified nucleotides. The method of claim 22, wherein the one or more primers comprise a plurality of 2'O-methyl modified nucleotides. The method of claim 23, wherein the one or more primers comprise up to 7 2'O-methyl modified nucleotides. The method of claim 1 or 2, wherein the temperature of the reaction is not returned to the temperature at which step (a) is performed during steps (b)-step (d). The method of claim 1 or claim 2, wherein one or more of the primers comprise a self-complementary portion. The method of claim 26, wherein the self-complementary portion forms a hairpin structure. The method of claim 27, wherein the hairpin structure comprises 5 to 10 base pairs.

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