Argonaute-mediated primer-free index amplification method and application thereof

Abstract

The invention discloses an Argonaute mediated primer-free index amplification method and an application of the Argonaute mediated primer-free index amplification method. The Argonaute-mediated primer-free index amplification method provided by the invention comprises the following steps: designing and synthesizing gDNA according to a target sequence; incubating Argonaute with cleavage activity to form an Argonaute / gDNA compound, and then cleaving the target sequence (short single-stranded DNA, marked as an ab chain, at the 5'end of a tangency point can be released) by using the Argonaute / gDNA compound; adding a single-stranded template (reverse complementary sequences of ab chains are arranged at the two ends, and a sequence located at the 5'end of a tangency point in gDNA is arranged in the middle), DNA polymerase, dNTPs and a reaction buffer solution into the cleavage product, and reacting to realize index amplification of the product. Through an endogenous primer self-generation mechanism, the core problems of complex primer design, serious non-specific interference and the like in the traditional technology are solved, and the Argonaute and the strand displacement polymerase have a synergistic effect, so that primer-free index amplification is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an Argonaute-mediated primerless exponential amplification method and its applications. Background Art

[0002] As a core technology in the fields of molecular diagnosis, gene sequencing, and synthetic biology, the development of nucleic acid amplification technology has always centered around how to efficiently and specifically amplify target sequences. Traditional technologies such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) have demonstrated practicality in specific scenarios, but their core reliance on primer-mediated target recognition mechanisms has led to a series of technical bottlenecks that are difficult to overcome.

[0003] Taking the PCR technology as an example, it achieves DNA amplification through three-step cycles of high-temperature denaturation (95 °C), low-temperature annealing (50–65 °C), and medium-temperature extension (72 °C). This process highly depends on a pair of specific primers (usually 18–25 nt). Primer design requires verification of sequence specificity through the BLAST tool and strict control of the GC content (40–60 %) to avoid the formation of secondary structures or primer dimers. However, the primer design cycle is long, the error rate is high, and the amplification efficiency of low-abundance targets decreases significantly. In addition, the dependence of PCR on precise temperature control equipment limits its application in point-of-care testing (POCT). In COVID-19 nucleic acid testing, although PCR is widely used, the rapid mutation of the viral genome often leads to primer off-targeting, forcing frequent design updates, highlighting the vulnerability of primer-dependent technologies.

[0004] Compared with the multi-temperature-step cycles of PCR, the LAMP technology achieves amplification under isothermal conditions (60–65 °C) through a strand-displacement DNA polymerase (such as Bst enzyme). Its core lies in using 4–6 primers (including inner primers, outer primers, and loop primers) to recognize 6–8 regions of the target, forming circular amplification products. However, the primer design of LAMP is more complex: the inner primers need to contain reverse complementary sequences, which are prone to forming secondary structures, and the design error tolerance of the dedicated software (such as Primer Explorer) is low (mismatch tolerance <2 nt). In practical applications, non-specific amplification caused by cross-reaction between primers and signal interference during multiplex detection severely limit its reliability. For example, in malaria-endemic areas, although LAMP has been used for the detection of the 18S rRNA gene of Plasmodium, misjudgments frequently occur due to the difficulty of primer design in differentiating closely related species.

[0005] The RPA technology takes a different approach. It uses a recombinase (such as T4 UvsX) to form a complex with a long primer (>30 nt), which invades double-stranded DNA and initiates amplification at a constant temperature (37-42 °C). This technology achieves target recognition through the strand exchange activity of the recombinase, avoiding the need for traditional thermal cycling. However, the synthesis cost of long primers is high (requiring chemical modification and HPLC purification), and high GC content easily leads to the loss of recombinase activity. In addition, the sensitivity of RPA is limited by the thermal sensitivity of the recombinase, and the background signal caused by non-specific binding (signal-to-noise ratio <2) also requires fluorescence probe-assisted interpretation. In the field detection of Ebola virus, although RPA is favored for its simple equipment, its limitations are exposed due to the inability of primers to quickly adapt to virus mutations.

[0006] In summary, the common defects of traditional technologies are concentrated in the design complexity, equipment dependence, and sensitivity limitations brought about by primer dependence. The multi-temperature requirements of PCR, the multi-primer design burden of LAMP, and the long primer synthesis cost of RPA all highlight the double-edged sword effect of primers as "molecular keys" - they are both the core tools for target recognition and the main bottlenecks for technology optimization. In addition, problems such as primer dimers, non-specific amplification, and low detection efficiency of low-abundance targets further restrict the application of these technologies in precision medicine and point-of-care testing. Against this background, developing a nucleic acid amplification technology that does not require primer design and has both high sensitivity and specificity has become the key direction to break through the current predicament.

[0007] TtAgo (Thermus thermophilus Argonaute) is a prokaryotic Argonaute protein derived from Gram-negative thermophilic bacteria. Through the precise guidance of 5'-phosphorylated single-stranded guide DNA (gDNA), it can achieve DNA-guided specific nucleic acid cleavage function at high temperatures of 65-85 °C. Compared with the CRISPR / Cas system, TtAgo exhibits significant technical advantages: First, its mechanism of action is not restricted by protospacer adjacent motif (PAM), and it can target any DNA sequence, while the CRISPR / Cas system relies on the recognition of specific PAM sequences; Second, TtAgo has the ability to accurately recognize single-base resolution and has no strict restrictions on the spatial position of mutation sites, while the CRISPR / Cas system is highly sensitive to base mismatches in the region adjacent to PAM, greatly limiting its application scope in mutation detection; Third, TtAgo can be compatible with multiple gDNA guide sequences at the same time, achieving parallel detection of multiple targets without signal crosstalk, while some CRISPR / Cas systems are prone to cross-reactions during multi-target detection; Fourth, TtAgo has excellent high-temperature resistance and maintains high cutting activity in the range of 65-85 °C. The cutting efficiency at low temperatures (such as 55 °C) can be further optimized by adding protein denaturants (such as DTT); Fifth, TtAgo only requires short-chain gDNA (15-20 nt) as a guide molecule, and compared with the long-chain RNA guide sequence (about 100 nt) relied on by the CRISPR / Cas system, its preparation cost is lower and its stability is significantly improved. Summary of the Invention

[0008] The object of the present invention is to provide an Argonaute-mediated primerless exponential amplification method and its application.

[0009] In the first aspect, the present invention claims to protect an Argonaute-mediated primerless exponential amplification method.

[0010] The Argonaute-mediated primerless exponential amplification method claimed by the present invention may include the following steps: (A1) Design and synthesize a single-stranded guide DNA with a phosphorylated modification at the 5' end according to the target sequence, denoted as gDNA; the gDNA is reverse complementary to a partial sequence (here the partial sequence is a continuous sequence) in the target sequence; (A2) Incubate the Argonaute with cutting activity with the gDNA obtained in (A1) to obtain an Argonaute / gDNA complex; (A3)Mix the Argonaute / gDNA complex obtained in (A2) with the target sequence, and complete the specific cleavage of the target sequence by reacting the Argonaute (for example, the cleavage site of TtAgo is the phosphodiester bond between the 10th and 11th bases at the 5' end of the gDNA), and release the part of the cleaved single strand located at the 5' end of the cleavage site; (A4)Add a single-stranded template, DNA polymerase, dNTPs, and reaction buffer to the reaction product obtained in step (A3) to obtain a reaction system; The single-stranded template consists of the following three parts in sequence from the 5' end to the 3' end: The first part: the reverse complementary sequence of the ab strand (denoted as the a'b' strand); The second part: the nucleotide sequence located at the 5' end of the gDNA in the gDNA (denoted as the c' strand, if the Argonaute is TtAgo, then the c' strand is the 1-10th amino acid sequence of the gDNA starting from the 5' end); The third part: the reverse complementary sequence of the ab strand (i.e., the a'b' strand).

[0011] Among them, the fragment composed of the second part (i.e., the c' strand) and the third part (i.e., the a'b' strand near the 3' end) has the same sequence as the gDNA. Therefore, in the subsequent amplification reaction, the new double-strands generated from the single-stranded template can be repeatedly specifically recognized and cleaved by the Argonaute / gDNA complex.

[0012] React the reaction system to achieve exponential amplification of the product.

[0013] In step (A4), the ab strand is complementary paired with the a'b' strand in the single-stranded template, and extends to form a new double-strand under the action of the DNA polymerase. The new double-strand is repeatedly specifically recognized and cleaved by the Argonaute / gDNA complex, and the released ab strand and cab strand (i.e., the reverse complementary sequence of the sequence obtained by sequentially connecting the c' strand and the a'b' strand) can re-enter and be complementary paired with the single-stranded template, and extend to form a new double-strand under the action of the DNA polymerase. Since then, a closed-loop dynamic process is formed to achieve exponential accumulation of the amplification products (i.e., the ab strand and the cab strand).

[0014] Furthermore, the 3' end of the single-stranded template can be modified with groups such as C3 spacer. The purpose is to block the 3' end to prevent non-specific amplification.

[0015] In one embodiment of the present invention, in step (A2), the Argonaute is TtAgo.

[0016] Further, in step (A4), the DNA polymerase may be a strand displacement polymerase.

[0017] In one embodiment of the present invention, the strand displacement polymerase is Vent DNA polymerase.

[0018] Further, in step (A2), the incubation may be at 75 °C for 30 min.

[0019] Further, in step (A3), the reaction may be at 85 °C for 1 h.

[0020] Further, in step (A4), the reaction conditions may be: 55 °C for 30 s, 85 °C for 45 s, for 120 cycles.

[0021] The Argonaute-mediated primerless exponential amplification method claimed in the present invention may include the following steps: (A1) described above; (A2) described above; and The following step (A34) formed by combining (A3) and (A4) described above: Mix the Argonaute / gDNA complex obtained in (A2) with the target sequence, the single-stranded template, the DNA polymerase, the dNTPs and the reaction buffer to obtain a reaction system, and perform a reaction on the reaction system to achieve exponential amplification of the product.

[0022] Further, in step (A34), the reaction conditions are: 55 °C for 30 s, 85 °C for 45 s, for 120 cycles.

[0023] The target sequence can be either double-stranded or single-stranded. The length of the target sequence can be about 80 - 120 bp or about 80 - 120 nt.

[0024] In one embodiment of the present invention, the target sequence is a double-stranded DNA with a length of 91 bp. Specifically, the target sequence is a double-stranded DNA formed by reverse complementarity of the forward strand shown in SEQ ID No.1 and the reverse strand shown in SEQ ID No.2. Correspondingly, the sequence of the gDNA is as shown in SEQ ID No.3 (phosphorylated modification at the 5' end). The sequence of the ab strand is as shown in SEQ ID No.4. The sequence of the single-stranded template is as shown in SEQ ID No.7 (modified with a C3 spacer group at the 3' end).

[0025] In one embodiment of the present invention, the system for performing the incubation in step (A2) contains 0.33 μM of TtAgo and 1.67 μM of gDNA, as well as 10 mM of Mg 2+ and a reaction buffer (such as 10× ThermoPol reaction buffer), with the balance being water. The reaction system of step (A3) is obtained by adding the target sequence, the reaction buffer (such as 10× ThermoPol reaction buffer), and water to the system after the reaction in step (A2) above, where the system after the reaction in step (A2) above accounts for a volume ratio of 6 / 10, and the concentration of the target sequence is 100 nM. The reaction system of step (A4) is obtained by adding the single-stranded template, the DNA polymerase, the dNTPs, the reaction buffer (such as 10× ThermoPol reaction buffer), SYBR Green I, and water to the system after the reaction in step (A3) above, where the final concentration of the single-stranded template is 0.1 μM, the final concentration of the DNA polymerase is 0.1 U / μL, the final concentration of the dNTPs is 0.25 mM, the final concentration of TtAgo is 0.1 μM, and the final concentration of gDNA is 0.5 μM.

[0026] In another embodiment of the present invention, the reaction system in step (A34) is obtained by adding the target sequence, the single-stranded template, the DNA polymerase, the dNTPs, the reaction buffer (such as 10× ThermoPol reaction buffer), SYBR Green I, and water to the system after the reaction in step (A2) above, where the concentration of the target sequence is 1 nM, the final concentration of the single-stranded template is 0.1 μM, the final concentration of the DNA polymerase is 0.1 U / μL, the final concentration of the dNTPs is 0.25 mM, the final concentration of TtAgo is 0.1 μM, and the final concentration of gDNA is 0.5 μM.

[0027] In a second aspect, the present invention claims the use of the method described in the first aspect above in detecting whether a target sequence has mutated.

[0028] Furthermore, the mutation can be an insertion, deletion, or substitution of a single base or multiple bases; the multiple bases are two or more (including two) bases.

[0029] In a third aspect, the present invention claims a method for detecting whether a specific mutation has occurred in a target sequence to be detected.

[0030] The method for detecting whether a specific mutation has occurred in a target sequence to be detected claimed by the present invention may include the following steps: (B1)Design and synthesize a phosphorylated single-stranded guide DNA with its 5'-end, denoted as gDNA, based on the mutant target sequence with a specific mutation; the gDNA is reverse complementary to a partial sequence (here the partial sequence is a continuous sequence) in the mutant target sequence; the partial sequence includes the nucleotide at the specific mutation position. (B2)Incubate the Argonaute with cleavage activity with the gDNA obtained in (B1) to obtain an Argonaute / gDNA complex. (B3)Mix the Argonaute / gDNA complex obtained in (B2) with the target sequence to be detected and incubate at a specific temperature; the specific temperature is the temperature at which the Argonaute can exert its cleavage activity. (B4)Add a single-stranded template, DNA polymerase, dNTPs and reaction buffer to the system processed in step (B3) to obtain a reaction system. The single-stranded template consists of the following three parts in sequence from the 5'-end to the 3'-end: The first part: the reverse complementary sequence of the ab strand (denoted as the a'b' strand); the ab strand is the part of the single strand at the 5'-end of the cleavage site where the mutant target sequence is specifically cleaved by the Argonaute / gDNA complex obtained in (B2) (the cleavage site of TtAgo is the phosphodiester bond between the 10th and 11th bases at the 5'-end of the gDNA). The second part: the nucleotide sequence at the 5'-end of the cleavage site in the gDNA (denoted as the c' strand; if the Argonaute is TtAgo, the c' strand is the 1st - 10th amino acid sequence of the gDNA starting from the 5'-end). The third part: the reverse complementary sequence of the ab strand (i.e., the a'b' strand).

[0031] Among them, the fragment composed of the second part (i.e., the c' strand) and the third part (i.e., the a'b' strand near the 3'-end) has the same sequence as the gDNA.

[0032] Perform an amplification reaction on the reaction system; monitor whether exponential amplification of the product occurs during the reaction process, and then determine whether the specific mutation has occurred in the target sequence to be detected as follows: if exponential amplification of the product occurs, the target sequence to be detected has occurred or is a candidate for the specific mutation; if exponential amplification of the product does not occur, the target sequence to be detected has not occurred or is a candidate for not having the specific mutation.

[0033] In this method, since TtAgo has single-base resolution (that is, TtAgo has extremely high requirements for the complementary pairing of gDNA and the target sequence. Only when the two are completely matched can the cleavage activity be activated. A single-base mismatch will cause a significant decrease in cleavage efficiency or even complete failure), therefore: if the specific mutation has occurred in the target sequence to be detected, after the gDNA designed according to the mutated target sequence forms a complex with TtAgo, it can specifically recognize and cleave the mutated target sequence, and then achieve subsequent exponential amplification of the product; while for the sequence without the specific mutation, it cannot be effectively recognized and cleaved, ultimately resulting in the inability to achieve exponential accumulation of the product throughout the process.

[0034] Furthermore, the 3' end of the single-stranded template can be modified with groups such as C3 spacer. The purpose is to block the 3' end and prevent non-specific amplification.

[0035] In one embodiment of the present invention, in step (B2), the Argonaute is TtAgo.

[0036] Furthermore, in step (B4), the DNA polymerase can be a strand displacement polymerase.

[0037] In one embodiment of the present invention, the strand displacement polymerase is Vent DNA polymerase.

[0038] Furthermore, in step (B2), the incubation can be incubation at 75 °C for 30 min.

[0039] Furthermore, in step (B3), the reaction can be incubation at 85 °C for 1 h.

[0040] Furthermore, in step (B4), the reaction conditions can be: 55 °C for 30 s, 85 °C for 45 s, 120 cycles.

[0041] The method for detecting whether a specific mutation has occurred in a target sequence to be detected, which the present invention claims to protect, may include the following steps: As described above (B1); As described above (B2); and The following step (B34) formed by combining the foregoing (B3) and the foregoing (B4): Mix the Argonaute / gDNA complex obtained in (B2) with the target sequence to be tested, the single-stranded template, the DNA polymerase, the dNTPs, and the reaction buffer to obtain a reaction system, and perform an amplification reaction on the reaction system; monitor whether exponential amplification of the product occurs during the reaction process, and then determine whether the specific mutation has occurred in the target sequence to be tested as follows: If exponential amplification of the product occurs, the specific mutation has occurred or is a candidate to have occurred in the target sequence to be tested; if exponential amplification of the product does not occur, the specific mutation has not occurred or is a candidate not to have occurred in the target sequence to be tested.

[0042] Further, in step (B34), the conditions for the amplification reaction are: 55°C for 30 s, 85°C for 45 s, 120 cycles.

[0043] Further, in steps (B4) and (B34), the monitoring of whether exponential amplification of the product occurs can be achieved as follows: Add a fluorescent dye to the reaction system simultaneously before the reaction; Further, the fluorescent dye is SYBR Green I.

[0044] Further, the target sequence can be either double-stranded or single-stranded. The length of the target sequence can be 80 - 120 bp or 80 - 120 nt.

[0045] In an embodiment of the present invention, the target sequence is a double-stranded DNA with a length of 91 bp. Specifically, the target sequence is a double-stranded DNA formed by reverse complementarity of the forward strand shown in SEQ ID No.1 and the reverse strand shown in SEQ ID No.2. Correspondingly, the sequence of the gDNA is as shown in SEQ ID No.3 (phosphorylated modification at the 5' end). The sequence of the ab strand is as shown in SEQ ID No.4. The sequence of the single-stranded template is as shown in SEQ ID No.7 (modified with a C3 spacer group at the 3' end).

[0046] Further, the length of the gDNA is 16 - 25 nt. In an embodiment of the present invention, the length of the gDNA is 17 nt.

[0047] Further, the length of the ab strand is 15 - 19 nt. In an embodiment of the present invention, the length of the ab strand is 19 nt.

[0048] In an embodiment of the present invention, the system for performing the incubation in step (B2) contains 0.33 μM of TtAgo and 1.67 μM of gDNA, and 10 mM of Mg2+ and a reaction buffer (such as 10× ThermoPol reaction buffer), with the balance being water. The reaction system of step (B3) is obtained by adding the target sequence, the reaction buffer (such as 10× ThermoPol reaction buffer), and water to the system after the reaction in the above step (B2), wherein the system after the reaction in the above step (B2) accounts for a volume ratio of 6 / 10, and the concentration of the target sequence is 100 nM. The reaction system of step (B4) is obtained by adding the single-stranded template, the DNA polymerase, the dNTPs, the reaction buffer (such as 10× ThermoPol reaction buffer), SYBR Green I, and water to the system after the reaction in the above step (B3), wherein the final concentration of the single-stranded template is 0.1 μM, the final concentration of the DNA polymerase is 0.1 U / μL, the final concentration of the dNTPs is 0.25 mM, the final concentration of the TtAgo is 0.1 μM, and the final concentration of the gDNA is 0.5 μM.

[0049] In another embodiment of the present invention, the reaction system in step (B34) is obtained by adding the target sequence, the single-stranded template, the DNA polymerase, the dNTPs, the reaction buffer (such as 10× ThermoPol reaction buffer), SYBR Green I, and water to the system after the reaction in the above step (B2), wherein the concentration of the target sequence is 1 nM, the final concentration of the single-stranded template is 0.1 μM, the final concentration of the DNA polymerase is 0.1 U / μL, the final concentration of the dNTPs is 0.25 mM, the final concentration of the TtAgo is 0.1 μM, and the final concentration of the gDNA is 0.5 μM.

[0050] Fourthly, the present invention claims any one of the following kits: (C1) A kit for performing Argonaute-mediated primerless exponential amplification, containing the gDNA, the Argonaute, the single-stranded template, and the DNA polymerase described in the first aspect above.

[0051] (C2) A kit for detecting whether a specific mutation occurs in a target sequence to be detected, containing the gDNA, the Argonaute, the single-stranded template, the DNA polymerase, and the fluorescent dye described in the third aspect above.

[0052] Furthermore, the kit may further contain an amplification buffer and dNTPs.

[0053] Experiments have proved that the present invention constructs an endogenous primer-driven amplification system based on the TtAgo protein: the TtAgo / gDNA complex releases the initial functional endogenous primer strand by targeting and cleaving the target DNA; this primer is complementary to the single-stranded template added exogenously to guide the polymerase to extend and generate a double-stranded structure. While the thermal cycle (denaturation - annealing - extension) drives the accumulation of amplification products, it triggers the secondary cleavage of the newly formed double-stranded by TtAgo and primer regeneration, and the released primers feedback to the amplification cascade, forming a self-sustaining cyclic system of cleavage-extension coupling. SYBR Green I real-time fluorescence quantitative analysis confirms that this system realizes the exponential synthesis of DNA products.

[0054] Compared with the prior art, the present invention has the following advantages: (1) The present invention completely abandons the design of exogenous primers and automatically generates endogenous primers. The TtAgo / gDNA complex directly recognizes and cleaves the target to obtain primers, completely eliminating the links of primer design, synthesis and verification. (2) The present invention has zero risk of primer dimers and non-specificity. The synergistic effect of the targeted cleavage of TtAgo and the proofreading activity (3'→5' exonuclease) of Vent DNA polymerase forms a double-specificity guarantee. (3) The present invention uses exponential self-driven amplification to achieve the accumulation of products 2 n with stable amplification efficiency; strand displacement efficiently unwinds, and the continuous synthesis ability of Vent DNA polymerase overcomes the template secondary structure, improving the sensitivity. (4) The present invention is compatible with SNPs, insertions / deletions mutations and does not require re-designing primers. Subsequently, it can be applied to amplification detection kits to avoid false positives and improve the detection rate.

[0055] In summary, through the endogenous primer self-generation mechanism, the present invention solves the core problems in traditional technologies such as complex primer design and serious non-specific interference. The synergistic effect of Argonaute protein and strand displacement polymerase realizes primer-free exponential amplification, providing a standardized and low-cost technical platform for molecular diagnosis, environmental monitoring and synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is the schematic diagram of the Argonaute-mediated primer-free exponential amplification method provided by the present invention.

[0057] Figure 2 It is the polyacrylamide gel electrophoresis result in the screening of two schemes of the present invention. The concentrations noted in the figure are the concentrations of ab strands in the reaction systems of the two schemes.

[0058] Figure 3 It is the real-time fluorescence quantitative amplification curve in the screening of Scheme I of the present invention.

[0059] Figure 4 It is the real-time fluorescence quantitative amplification curve in the screening of Scheme II of the present invention.

[0060] Figure 5 This is the real-time fluorescence quantitative amplification curve of the practical application case of Solution II of the present invention. Specific embodiments

[0061] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0062] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0063] The TtAgo used in the following embodiments was purchased from NEB, and the product number is: M0665S. The Vent DNA polymerase was purchased from NEB, and the product number is: M0254V.

[0064] Example 1, Argonaute-mediated primer-free exponential amplification method and its application I. Establishment of Argonaute-mediated primer-free exponential amplification method The schematic diagram of the Argonaute-mediated primer-free exponential amplification method provided by the present invention is as Figure 1 shown. The Argonaute used in this embodiment is TtAgo, and the DNA polymerase used is Vent DNA polymerase.

[0065] Introduction to TtAgo protein: It is a prokaryotic protein from Gram-negative Thermus thermophilus, which can maintain its activity at a high temperature of 95 °C and specifically cleaves the target DNA complementary to the guide DNA (gDNA). The cleavage site is the phosphodiester bond between the 10th and 11th bases at the 5' end of the target DNA. The advantage of TtAgo protein is that it does not require a specific protospacer adjacent motif (PAM), which makes it more flexible in designing guide sequences and capable of targeting more DNA sequences. As a multi-turnover enzyme, a single TtAgo-gDNA complex can cleave multiple nucleic acid strands, improving the detection efficiency. TtAgo has the precise recognition ability of single-base resolution, that is, TtAgo has extremely high requirements for the complementary pairing of gDNA and the target sequence, and the cleavage activity can only be activated when the two are completely matched. A single-base mismatch will lead to a significant decrease in cleavage efficiency or even complete inactivation. This protein can recognize and cleave the target nucleic acid sequence that is completely complementary to its guide strand with extremely high precision, and even a single-base mismatch (SNV, single nucleotide variant) between the target and the guide strand can be effectively distinguished.

[0066] Vent DNA polymerase is a DNA polymerase with strand displacement activity and 3´→5´ exonuclease activity.

[0067] The Argonaute-mediated primerless exponential amplification method provided by the present invention will be introduced in detail below by taking TtAgo and Vent DNA polymerase as examples, as follows: 1. Design and preparation of guide DNA (gDNA) Target sequence: a double-stranded nucleotide sequence containing the region to be amplified with a length of 80 - 120 bp (the forward strand is denoted as the abcd strand), or a single-stranded nucleotide sequence containing the region to be amplified with a length of 80 - 120 nt (denoted as the abcd strand). The target sequence is divided into three parts, in order from the 5' end to the 3' end (if the target sequence is double-stranded, it is for the forward strand): The first part: denoted as the ab strand; The second part: denoted as the c strand; The third part: denoted as the d strand.

[0068] Among them, the region to be amplified is located in the first part (i.e., the ab strand) and the second part (i.e., the c strand).

[0069] Design and synthesize a single-stranded guide DNA (i.e., gDNA) with a phosphorylated modification at the 5'-end (the length of the gDNA is usually 16-25 nt). The designed gDNA here needs to meet the following requirements: the gDNA is reverse complementary to a partial sequence of the target sequence or the forward strand in the target sequence (the partial sequence here is a continuous sequence, specifically the part of the ab strand near the 3'-end and the entire c strand), and when TtAgo cuts the target sequence subsequently, the cleavage site is exactly between the ab strand and the c strand, and after cleavage, the ab strand (with a length usually of 15-19 nt) is released.

[0070] 2. Pre-incubation of the TtAgo / gDNA complex Incubate TtAgo with the gDNA obtained in step 1 according to Table 1. The incubation conditions are 75 °C for 30 min to obtain the TtAgo / gDNA complex.

[0071]

[0072] Note: The 10× ThermoPol reaction buffer is purchased from NEB, and the catalog number is B9004S.

[0073] 3. TtAgo cleavage of the target to generate endogenous primers Mix the TtAgo / gDNA complex obtained in step 2 with the target sequence to obtain the reaction system shown in Table 2, and incubate at 85 °C for 1 h.

[0074]

[0075] Note: The 10× ThermoPol reaction buffer is purchased from NEB, catalog number: B9004S.

[0076] In this step, TtAgo specifically cleaves the target sequence or the forward strand in the target sequence, and releases the part of the cleaved single strand at the 5'-end of the cleavage site, that is, the ab strand.

[0077] 4. Single-stranded template integration and exponential amplification (1) Design and synthesize a single-stranded template (a'b'c'a'b'), which consists of the following three parts in sequence from the 5'-end to the 3'-end: The first part: the reverse complementary sequence of the ab strand (denoted as the a'b' strand); The second part: the nucleotide sequence at the 5'-end of the cleavage site in the gDNA (i.e., the 1-10 amino acid sequence of the gDNA from the 5'-end, denoted as the c' strand, which is reverse complementary to the c strand mentioned above); The third part: the reverse complementary sequence of the ab strand (i.e., the a'b' strand).

[0078] And the 3'-end of the single-stranded template is decorated with a C3 spacer group. The purpose is to block the 3'-end and prevent non-specific amplification.

[0079] Among them, the fragment composed of the second part (i.e., the c' strand) and the third part (i.e., the a'b' strand near the 3'-end) has the exact same sequence as the gDNA. Therefore, in the subsequent amplification reaction, the new double-strands generated from the single-stranded template can be repeatedly and specifically recognized and cleaved by the TtAgo / gDNA complex.

[0080] (2) Prepare the mix as shown in Table 3.

[0081]

[0082] Note: The 10× ThermoPol reaction buffer is purchased from NEB, product number B9004S, and the 20× SYBR Green I is purchased from Thermo Fisher Scientific, product number S7563.

[0083] (3) Add the mix (10 μL) obtained in step (2) to the system (10 μL) after the reaction in step 3 to obtain a new reaction system (20 μL). Then set the following reaction program for the amplification reaction: 55 °C for 30 s → 85 °C for 45 s, for a total of 120 cycles.

[0084] In this step, the ab strand released in step 3 is complementary to the a'b' strand in the single-stranded template and extends under the action of Vent DNA polymerase to form a new double-strand (the forward strand is the abcab strand, which is reverse complementary to the single-stranded template a'b'c'a'b' mentioned above). The new double-strand is repeatedly and specifically recognized and cleaved by the TtAgo / gDNA complex (the cleavage site is between the ab strand near the 5'-end and the c strand in the forward strand), releasing the ab strand and the cab strand (i.e., formed by sequentially connecting the c strand and the ab strand), which can re-enter and be complementary to the single-stranded template (a'b'c'a'b' strand) and extend under the action of Vent DNA polymerase to form a new double-strand. Since then, a closed-loop dynamic process is formed to achieve the exponential accumulation of the amplification products (i.e., the ab strand and the cab strand).

[0085] During the reaction process, the exponential amplification of the product can be verified by monitoring the SYBR Green I dynamic amplification curve.

[0086] II. Application Cases The target sequence is derived from the EGFR gene. EGFR (Epidermal Growth Factor Receptor) is a receptor tyrosine kinase gene that regulates cell proliferation, survival, and differentiation by activating downstream signaling pathways (such as MAPK, PI3K-AKT). Abnormalities in its function (mutation / overexpression) are closely related to various cancers. The exponential amplification of specific fragments of EGFR (such as mutation hotspots) can be achieved through the method established in Step 1, which can subsequently be used to detect whether a specific mutation has occurred in the mutation hotspot region of the EGFR gene, avoiding false positives and improving the detection rate.

[0087] The specific target sequence is as follows: Forward strand (5’-3’): TCA AGA TCA CAG ATT TTG GGC GGG CCA AAC TGC TGG GTG CGGAAG AGA AAG AAT ACC ATG CAG AAG GAG GCA AAG TAA GGA GGT GGC TTT A (SEQ IDNo.1).

[0088] Reverse strand (5’-3’): TAA AGC CAC CTC CTT ACT TTG CCT CCT TCT GCA TGG TAT TCTTTC TCT TCC GCA CCC AGC AGT TTG GCC CGC CCA AAA TCT GTG ATC TTG A (SEQ IDNo.2).

[0089] (1) Scheme design A total of two schemes are designed as follows. By designing different gDNAs, different cleavage sites are generated, and the lengths of the endogenous primer strands (i.e., the ab strands mentioned above) that are sheared off are different. The length of the ab strand in Scheme I is controlled to be 15 nt, and the length of the ab strand in Scheme II is controlled to be 19 nt.

[0090] Solution I: Design gDNA1 for the above target sequence, and the sequence is as follows (5’-3’): gDNA1: p-GCC CGC CCA A AA TCT GT.

[0091] gDNA1 was synthesized by Shanghai Sangon Biotech Co., Ltd., purified by HPLC and verified by mass spectrometry, and stored at -20 °C for later use.

[0092] Corresponding to this scheme, the sequences of the ab strand, c strand, and d strand in the forward strand of the target sequence are as follows (5’-3’): ab strand: TCA AGA TCA CAG ATT (15 nt); c strand: TTG GGC GGG C; d strand: CA AAC TGC TGG GTG CGG AAG AGA AAG AAT ACC ATG CAG AAG GAG GCAAAG TAA GGA GGT GGC TTT A.

[0093] Corresponding to this scheme, the sequence of the single-stranded template (i.e., a'b'c'a'b' strand) is as follows (5'-3'): a'b'c'a'b' strand: AATCTGTGATCTTGA GCCCGCCCAA AATCTGTGATCTTGA-C3.

[0094] Solution II: Design gDNA2 for the above target sequence, and the sequence is as follows (5'-3'): gDNA2: p-TTTGGCCCGCCCAAAAT (SEQ ID No.3).

[0095] gDNA2 was synthesized by Shanghai Sangon Biotech Co., Ltd., purified by HPLC and verified by mass spectrometry, and stored at -20°C for later use.

[0096] Corresponding to this scheme, the sequences of the ab strand, c strand and d strand in the forward strand of the target sequence are as follows (5'-3'): ab strand: TCA AGA TCA CAG ATT TTG G (19nt) (SEQ ID No.4); c strand: GC GGG CCA AA (SEQ ID No.5); d strand: C TGC TGG GTG CGG AAG AGA AAG AAT ACC ATG CAG AAG GAG GCA AAG TAAGGA GGT GGC TTT A (SEQ ID No.6).

[0097] Corresponding to this scheme, the sequence of the single-stranded template (i.e., a'b'c'a'b' strand) is as follows (5'-3'): a'b'c'a'b' strand: CCAAAATCTGTGATCTTGA TTTGGCCCGC CCAAAATCTGTGATCTTGA-C3 (SEQ ID No.7).

[0098] (2) Screening of the two schemes For the two schemes, first, prepare the TtAgo / gDNA complex as described in Step 1-2, skip Step 1-3, artificially synthesize the respective ab strands (sequences as before) and single-stranded templates (a'b'c'a'b') (sequences as before) corresponding to the two schemes, then prepare the mix according to Step 1-4(2), and finally prepare the reaction systems shown in Table 4 for the two schemes respectively (three parallels with the amounts of ab strands set at 0, 1 nM, and 10 nM).

[0099]

[0100] Set the following reaction program for the amplification reaction: 55°C for 30 s → 85°C for 45 s, for a total of 120 cycles.

[0101] During the experiment, on the one hand, detect the final reaction products by polyacrylamide gel electrophoresis; on the other hand, verify whether exponential amplification of the products occurs in the two schemes by monitoring the SYBR Green I dynamic amplification curve.

[0102] The results show that: The 10% PAGE verification bands ( Figure 2 ) show that bright amplification bands are produced in the final products of both Scheme I and Scheme II. It is worth noting that in the negative control system without ab strands (the lane marked "0"), Scheme II shows a cleaner background fluorescence signal, and the number of non-specific bands is reduced compared to Scheme I.

[0103] Real-time fluorescence quantitative kinetic curve analysis ( Figure 3 and Figure 4 ) shows that the reaction system of Scheme II presents a typical S-shaped amplification curve with a clear exponential growth phase, indicating that it can effectively guide the exponential amplification of the template. This result is consistent with the results of polyacrylamide gel electrophoresis detection.

[0104] The above results indicate that Scheme II (with an ab strand length of 19 nt) has better amplification kinetic characteristics and product specificity.

[0105] (3) Practical application of Scheme II The target sequence from the EGFR gene is detected using the above-mentioned Scheme II. The specific experimental steps are as follows: First, prepare the TtAgo / gDNA complex as described in 1-2 of Step 1 above, and then prepare the reaction system as shown in Table 5. Set the reaction program for amplification reaction: 55°C for 30 s → 85°C for 45 s, for a total of 120 cycles (equivalent to combining the component systems in 3-4 of Step 1 above into one system, reducing the step of incubating at 85°C for 1 h in Step 3, and detecting with the initial concentration of the target sequence reduced from 500 nM to 10 nM).

[0106]

[0107] The results show that as Figure 5 shown, the experimental group presented a typical S-shaped amplification curve, achieving exponential amplification. This indicates the feasibility of this scheme for detecting the target sequence of the EGFR gene. And the step of incubating at 85°C for 1 h in Step 3 is not necessary. And effective detection can still be achieved for the target sequence with a concentration of only 10 nM, showing that the detection method of the present invention has high sensitivity.

[0108] Since TtAgo has single-base resolution, that is, TtAgo has extremely high requirements for the complementary pairing of gDNA and the target sequence. Only when the two are completely matched can the cleavage activity be activated, and a single-base mismatch will cause a significant decrease in cleavage efficiency or even complete failure. Therefore, the Argonaute-mediated primerless exponential amplification method established above in the present invention can be used to detect whether a specific mutation (including single-base or multi-base insertion, deletion, or substitution) has occurred in the target sequence to be detected. Among them, gDNA is designed and synthesized according to the mutated target sequence with a specific mutation. If the specific mutation has occurred in the target sequence to be detected, after the gDNA designed according to the mutated target sequence forms a complex with TtAgo, it can specifically recognize and cleave the mutated target sequence, thereby releasing the endogenous primer (i.e., the ab strand), and then realizing the subsequent exponential amplification of the product; while for the sequence without the specific mutation, it cannot be effectively recognized and cleaved, and the ab strand cannot be released, so the subsequent exponential amplification of the product cannot be realized.

[0109] Specifically, to detect whether a specific mutation has occurred in the target sequence to be detected, the following steps are carried out: (a1) Referring to what is described in 1 of Step 1 above, design and synthesize gDNA according to the mutated target sequence with a specific mutation (controlling the length of the ab strand to be 19 nt); the gDNA is reverse complementary to a partial sequence in the mutated target sequence (here the partial sequence is a continuous sequence, specifically the part of the ab strand near the 3' end and the entire c strand); the partial sequence includes the nucleotide at the specific mutation position; (a2)As described in Step 1-2 above, incubate TtAgo with the gDNA obtained in (a1) to obtain the TtAgo / gDNA complex; (a3)As described in Step 1-3 above, mix the TtAgo / gDNA complex obtained in (a2) with the target sequence to be detected and perform an incubation reaction; (a4)As described in Step 1-4 above, synthesize the corresponding single-stranded template (a'b'c'a'b'), prepare the mix, and then add the mix to the system processed in step (a3) to obtain a reaction system and perform an amplification reaction; monitor whether exponential amplification of the product occurs during the reaction, and then determine whether the specific mutation has occurred in the target sequence to be detected as follows: if exponential amplification of the product occurs, the specific mutation has occurred in the target sequence to be detected; if exponential amplification of the product does not occur, the specific mutation has not occurred in the target sequence to be detected.

[0110] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. An Argonaute-mediated primer-free exponential amplification method comprising the following steps: (A1) designing and synthesizing a single-stranded guide DNA with a phosphorylated 5' end according to the target sequence, which is referred to as gDNA; the gDNA is reverse complementary to a portion of the target sequence; (A2) incubating Argonaute with cleavage activity with the gDNA obtained in (A1) to obtain an Argonaute / gDNA complex; (A3) mixing the Argonaute / gDNA complex obtained in (A2) with the target sequence, and allowing the Argonaute to specifically cleave the target sequence and release the cleaved single-stranded portion located at the 5' end of the cleavage site, which is recorded as ab strand; (A4) adding a single-stranded template, DNA polymerase, dNTPs and reaction buffer to the reaction product obtained in step (A3) to obtain a reaction system; the single-stranded template is composed of the reverse complementary sequence of the ab chain, the nucleotide sequence located at the 5' end of the cleavage site in the gDNA and the reverse complementary sequence of the ab chain from the 5' end to the 3' end; and reacting the reaction system to achieve exponential amplification of the product.

2. The method according to claim 1, characterized in that: In step (A2), the Argonaute is TtAgo; and / or In step (A4), the DNA polymerase is a strand displacement polymerase; Further, the strand displacement polymerase is Vent DNA polymerase; and / or In step (A2), the incubation is 75° C. for 30 min; and / or In step (A3), the reaction is incubated at 85°C for 1 hour; and / or In step (A4), the reaction conditions are: 55°C for 30 s, 85°C for 45 s, and 120 cycles.

3. An Argonaute-mediated primer-free exponential amplification method comprising the following steps: The step (A1) as claimed in claim 1 or 2; The step (A2) recited in claim 1 or 2; and The following step (A34) is formed by combining step (A3) and step (A4) described in claim 1 or 2: mixing the Argonaute / gDNA complex obtained in step (A2) with the target sequence, the single-stranded template, the DNA polymerase, the dNTPs and the reaction buffer to obtain a reaction system, and reacting the reaction system to achieve exponential amplification of the product; Furthermore, in step (A34), the reaction conditions are: 55° C. for 30 s, 85° C. for 45 s, and 120 cycles.

4. Use of the method according to any one of claims 1 to 3 in detecting whether a target sequence has mutated.

5. The use according to claim 4, characterized in that: The mutation is the insertion, deletion or substitution of a single base or multiple bases.

6. A method for detecting whether a target sequence to be tested has a specific mutation, comprising the following steps: (B1) designing and synthesizing a single-stranded guide DNA with a phosphorylation modification at the 5' end according to a target sequence after a specific mutation, which is referred to as gDNA; the gDNA is reverse complementary to a partial sequence in the target sequence after a specific mutation; the partial sequence includes the nucleotide at the specific mutation position; (B2) incubating Argonaute with cleavage activity with the gDNA obtained in (B1) to obtain an Argonaute / gDNA complex; (B3) mixing the Argonaute / gDNA complex obtained in (B2) with the target sequence to be detected, and incubating at a specific temperature; the specific temperature is a temperature at which the Argonaute can exert its cleavage activity; (B4) adding a single-stranded template, DNA polymerase, dNTPs and reaction buffer to the system treated in step (B3) to obtain a reaction system; the single-stranded template is composed of the reverse complementary sequence of the ab chain, the nucleotide sequence located at the 5' end of the cleavage site in the gDNA and the reverse complementary sequence of the ab chain from the 5' end to the 3' end; the ab chain is the portion of the single-stranded cleavage located at the 5' end of the cleavage site of the target sequence after the mutation specifically cleaved by the Argonaute / gDNA complex obtained in (B2); The reaction system is subjected to an amplification reaction; during the reaction, whether exponential amplification of the product occurs is monitored, and then whether the target sequence to be tested has undergone the specific mutation is determined as follows: if exponential amplification of the product occurs, the target sequence to be tested has undergone the specific mutation or the candidate has undergone the specific mutation; if exponential amplification of the product does not occur, the target sequence to be tested has not undergone the specific mutation or the candidate has not undergone the specific mutation.

7. The method according to claim 6, characterized in that: In step (B2), the Argonaute is TtAgo; and / or In step (B4), the DNA polymerase is a strand displacement polymerase; Further, the strand displacement polymerase is Vent DNA polymerase; and / or In step (B2), the incubation is 75° C. for 30 min; and / or In step (B3), the incubation is performed at 85° C. for 1 h; and / or In step (B4), the conditions of the amplification reaction are as follows: 55°C for 30 s, 85°C for 45 s, and 120 cycles.

8. A method for detecting whether a target sequence to be tested has a specific mutation, comprising the following steps: The step (B1) as claimed in claim 6 or 7; The step (B2) recited in claim 6 or 7; and The following step (B34) is formed by combining step (B3) and step (B4) described in claim 6 or 7: mixing the Argonaute / gDNA complex obtained in step (B2) with the target sequence to be detected, the single-stranded template, the DNA polymerase, the dNTPs and the reaction buffer to obtain a reaction system, and subjecting the reaction system to an amplification reaction; monitoring whether exponential amplification of the product occurs during the reaction, and then determining whether the target sequence to be detected has undergone the specific mutation as follows: if exponential amplification of the product occurs, the target sequence to be detected has undergone the specific mutation or the candidate has undergone the specific mutation; if exponential amplification of the product does not occur, the target sequence to be detected has not undergone the specific mutation or the candidate has not undergone the specific mutation; Furthermore, in step (B34), the conditions of the amplification reaction are: 55°C for 30 s, 85°C for 45 s, and 120 cycles.

9. The method according to any one of claims 6 to 8, characterized in that: In step (B4) and step (B34), the monitoring of whether exponential amplification of the product occurs can be achieved by: adding a fluorescent dye to the reaction system simultaneously before performing the amplification reaction; Furthermore, the fluorescent dye is SYBR Green I.

10. Any of the following kits: (C1) A kit for performing Argonaute-mediated primer-free exponential amplification, comprising the gDNA described in any one of claims 1 to 3, the Argonaute, the single-stranded template and the DNA polymerase; (C2) A kit for detecting whether a target sequence to be tested has a specific mutation, comprising the gDNA described in any one of claims 6 to 9, the Argonaute, the single-stranded template, the DNA polymerase and the fluorescent dye.

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