CN112481257A

CN112481257A - Reagent for improving replication efficiency of adeno-associated virus inverted terminal repeat (inverted terminal repeat) sequence, adeno-associated virus DNA replication method and application

Info

Publication number: CN112481257A
Application number: CN202011442675.3A
Authority: CN
Inventors: 刘兆成; 马勇; 郑岷雪; 赵国栋; 熊尚岷; 张春
Original assignee: Gwp Biotechnology Co ltd
Current assignee: Gwp Biotechnology Co ltd
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-03-12
Anticipated expiration: 2040-12-11
Also published as: CN112481257B

Abstract

The invention provides a reagent for improving replication efficiency of an inverted terminal repeat sequence of an adeno-associated virus, wherein the reagent is an oligonucleotide chain and sequentially comprises: a specific sequence comprising at least a first sequence for complementary pairing with part or all of a sequence segment of a first side stem near the 5' end of a long stem palindrome of a T-shaped hairpin of ITR sequences, and a second sequence for complementary pairing with part of a first non-complementary pairing sequence segment adjacent to the first side stem of the T-shaped hairpin; a terminal modification structure to prevent extension of the 3' end of the specific sequence by a nucleic acid polymerase. The invention also provides an adeno-associated virus DNA replication method and application. The reagent competes with the T-shaped hairpin structure of the ITR sequence of the adeno-associated virus to form a hybridization product of a specific sequence and the single-stranded DNA template of the adeno-associated virus, and simultaneously the T-shaped hairpin structure in the template is unzipped, so that the blocking effect of the T-shaped hairpin structure in the template on nucleic acid polymerase is eliminated in the primer extension process.

Description

Reagent for improving replication efficiency of adeno-associated virus inverted terminal repeat (inverted terminal repeat) sequence, adeno-associated virus DNA replication method and application

Technical Field

The invention relates to the technical field of molecular biology, in particular to a reagent for improving the replication efficiency of an inverted terminal repeat sequence of adeno-associated virus, an adeno-associated virus DNA replication method and application.

Background

Adeno-associated virus (AAV) is a replication-defective DNA virus, consisting of only 3 coat proteins and a single DNA strand. Its replication requires the assistance of a helper virus such as adenovirus or herpes virus. The AAV genome is linear single-stranded DNA, each consisting of 4680 bases, including Inverted Terminal Repeat (ITR) sequences of 145nt each at both ends and 2 open reading frames in the middle. ITRs at both ends are cis-acting viral elements essential for AAV replication and packaging. The content of CG in the ITR sequence reaches more than 70 percent. Adjacent to the internal open reading frame in the ITR sequence is a non-complementary pairing sequence with the length of 20nt, and the following 125nt forms a three-segment palindrome structure to form a T-shaped hairpin structure together. Two open reading frames in the ITR sequence are Rep gene and Cap gene, which respectively code Rep protein and Cap protein related to AAV replication. Recombinant AAV (rAAV) expression vectors formed by cutting Rep and Cap gene sequences and inserting other target gene DNAs have been widely used in the research of life sciences and clinical medicine. The rAAV vector has the advantages of high safety, low immunogenicity, stable physical properties, wide spectrum of infected cells and the like, and is considered to be one of the most potential gene therapy vectors.

In actual research work, rAAV vectors need to be replicated and amplified in host cells such as e.coli as DNA plasmids. However, the special T-shaped hairpin structure with high thermal stability greatly increases the error rate of point mutation or deletion of ITR sequences in the replication process. Because of the critical role that ITRs play in the packaging of rAAV viral particles, the presence of mutated or deleted ITRs increases the production of viral particles with defects (including empty viral particles with unpackaged DNA, viral particles with host cell proteins, and viral particles with packed non-rAAV DNA, etc.), which in turn decreases the quality and yield of the viral particles produced. In particular, when rAAV vectors are used as gene therapy drugs or nucleic acid vaccines, in order to ensure the safety of such medical products that are directly used in humans, it is necessary to ensure that rAAV DNA, which is an active pharmaceutical ingredient, is completely identical in different batches of product.

At present, the main method for identifying the accuracy of ITR in rAAV vector is restriction enzyme digestion, which is judged by the size and number of digestion fragments. However, due to the low resolution of gel electrophoresis, it is difficult to detect point mutations or small deletions, and thus the accuracy of identification cannot be sufficiently ensured. While direct sequencing of the ITR region provides sufficient resolution, the T-hairpin structure with ultra-high thermostability has strong inhibitory effect on conventional Sanger sequencing, often leading to sequencing failure. Meanwhile, the T-shaped hairpin structure can inhibit PCR reaction (such as accurate quantification of rAAV DNA in the production process of rAAV vector) with ITR as a target sequence, so that the PCR reaction efficiency is greatly reduced and even amplification fails, and further subsequent work is influenced. The adverse effect of the T-hairpin on the sequencing and PCR reactions of the ITRs is due to the rapid temperature drop during the annealing phase of the reaction, and the formation of the T-hairpin precedes the hybridization product of the primer and template due to kinetic advantages. The T-shaped hairpin structure may hinder primer extension by nucleic acid polymerase during the primer extension phase, resulting in premature termination of the reaction or reduced reaction efficiency. To overcome the above difficulties, there are two common methods. One method is to greatly increase the template dosage of sequencing or PCR reaction, but in many scenes, rAAV templates are very precious, and increasing the template dosage is difficult to realize. Another method is to add additives capable of reducing the stability of the intramolecular secondary structure, such as compounds like dimethyl sulfoxide (DMSO) or betaine (betaine), to the sequencing or PCR reaction system, but such compounds themselves will increase the error rate of sequencing and PCR amplification, making it difficult to ensure that the obtained result faithfully reflects the sequence of the original template. In addition, both of the above methods can greatly increase the experimental cost. Therefore, the development of an experimental method which does not improve the error rate per se, has low cost and can effectively solve the difficulty of the ITR sequence in sequencing and PCR reaction has great significance for popularizing the application of the rAAV vector in the basic research of life science and the clinical medicine field.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a reagent for improving the replication efficiency of an ITR sequence of the adeno-associated virus, destroying the thermal stability of a T-shaped hairpin structure in a single-chain template of the adeno-associated virus and eliminating the adverse effect of the T-shaped hairpin structure on the corresponding molecular biology technology of the adeno-associated virus.

In order to achieve the above object, the present invention is achieved by the following technical solutions.

The invention provides a reagent for improving replication efficiency of an ITR sequence of adeno-associated virus, wherein the reagent is an oligonucleotide chain and sequentially comprises:

a specific sequence comprising at least a first sequence for complementary pairing with part or all of a sequence segment of the first side stem near the 5' end of the long stem palindrome of the T-shaped hairpin structure of the ITR sequence, and a second sequence for complementary pairing with part of the first non-complementary pairing sequence segment adjacent to the first side stem of the T-shaped hairpin structure; the first sequence is adjacent to the second sequence;

a terminal modification structure to prevent extension of the 3' end of the specific sequence by a nucleic acid polymerase.

Preferably, the specific sequence further comprises a third sequence complementarily paired with part or all of the sequence segment of the second side stem of the T-shaped hairpin short stem palindrome adjacent to the first side stem.

Preferably, the terminal modification structure comprises a minor groove binder, an inverted DNA nucleotide, a C3 spacer, and an oligonucleotide strand that does not hybridize to an adeno-associated viral template.

Preferably, the reagent synthesis monomers include deoxyribonucleotides, ribonucleotides, locked nucleotides, non-natural nucleotides; several modes of linkage between monomers include phosphodiester linkage or peptide linkage.

Preferably, the total length of the first sequence and the third sequence is 4-46 nt; the length of the second sequence is 4-46 nt.

The second purpose of the invention is to provide a method for replicating adeno-associated virus DNA, wherein the agent for improving the replication efficiency of the adeno-associated virus ITR sequence is added into a replication system of the adeno-associated virus DNA, and the base pairing in a T-shaped hairpin structure formed by a single-stranded ITR sequence of the adeno-associated virus DNA is destroyed by the agent, so as to reduce the inhibition effect of the T-shaped hairpin structure in molecules on nucleic acid polymerase.

Preferably, the method specifically comprises the following steps: adding the reagent into the reaction system before the replication reaction of the adeno-associated virus DNA begins; the initial reaction concentration of the reagent is 0.01-5 mu M.

Preferably, when two complementary paired templates are included in the replication reaction system, two reagents are added to match the two complementary paired templates, respectively, or one reagent is added to match one of the templates.

Preferably, the method further comprises the steps of: the total length of the specific sequence of the reagent is varied to adjust the reagent to counteract the blocking effect of the T-hairpin structure on the nucleic acid polymerase.

The third purpose of the present invention is to provide the reagent for improving the replication efficiency of the ITR sequence of the adeno-associated virus or the application of the DNA replication method of the adeno-associated virus in PCR reaction or sequencing reaction using the adeno-associated virus as a template.

Compared with the prior art, the invention has the beneficial effects that:

the reagent for improving the replication efficiency of the ITR sequence of the adeno-associated virus comprises a specific sequence and a terminal modification structure. The specificity sequence at least comprises a first sequence and a second sequence, and is used for competing with a T-shaped hairpin structure of an ITR sequence of the adeno-associated virus to form a hybridization product of the specificity sequence and an adeno-associated virus single-stranded DNA template, and simultaneously melting the T-shaped hairpin structure in the template, so that the inhibition effect of the T-shaped hairpin structure in the template on nucleic acid polymerase is eliminated in the primer extension process, and the molecular biology technology work of synthesizing a new adeno-associated virus nucleic acid molecule by the polymerase is smoothly carried out. In addition, the melting agent has simple structure, low manufacturing cost and high melting efficiency.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood and to be implemented according to the content of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings. The detailed description of the present invention is given in detail by the following examples and the accompanying drawings.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:

FIG. 1 is a schematic diagram of the ITR sequence of a rAAV matched to a melting agent in accordance with one embodiment of the present invention;

FIG. 2 is a flow chart of a PCR reaction with a melting reagent using an ITR sequence of a rAAV as a template in an embodiment of the present invention;

FIG. 3 is a schematic diagram of the structure of two complementary strands of a rAAV vector plasmid containing an ITR sequence region according to an embodiment of the present invention;

FIG. 4 is a schematic diagram showing the matching relationship between two complementary single strands of an rAAV vector plasmid and a forward Disruptor F5 and a reverse Disruptor R5, respectively, in an embodiment of the present invention;

FIG. 5 shows the result of a fluorescent quantitative PCR reaction to amplify the ITR sequence of a commercially available rAAV vector plasmid as a template when an experimental set 5 pair of melting reagents of different concentrations is added in an embodiment of the present invention;

FIG. 6 shows the results of a 3% agarose gel electrophoresis experiment performed on the final fluorescent quantitative PCR product of the experiment of FIG. 5 according to an embodiment of the present invention;

FIG. 7 shows the result of rAAV-ITR fluorescence quantitative PCR experiments without adding a melting reagent and with 6 pairs of melting reagents having the same length of the first sequence and the second sequence according to an embodiment of the present invention;

FIG. 8 shows the results of a 3% agarose gel electrophoresis experiment performed on the final fluorescent quantitative PCR product of the experiment of FIG. 7;

FIG. 9 is a schematic diagram showing the matching relationship between two complementary single strands of an rAAV vector plasmid and a forward Disruptor F12 and a reverse Disruptor R12, respectively, in an embodiment of the present invention;

FIG. 10 shows the result of rAAV-ITR fluorescence quantitative PCR experiments without adding a melting reagent and with 10 pairs of melting reagents, each pair of melting reagents having a first sequence length of 20nt and a second sequence length different from each other, according to one embodiment of the present invention;

FIG. 11 shows the results of 3% agarose gel electrophoresis of the final fluorescent quantitative PCR product of the experiment of FIG. 10.

In the figure: 11. long stem palindrome; 111. a first lateral stem; 12. first short stem palindrome; 121. a second lateral stalk; 13. second short stem palindrome; 14. a first non-complementary pairing sequence;

20. a melting agent; 21. a first sequence; 22. a second sequence; 23. and (3) a terminal modification structure.

Detailed Description

The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings, which will enable those skilled in the art to practice the present invention with reference to the accompanying specification. In the drawings, the shape and size may be exaggerated for clarity, and the same reference numerals will be used throughout the drawings to designate the same or similar components. In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, and the like are used based on the orientation or positional relationship shown in the drawings. In particular, "height" corresponds to the dimension from top to bottom, "width" corresponds to the dimension from left to right, and "depth" corresponds to the dimension from front to back. These relative terms are for convenience of description and are not generally intended to require a particular orientation. Terms concerning attachments, coupling and the like (e.g., "connected" and "attached") refer to a relationship wherein structures are secured or attached, either directly or indirectly, to one another through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise.

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.

Example 1

During the annealing phase of the PCR or sequencing reaction, the temperature is rapidly reduced, and the T-shaped hairpin structure of the ITR sequence of the rAAV is formed before the hybridization product of the primer and the template due to the kinetic advantage. The T-shaped hairpin structure has high thermal stability, and can prevent the primer from being extended by nucleic acid polymerase in the extension stage, thereby causing premature termination of the reaction or reduction of the reaction efficiency.

The invention provides a reagent for improving replication efficiency of an ITR sequence of adeno-associated virus, wherein the reagent (a unwinding agent, English disproptor) is an oligonucleotide chain, and sequentially comprises:

a specific sequence comprising at least a first sequence for complementary pairing with part or all of a sequence segment of a first side stem of a T-shaped hairpin structure palindrome of the ITR sequence near the 5' end, and a second sequence for complementary pairing with part of a first non-complementary pairing sequence segment of the T-shaped hairpin structure adjacent to the first side stem; and the first sequence is adjacent to the second sequence; specifically, as shown in fig. 1, the ITR sequence consists of 145 bases, of which 125nt forms a double-stranded long hairpin-palindrome consisting of one long stem palindrome 11 and two short stem palindromes (first short stem palindrome 12 and second short stem palindrome 13), the remaining 20nt not forming complementary pair structures, together forming a T-shaped hairpin structure; the side stem of the long stem palindrome 11 of the T-shaped hairpin structure close to the 5 ' end of the ITR sequence is a first side stem 111, the short stem palindrome of the T-shaped hairpin structure close to the 5 ' end of the ITR sequence is a first short stem palindrome 12, and the short stem palindrome of the T-shaped hairpin structure close to the 3 ' end of the ITR sequence is a second short stem palindrome 13; the side stem of the first short stem palindrome close to the first side stem is a second side stem 121; a segment of non-complementary matched sequence adjacent to the 5' end of the first flanking stem 111, denoted as first non-complementary matched sequence 14; the specific sequence of the melting agent 20 comprises at least a first sequence 21 and a second sequence 22, the first sequence 21 is used for competition with the long stem palindrome of the T-shaped hairpin structure, the first sequence 21 is complementary-paired with the first side stem 111 of the long stem palindrome, thereby breaking the double strand of the long stem palindrome 11 in the original T-shaped hairpin structure; the second sequence 22 is complementary-paired with a portion of the first non-complementary pairing sequence 14, and the formed base pair structure makes the hybridization product formed by the melting reagent and the ITR sequence more stable; namely, the specificity sequence can solve the partial complementary pairing structure of the T-shaped hairpin structure of the ITR sequence of the adeno-associated virus, and the reagent is named as a melting agent; the following are all descriptions of the reagents of the present invention in terms of melting reagents, but the use of the reagents of the present invention is not limited by the written description of the melting reagents. By extending the length of the second sequence in the melting agent, the complementary pairing length of the melting agent and the first non-complementary pairing sequence 14 of the adeno-associated viral DNA is increased, so as to improve the thermal stability of the duplex product formed by the hybridization of the melting agent and the adeno-associated viral DNA single strand, and further increase the competitive power of the melting agent and the T-shaped hairpin structure of the ITR sequence, so as to prevent the T-shaped hairpin structure from being reformed by reverse reaction. When the melting reagent competes with the T-hairpin of the ITR sequence and forms a corresponding hybridization product, the single strand of adeno-associated virus DNA is left with 12 and 13 short palindromes, which are not sufficiently thermostable to prevent further extension by the nucleic acid polymerase.

A terminal modification structure to prevent extension of the 3' end of the specific sequence by a nucleic acid polymerase. Specifically, in the annealing stage, a primer and a template are complementarily combined to form a hybrid strand, then dNTP is added to the 3 'end of the primer in sequence in a complementary pairing mode under the action of nucleic acid polymerase, the hybrid double strand is continuously extended to form a new DNA double strand, and the terminal modification structure of the melting agent can prevent the 3' end of the specific sequence from being continuously extended under the action of the nucleic acid polymerase during the extension process. As shown in fig. 1, which is a schematic diagram of matching the ITR of the rAAV with a melting agent in one embodiment, the melting agent includes a first sequence 21, a second sequence 22, and a terminal modification structure 23.

In one embodiment, the specific sequence further comprises a third sequence complementary-paired with part or all of the sequence segment of the short stem palindrome of the T-shaped hairpin near the second side stem of the first side stem, i.e., the specific sequence further comprises a third sequence complementary-paired with part or all of the sequence segment of the second side stem 121 of the first short stem palindrome 12 (not shown in the figure), so as to increase the degree of melting of the T-shaped hairpin structure, reduce the short stem palindrome on the single strand of the adeno-associated virus DNA after melting, or eliminate the whole double-stranded region of the T-shaped hairpin structure.

In one embodiment, the terminal modification structure comprises minor groove binder (minor groove binder), inverted DNA nucleotide (inverted DNA nucleotide), C3 spacer (C3 spacer), oligonucleotide chain not hybridized with the template, and the like, which can prevent extension of the 3' end of the single-stranded nucleic acid by the nucleic acid polymerase. Specifically, in one embodiment, the minor groove binder is a chemical group that can non-covalently bind to the minor groove of double-stranded DNA, preventing extension of the 3' end of the specific sequence by a nucleic acid polymerase. In yet another embodiment, the C3 spacer, i.e., the 3-carbon spacer, prevents extension of the 3' end of the specific sequence by a nucleic acid polymerase by introducing a spacer in the melting reagent. In yet another embodiment, nucleic acid polymerase extension is prevented by placing an oligonucleotide strand at the 3' end of the specific sequence that is not complementary paired with the adeno-associated viral template.

In one embodiment, the reagent synthesis monomers include deoxyribonucleotides, ribonucleotides, and non-natural nucleotides. Wherein, non-natural nucleotide means an artificially synthesized, non-naturally occurring nucleotide, including but not limited to chemically modified nucleotides such as locked nucleotide(s), morpholino nucleotide(s), hexitol nucleotide(s), ribulose nucleotide(s), fluorinated nucleotide(s), alkyl nucleotide(s), phosphorothioate nucleotide(s), and the like. Several inter-monomer linkages include, but are not limited to, phosphodiester linkages or peptide linkages, including natural or synthetic.

In one embodiment, the total length of the first sequence and the third sequence is 4-46 nt. In one embodiment, the length of the second sequence is 4-46 nt. The total length of the first sequence and the third sequence and the length of the second sequence are limited to ensure the efficiency of melting the T-shaped hairpin double-stranded region of the ITR sequence by the melting agent and the stability of the single-stranded hybridization product with the adeno-associated virus DNA.

Example 2

The invention provides an adeno-associated virus DNA replication method, wherein the agent for destroying the T-shaped hairpin structure of the ITR sequence of adeno-associated virus is added into an adeno-associated virus DNA replication system, and the base pairing in the T-shaped hairpin structure of the single-stranded ITR sequence of the adeno-associated virus DNA is destroyed by the agent, so that the situation that the T-shaped hairpin structure of the ITR sequence obstructs the extension of a primer by nucleic acid polymerase in an extension stage is avoided, namely the obstruction effect of the T-shaped hairpin structure in a molecule on the nucleic acid polymerase is eliminated, and the replication efficiency of a nucleic acid fragment is improved.

In one embodiment, the method specifically comprises the steps of: adding the reagent into the reaction system before the adeno-associated virus DNA replication reaction begins; the initial reaction concentration of the reagent is 0.01-5 mu M.

Further, the method also comprises the following steps: the total length of the specific sequence of the reagent is varied to adjust the reagent to counteract the blocking effect of the T-hairpin structure on the nucleic acid polymerase. Specifically, the optimal length of the melting agent matched with the single-stranded template of the corresponding adeno-associated virus DNA is found by increasing or decreasing the total length of the specific sequence of the melting agent, so as to improve the degree of the T-shaped hairpin structure in the molecule of the corresponding single-stranded nucleic acid template melted by the melting agent.

When two complementary paired templates are included in the replication reaction system, two reagents respectively matched with the two complementary paired templates or one reagent matched with one template are added. When two reagents which are respectively matched with the two complementary matched templates are added, the nucleic acid replication efficiency of the two templates can be improved simultaneously; when a reagent matching a template is added, the efficiency of nucleic acid replication of the template is increased. Specifically, when two complementary DNA single-stranded templates exist in the reaction system, only one melting agent matched with one DNA single-stranded template can be added, or two melting agents respectively matched with two complementary paired template single strands can be simultaneously added, such as PCR reaction. When only one DNA single-stranded template is present in the reaction system, a compatible melting reagent is added, such as a sequencing reaction.

In one embodiment, taking a fluorescent quantitative PCR reaction as an example, the method specifically comprises the following steps:

(1) preparing required reagents, melting at room temperature, and fully and uniformly mixing;

(2) preparing a reaction solution which comprises polymerase, a mixture of four dNTPs, a forward primer, a reverse primer, a forward melting agent, a reverse melting agent, a certain commercially available rAAV carrier plasmid DNA template and PCR buffer solution;

(3) the PCR reaction was started. Wherein, the forward melting agent and the reverse melting agent are respectively used for matching two complementary single-stranded templates.

Specifically, the method comprises the following steps: taking out the required reagent in advance, melting at room temperature, and mixing well. The reaction solution was prepared according to the established reaction system: 1 XPCR buffer, 4mM Mg²⁺0.06U/μ L Taq polymerase, 0.3mM mixture of four dntps, 0.2 μ M forward primer, 0.2 μ M reverse primer, 0.1 μ M fluorescent probe, 0.2 to 5 μ M forward disturbator and the same concentration of reverse disturbator, and 3000 copies/reaction of a commercially available rAAV vector plasmid DNA. Experiments without disturbptor were used as controls. The reaction procedure for PCR was: taq polymerase was activated at 95 ℃ for 15 min, 45 cycles of denaturation at 95 ℃ for 10 sec, annealing at 54 ℃ for 30 sec and reading of fluorescence and extension at 72 ℃ for 20 sec, and cooling at 4 ℃ for 1 min. In one embodiment, fig. 3 is a DNA structure of a plasmid of a commercially available rAAV vector, and fig. 4 is a graph showing a matching relationship between a forward disturbtor and a reverse disturbtor, each of which has a total length of 36nt, and a DNA of a plasmid of a commercially available rAAV vector. The forward primer sequence is SEQ ID NO: 1; the reverse primer sequence is SEQ ID NO: 2; the fluorescent probe sequence is SEQ ID NO: 3; the forward disturbptor sequence is SEQ ID NO: 4; the reverse disturbptor sequence is SEQ ID NO: 5. the nucleotide sequence of SEQ ID NO: 1 is specifically GTAACCACGTGCGGACCGAG. The nucleotide sequence of SEQ ID NO: 2 is specifically TGTCGAGACTGCAGGCTCTAG. The nucleotide sequence of SEQ ID NO: 3 is specifically TCGAAAGCGGCCGCGACTAGT. The nucleotide sequence of SEQ ID NO: 4 is specifically GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCataa. The nucleotide sequence of SEQ ID NO: 5 is specifically GAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCCCTAaaaa. The end modification structures of the forward Disruptor and the reverse Disruptor are four bases which are not complementary with the template so as to prevent the corresponding melting agent from being extended by DNA polymerase, and the method is simple, effective and low in cost. Specific sequences of Forward and reverse DisruptorsThe total length is 36nt, the length of the first sequence and the second sequence of the corresponding specific sequence is 18nt, and the corresponding melting agent is complementarily paired with the first side stem part sequence and the first non-complementary pairing sequence part sequence section of the ITR sequence of the corresponding template.

The principle of PCR reaction is shown in FIG. 2, and a melting reagent is added to the reaction system, taking a single-stranded DNA template of a commercially available rAAV vector plasmid as an example. In the cooling and annealing stages, the primer is complementarily paired with the 3' end of a single-stranded DNA template of a certain commercially available rAAV vector plasmid; meanwhile, the melting agent competes with the T-shaped hairpin structure in the ITR sequence of the rAAV to form a hybrid product of the specific sequence and the template, and the primer continues to extend to synthesize the double-stranded product under the action of polymerase. In the process of continuing extending the primer, when different nucleic acid polymerases reach the pairing region of the Disproptor and the template, the pairing structure can be broken through different mechanisms (for example, Taq polymerase can gradually degrade the Disproptor through the 5 '-3' exonuclease activity of the Taq polymerase), and then the synthesis of a complete double-stranded product is completed. In FIG. 2, the first sequence of the melting agent is the lower part of the first side stem, the second sequence is the part of the first non-complementary counterpart sequence near the first side stem, and after the melting agent hybridizes with the DNA single-stranded template, two short stem palindromes, namely a first short stem palindrome 12 and a second short stem palindrome 13, remain, and the thermal stability of the two short stem palindromes is not enough to prevent further extension of the nucleic acid polymerase.

Example 3

The present invention provides a reagent for improving replication efficiency of an ITR sequence of an adeno-associated virus as described above or an application of the DNA replication method of an adeno-associated virus as described above to a PCR reaction or a sequencing reaction using an adeno-associated virus as a template. The melting agent or the adeno-associated virus DNA replication method can be applied to a molecular biological method for synthesizing a novel nucleic acid molecule by the nucleic acid polymerase, and the T-shaped hairpin structure in the adeno-associated virus DNA single-stranded template is melted by the melting agent, so that the blocking effect of the T-shaped hairpin structure with high thermal stability on the nucleic acid polymerase in the extension process is eliminated.

In order to illustrate the invention herein, specific examples are set forth below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any way.

Example 4

The addition of a melting agent can improve the PCR reaction efficiency of amplifying the ITR (rAAV-ITR) sequence of the rAAV vector plasmid

(1) Primers, probes and a Dispeptor for fluorescent quantitative PCR reaction are designed according to T-shaped hairpin structures predicted by MFold software of ITR sequences of certain commercially available rAAV vector plasmids. FIG. 4 is a diagram showing the match relationship between ITRs in two single strands of a commercially available rAAV vector plasmid and their peripheral sequences, as predicted by MFold software, and T-shaped hairpin structures, forward melting agent and reverse melting agent. The solid line marks the complementary pairing sequences of the forward and reverse primers; the complementary pairing sequence of the fluorescent probe is marked by a dotted line; grey fonts in the template mark sequences in the corresponding template sequences that are complementary paired to the corresponding melting agent.

Specifically, the sequence of each reactant in the fluorescent quantitative PCR reaction is as follows: the sequence of the forward primer is SEQ ID NO: 1; the sequence of the reverse primer is SEQ ID NO: 2; the fluorescent probe is characterized in that the 5 'end is marked with FAM fluorescent group, the 3' end is marked with BHQ1 quenching group, and the sequence is SEQ ID NO: 3; the sequence of forward dispatcher is SEQ ID NO: 4; the sequence of the reverse disturbptor is SEQ ID NO: 5. in the implementation case, the 3' end of the Disproptor adds four bases which are not complementary to the target sequence to prevent it from being extended by DNA polymerase, and this method is simple, economical and effective. The total length of the specific sequences of the forward and reverse dispriptors is 36nt, and the corresponding melting reagent is complementary-paired with the first side stem part sequence and the first non-complementary-pairing sequence part sequence of the ITR sequence of the corresponding template.

(2) Fluorescent quantitative PCR reaction: taking out the required reagent in advance, melting at room temperature, and mixing well. The reaction solution was prepared according to the established reaction system: 1 XPCR buffer, 4mM Mg²⁺0.06U/. mu.L Taq polymerase, 0.3mM four dNTP mix, 0.2. mu.M forward primer (SEQ ID NO: 1), 0.2. mu.M reverse primer (SEQ ID NO: 2), 0.1. mu.M fluorescent probe (SEQ ID NO: 3), forward Disproptor (SEQ ID NO: 4) and reverse Disproptor (SEQ ID NO: 5) at the same concentration, and 3000 copies/reaction of a commercially available rAAV vector plasmid DNA. Six experimental groups are respectively configured, and the initial reaction concentration of the forward Disproptor in the six experimental groups is 0.2 mu M, 0.5 mu M, 1 mu M, 2 mu M, 3 mu M and 5 mu M respectively. An additional experiment without Disproptor was used as a control. The reaction procedure for PCR was: taq polymerase was activated at 95 ℃ for 15 min, 45 cycles of denaturation at 95 ℃ for 10 sec, annealing at 54 ℃ for 30 sec and reading of fluorescence and extension at 72 ℃ for 20 sec, and cooling at 4 ℃ for 1 min.

(3) Electrophoretic analysis of fluorescent quantitative PCR products: mu.L of the PCR product and 1.5. mu.L of the loading buffer were mixed and electrophoresed on a 3% agarose gel at 90V for 50 minutes.

(4) FIG. 5 shows the results of fluorescent quantitative PCR reaction in which ITR sequences of commercially available rAAV vector plasmids were amplified using a pair of dispriptors having specific sequences of 36nt and different concentrations as templates, when added to the reaction system. FIG. 6 shows the results of 3% agarose gel electrophoresis of the final fluorescent quantitative PCR product of the experiment of FIG. 5. When the concentration of the Disproptor is 5 mu M, because the concentration of the oligonucleotides in the reaction system is too high, dimers are formed among the oligonucleotides, and a certain inhibition effect is generated on the PCR reaction. As shown in FIGS. 5 and 6, the PCR amplification reaction of ITR sequences of certain commercially available rAAV vector plasmid has poor PCR reaction effect under the condition without the PCR primer, the Ct value of the quantitative PCR result is high and reaches 42.3, and a product band cannot be observed in a gel electrophoresis experiment. When different concentrations of the same pair of specific sequences of the Disproptor with the length of 36nt are added into the reaction system, the Ct value detected by the PCR reaction is gradually reduced to 33.9 with the gradual increase of the concentration of the Disproptor to 3 mu M, and the brightness of the product band in the gel electrophoresis experiment is gradually enhanced. However, when the concentration of the Disproptor reaches 5 μ M, the Ct value is increased to 36.8 instead of the experimental condition of 3 μ M concentration, and the brightness of the product band in the gel electrophoresis experiment is also darkened. This result indicates that the higher the concentration of the irraptor added, the better the detection effect of the PCR reaction for the T-hairpin structure of the ITR sequence. However, when the concentration of the PCR product is too high, the PCR reaction is inhibited by the formation of oligonucleotide dimers due to the too high total concentration of the oligonucleotides in the reaction system. Therefore, when the PCR reaction of rAAV-ITR is inhibited by the Disruptor elimination T-shaped hairpin structure, the added Disruptor has an optimal concentration interval, and the effect is not better when the concentration of the Disruptor is higher.

Example 5

Effect of different length of melting Agents on PCR reaction of rAAV-ITR

(1) A group of dispriptors with different lengths is designed according to a T-shaped hairpin structure of a certain commercially available rAAV vector plasmid predicted by MFold software, and in this embodiment, a melting agent with a specific sequence including a first sequence and a second sequence is taken as an example, wherein the specific sequence of each dispriptor consists of two parts with equal length, namely the first sequence and the second sequence of the melting agent are equal in length. FIG. 4 is a diagram showing the matching relationship between the forward melting agent and the reverse melting agent, which are 18nt long in the first sequence and the second sequence (i.e., the length of the corresponding disproptor is 18+18), and the T-shaped hairpin structure of ITRs and their surrounding sequences in two single strands of a commercially available rAAV vector plasmid, which are predicted by the MFold software, and the forward melting agent and the reverse melting agent.

Specifically, in one embodiment, the first and second sequences of forward and reverse disproptor F1 and R1 are each 10nt in length (i.e., the corresponding disproptor is 10+10 in length) as the pair of melting agents of experimental group 1. Forward disturbptor F1 sequence is SEQ ID NO: 6; reverse distraptor R1 sequence is SEQ ID NO: 11.

SEQ ID NO: 6 is specifically GAGCGCGCAGAGAGGGAGTGaaat;

SEQ ID NO: 11 is specifically GAGCGCGCAGCTGCCTGCAGaaaa.

In yet another example, the forward Disruptor F2 and the reverse Disruptor R2 each have a length of 12nt (i.e., the corresponding Disruptor has a length of 12+12) as the pair of melting agents of Experimental group 2. Forward disturbptor F2 sequence is SEQ ID NO: 7; reverse distraptor R2 sequence is SEQ ID NO: 12.

SEQ ID NO: 7 is specifically GCGAGCGCGCAGAGAGGGAGTGGCatta;

SEQ ID NO: 12 is specifically GCGAGCGCGCAGCTGCCTGCAGGGaaaa.

In yet another example, the forward Disruptor F3 and the reverse Disruptor R3 each have a length of 14nt (i.e., the corresponding Disruptor has a length of 14+14) as the pair of melting agents of experiment group 3. Forward disturbptor F3 sequence is SEQ ID NO: 8; reverse distraptor R3 sequence is SEQ ID NO: 13.

SEQ ID NO: 8 is specifically GAGCGAGCGCGCAGAGAGGGAGTGGCCAtaaa;

SEQ ID NO: 13 is specifically GAGCGAGCGCGCAGCTGCCTGCAGGGGCaaat.

In yet another example, the forward Disruptor F4 and the reverse Disruptor R4 each have a length of 16nt for the first sequence and the second sequence (i.e., 16+16 for the corresponding Disruptor) as the pair of melting agents of Experimental group 4. Forward disturbptor F4 sequence is SEQ ID NO: 9; reverse distraptor R4 sequence is SEQ ID NO: 14.

SEQ ID NO: 9 is specifically GCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACaaat;

SEQ ID NO: 14 is specifically GCGAGCGAGCGCGCAGCTGCCTGCAGGGGCCCataa.

In yet another example, the forward Disruptor F5 and the reverse Disruptor R5 each have a length of 18nt (i.e., the corresponding Disruptor has a length of 18+18) as the pair of melting agents of Experimental group 5. Forward disturbptor F5 sequence is SEQ ID NO: 4; reverse distraptor R5 sequence is SEQ ID NO: 5.

SEQ ID NO: 4 is specifically GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCataa;

SEQ ID NO: 5 is specifically GAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCCCTAaaaa.

In yet another example, the forward Disruptor F6 and the reverse Disruptor R6 each have a length of 20nt (i.e., the corresponding Disruptor has a length of 20+20) as a pair of melting agents of Experimental group 6. Forward disturbptor F6 sequence is SEQ ID NO: 10; reverse distraptor R6 sequence is SEQ ID NO: 15.

SEQ ID NO: 10 is specifically GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCAaata; SEQ ID NO: 15 is specifically GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCCCTACGaaat.

In the above examples, the 3' end of each irruptor is augmented with four bases that do not complementarily pair with the target sequence to prevent extension by DNA polymerase, which is simple, cost effective and efficient.

(2) Fluorescent quantitative PCR reaction: taking out the required reagent in advance, melting at room temperature, and mixing well. The reaction solution was prepared according to the established reaction system: 1 XPCR buffer, 4mM Mg²⁺0.06U/. mu.L Taq polymerase, 0.3mM four dNTP mix, 0.2. mu.M forward primer (SEQ ID NO: 1), 0.2. mu.M reverse primer (SEQ ID NO: 2), 0.1. mu.M fluorescent probe (SEQ ID NO: 3); forward Disruptor (SEQ ID NO: 4 and one of SEQ ID NO: 6 to 10) and matched reverse Disruptor were each 2. mu.M to form pairs of melting agents for six groups, experiment 1 to experiment 6, respectively; and 3000 copies/reaction of a commercially available rAAV vector plasmid DNA. Experiments without disturbptor were used as controls. The reaction procedure for PCR was: taq polymerase was activated at 95 ℃ for 15 min, 45 cycles of denaturation at 95 ℃ for 10 sec, annealing at 54 ℃ for 30 sec and reading of fluorescence and extension at 72 ℃ for 20 sec, and cooling at 4 ℃ for 1 min.

(4) FIG. 7 is a comparison of the result of rAAV-ITR fluorescent quantitative PCR experiment with 2. mu.M of Disproptor pairs of different lengths added to the reaction system and the result of experiment without Disproptor. FIG. 8 shows the results of 3% agarose gel electrophoresis of the final fluorescent quantitative PCR product of the experiment of FIG. 7. Under the reaction condition without the PCR, the PCR reaction effect is poor, the Ct value of the quantitative PCR result is high and reaches 43.6, and a product band cannot be observed in a gel electrophoresis experiment. When 2 mu M of the irriptor with the length of the first sequence and the second sequence both being 10nt (namely the length of the irriptor is 10+10 correspondingly) is added into the reaction system, the PCR reaction effect is not obviously improved, the Ct value of the quantitative PCR result is only reduced to 43.0, and a product band can not be observed in a gel electrophoresis experiment. However, as the length of the Disproptor pair is further increased, the Ct value detected by the PCR reaction is greatly reduced, and the brightness of the product band observed in the gel electrophoresis experiment is gradually increased. When the pair of melting agents, which are respectively 18nt long in the forward direction disproptor and 18+18 long in the reverse direction disproptor and 20nt long in the second sequence (namely 20+20 long in the corresponding disproptor), is added into the reaction system, the Ct value of the quantitative PCR result is sequentially reduced to 34.8 and 34.5, which shows a trend towards stability, and the brightness of the observed product band in the gel electrophoresis experiment is basically kept unchanged. This result indicates that, for the pair of melting reagents having the first sequence with the same length as the second sequence, it is not as long as possible, and under the experimental conditions, the effect of increasing the efficiency of rAAV-ITR PCR reaction is substantially maximized when the lengths of the first sequence and the second sequence in the forward direction Disraptor and the reverse direction Disraptor are both 20nt (i.e., the length of the corresponding Disraptor is 20+ 20).

Example 6

Effect of varying only the second sequence length of the melting reagent on the PCR reaction of rAAV-ITRs

(1) A group of dispriptors with different lengths are designed according to a T-shaped hairpin structure of a certain commercial rAAV vector plasmid predicted by MFold software. Each irraptor sequence consists of two parts: the first sequence has a fixed length of 20nt, and the second sequence has a variable length. FIG. 9 is a diagram showing the matching relationship between the forward melting agent and the reverse melting agent, which is predicted by MFold software, for ITRs in two single strands of a commercially available rAAV vector plasmid and their surrounding sequences, with the forward melting agent and the reverse melting agent, wherein the first sequence length is 20nt and the second sequence length is 12 nt.

Specifically, in one embodiment, the first sequences of forward Disruptor F7 and reverse Disruptor R7 are both 20nt, and the second sequences of forward Disruptor F7 and reverse Disruptor R7 are both 4nt in length (i.e., the corresponding Disruptor is 20+4 in length), as the pair of melting agents of experiment group 7. Forward disturbptor F7 sequence is SEQ ID NO: 16; reverse distraptor R7 sequence is SEQ ID NO: 25.

SEQ ID NO: 16 is specifically GTGAGCGAGCGAGCGCGCAGAGAGtaaa;

SEQ ID NO: 25 is specifically GTGAGCGAGCGAGCGCGCAGCTGCaaaa.

In yet another example, the first sequences in the forward Disruptor F8 and the reverse Disruptor R8 are both 20nt, and the second sequences in the forward Disruptor F8 and the reverse Disruptor R8 are both 6nt in length (i.e., the corresponding Disruptors are 20+6 in length), as pairs of unzipping agents for the experimental group 8. Forward disturbptor F8 sequence is SEQ ID NO: 17; reverse distraptor R8 sequence is SEQ ID NO: 26.

SEQ ID NO: 17 is specifically GTGAGCGAGCGAGCGCGCAGAGAGGGtaaa;

SEQ ID NO: 26 is specifically GTGAGCGAGCGAGCGCGCAGCTGCCTaata.

In yet another example, the first sequences in the forward Disruptor F9 and the reverse Disruptor R9 are both 20nt, and the second sequences in the forward Disruptor F9 and the reverse Disruptor R9 are both 8nt in length (i.e., the corresponding Disruptors are 20+8 in length), as pairs of unzipping agents for the experimental group 9. Forward disturbptor F9 sequence is SEQ ID NO: 18; reverse distraptor R9 sequence is SEQ ID NO: 27.

SEQ ID NO: 18 is specifically GTGAGCGAGCGAGCGCGCAGAGAGGGAGaata;

SEQ ID NO: 27 is specifically GTGAGCGAGCGAGCGCGCAGCTGCCTGCtaaa.

In yet another example, the first sequences in the forward Disruptor F10 and the reverse Disruptor R10 are both 20nt, and the second sequences in the forward Disruptor F10 and the reverse Disruptor R10 are both 10nt in length (i.e., the corresponding Disruptors are 20+10 in length), as pairs of unzipping agents for the experimental group 10. Forward disturbptor F10 sequence is SEQ ID NO: 19; reverse distraptor R10 sequence is SEQ ID NO: 28.

SEQ ID NO: 19 is in particular GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGaata;

SEQ ID NO: 28 is specifically GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGaaaa.

In yet another example, the first sequences in the forward Disruptor F11 and the reverse Disruptor R11 are both 20nt, and the second sequences in the forward Disruptor F11 and the reverse Disruptor R11 are both 11nt in length (i.e., the corresponding Disruptors are 20+11 in length), as pairs of unzipping agents for the experimental group 11. Forward disturbptor F11 sequence is SEQ ID NO: 20; reverse distraptor R11 sequence is SEQ ID NO: 29.

SEQ ID NO: 20 is specifically GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGaatt;

SEQ ID NO: 29 is specifically GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGaaaa.

In yet another example, the first sequences in the forward Disruptor F12 and the reverse Disruptor R12 are both 20nt, and the second sequences in the forward Disruptor F12 and the reverse Disruptor R12 are both 12nt in length (i.e., the corresponding Disruptors are 20+12 in length), as pairs of unzipping agents for the experimental group 12. Forward disturbptor F12 sequence is SEQ ID NO: 21; reverse distraptor R12 sequence is SEQ ID NO: 30.

SEQ ID NO: 21 is specifically GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCatta;

SEQ ID NO: 30 is specifically GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGaaaa.

In yet another example, the first sequences in the forward Disruptor F13 and the reverse Disruptor R13 are both 20nt, and the second sequences in the forward Disruptor F13 and the reverse Disruptor R13 are both 14nt in length (i.e., the corresponding Disruptors are 20+14 in length), as pairs of unzipping agents for experimental group 13. Forward disturbptor F13 sequence is SEQ ID NO: 22; reverse distraptor R13 sequence is SEQ ID NO: 31.

SEQ ID NO: 22 is specifically GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAtaaa;

SEQ ID NO: 31 is specifically GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCaaat.

In yet another example, the first sequences in both forward Disruptor F14 and reverse Disruptor R14 are 20nt in length, and the second sequences in both forward Disruptor F14 and reverse Disruptor R14 are 16nt in length (i.e., 20+16 in length), as pairs of unzipping agents for experimental group 14. Forward disturbptor F14 sequence is SEQ ID NO: 23; reverse distraptor R14 sequence is SEQ ID NO: 32.

SEQ ID NO: 23 is specifically GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACaaat;

SEQ ID NO: 32 is specifically GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCCCataa.

In yet another example, the first sequences in the forward Disruptor F15 and the reverse Disruptor R15 are both 20nt, and the second sequences in the forward Disruptor F15 and the reverse Disruptor R15 are both 18nt in length (i.e., the corresponding Disruptors are 20+18 in length), as pairs of unzipping agents for the experimental group 15. Forward disturbptor F15 sequence is SEQ ID NO: 24; reverse distraptor R15 sequence is SEQ ID NO: 33.

SEQ ID NO: 24 is specifically GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCataa;

SEQ ID NO: 33 are specifically GTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCCCTAaaaa.

(2) Fluorescent quantitative PCR reaction: taking out the required reagent in advance, melting at room temperature, and mixing well. The reaction solution was prepared according to the established reaction system: 1 XPCR buffer, 4mM Mg²⁺0.06U/. mu.L Taq polymerase, 0.3mM four dNTP mix, 0.2. mu.M forward primer (SEQ ID NO: 1), 0.2. mu.M reverse primer (SEQ ID NO: 2), 0.1. mu.M fluorescent probe (SEQ ID NO: 3), forward Disproptor (SEQ ID NO: 10 and one of SEQ ID NO: 16 to 24) and matched reverse Disproptor each 2. mu.M to form ten sets of melting agent pairs for experiment set 6 to experiment set 15, respectively; and 3000 copies/reaction of a commercially available rAAV vector plasmid DNA. An additional experiment without Disproptor was used as a control. The reaction procedure for PCR was: taq polymerase was activated at 95 ℃ for 15 min, 45 cycles of denaturation at 95 ℃ for 10 sec, annealing at 54 ℃ for 30 sec and reading of fluorescence and extension at 72 ℃ for 20 sec, and cooling at 4 ℃ for 1 min.

(4) FIG. 10 is a comparison of the rAAV-ITR fluorescent quantitative PCR experiment result with the experiment result without the Disruptor when the ten experiment groups of different melting agent pairs are added into the reaction system. FIG. 11 shows the results of a 3% agarose gel electrophoresis experiment performed on the final fluorescent quantitative PCR product of the experiment of FIG. 10. Under the reaction condition without the PCR, the PCR reaction effect is poor, amplification signals are not detected in quantitative PCR reaction, and a product band is not observed in a gel electrophoresis experiment. When 2. mu.M of the melting agent pair of experiment 7 and 2. mu.M of the melting agent pair of experiment 8 were added to the reaction system, the Ct values of the quantitative PCR results were 43.5 and 43.9, respectively, and no product band was observed in the gel electrophoresis experiment. When 2 μ M of the melting agent pairs of experiment group 9, experiment group 10 and experiment group 11 were added to the reaction system, the PCR reaction effect was not significantly improved: quantitative PCR results Ct values only dropped to about 42.0, 42.3 and 41.0, and only very low intensity product bands were observed in gel electrophoresis experiments. However, when 2 μ M of the experimental group 12 melting agent pair was added to the reaction system, the PCR reaction effect was significantly improved: the Ct value of the quantitative PCR result is greatly reduced to 36.8, and the brightness of a product band observed in a gel electrophoresis experiment is also obviously enhanced. As the length of the second sequence of the Disproptor is further increased, the Ct value detected by the PCR reaction is further reduced, and the brightness of the product band observed in the gel electrophoresis experiment is further enhanced. When the experimental group 14, the experimental group 15 and the experimental group 6 melting reagent pairs were added to the reaction system, the Ct values of the quantitative PCR results were sequentially decreased to 34.6, 34.6 and 34.9, and the brightness of the product bands observed in the gel electrophoresis experiment was also substantially maintained. The result shows that the length of the second sequence in the melting agent is not longer and better, under the experimental condition, the effect of the melting agent on improving the efficiency of rAAV-ITR PCR reaction is maximized when the length of the second sequence of each melting agent in the melting agent pair is 16nt, and the capability of improving the efficiency of rAAV-ITR PCR reaction cannot be further improved by further lengthening the length of the second sequence.

The sequences involved in the present invention are shown in table one.

Watch 1

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; those skilled in the art can readily practice the invention as shown and described in the drawings and detailed description herein; however, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the scope of the invention as defined by the appended claims; meanwhile, any changes, modifications, and evolutions of the equivalent changes of the above embodiments according to the actual techniques of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. An agent for increasing the replication efficiency of an inverted terminal repeat of an adeno-associated virus, wherein the agent is an oligonucleotide chain comprising in sequence:

a specific sequence comprising at least a first sequence for complementary pairing with part or all of a sequence segment of a first side stem near the 5' end of a long stem palindrome of a T-shaped hairpin of ITR sequences, and a second sequence for complementary pairing with part of a first non-complementary pairing sequence segment adjacent to the first side stem of the T-shaped hairpin; the first sequence is adjacent to the second sequence;

2. The agent according to claim 1, wherein the specific sequence further comprises a third sequence complementary-paired to a part or all of the sequence segment of the short stem palindrome of the T-shaped hairpin adjacent to the second side stem of the first side stem.

3. The reagent of claim 1, wherein the terminal modification structure comprises a minor groove binder, an inverted DNA nucleotide, a C3 spacer, and an oligonucleotide chain that does not hybridize to the adeno-associated virus template.

4. The agent for increasing the replication efficiency of an inverted terminal repeat of an adeno-associated virus according to claim 1, wherein the synthetic monomers of the agent comprise deoxyribonucleotides, ribonucleotides, non-natural nucleotides; several modes of linkage between monomers include phosphodiester linkage or peptide linkage.

5. The agent for increasing the replication efficiency of an adeno-associated virus inverted terminal repeat according to claim 2, wherein the total length of the first sequence and the third sequence is 4 to 46 nt; the length of the second sequence is 4-46 nt.

6. A method for replicating adeno-associated virus DNA, wherein the agent according to any one of claims 1 to 5 for improving the replication efficiency of the inverted terminal repeat of adeno-associated virus DNA is added to the adeno-associated virus DNA replication system, and the agent destroys base pairing in a T-shaped hairpin structure formed by single-stranded ITR sequences of the adeno-associated virus DNA, so as to reduce the inhibition effect of the T-shaped hairpin structure on nucleic acid polymerase.

7. The method of claim 6, comprising the steps of: adding the reagent into the reaction system before the adeno-associated virus DNA replication reaction begins; the initial reaction concentration of the reagent is 0.01-5 mu M.

8. The method of claim 6, wherein when two complementary paired templates are included in the replication reaction system, two reagents matching the two complementary paired templates respectively or one reagent matching one of the templates are added.

9. The method of claim 6, further comprising the steps of: the total length of the specific sequence of the reagent is varied to adjust the reagent to counteract the blocking effect of the T-hairpin structure on the nucleic acid polymerase.

10. Use of the reagent according to any one of claims 1 to 5 for increasing the efficiency of replication of an inverted terminal repeat of an adeno-associated virus or the method for DNA replication of an adeno-associated virus according to any one of claims 6 to 9 in a PCR reaction or a sequencing reaction using an adeno-associated virus as a template.