Method for detecting integrity of a DNA template

Abstract

This invention discloses a method for detecting DNA template integrity. The method can compare template integrity under different conditions, establish batch-to-batch consistency standards, and has high sensitivity. This method does not rely on visual judgment of whether the main band is clear, or whether there are abnormal migration rates, tailing, or diffusion to detect template integrity. Compared with existing agarose gel electrophoresis methods, this method effectively avoids the problems of strong subjectivity and lack of quantification.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for detecting the integrity of a DNA template. Background Technology

[0002] In vitro transcription (IVT) is one of the core technologies for producing messenger RNA (mRNA) and has wide applications in mRNA vaccines, gene therapy, and recombinant protein expression. IVT utilizes bacteriophage RNA polymerases (e.g., T7, T3, or SP6) to efficiently synthesize target mRNA in vitro using linearized DNA as a template. The IVT DNA template refers to a linear double-stranded DNA molecule containing a specific bacteriophage promoter sequence (e.g., T7, T3, or SP6) and the target gene sequence. It serves as the direct substrate for RNA polymerases to synthesize specific RNAs (e.g., mRNA, gRNA) in a cell-free system in vitro, and is generally linearized and double-stranded. During IVT, the quality of the IVT DNA template, especially its integrity, directly determines the efficiency of the IVT reaction, the uniformity and functionality of the mRNA product, and thus affects the quality and safety of the final product.

[0003] In mRNA preparation, IVT template integrity is primarily assessed using tools such as agarose gel electrophoresis, capillary electrophoresis, and fragment analysis software. Agarose gel electrophoresis is the most basic and intuitive method, comparing the sample with a marker to observe whether the target band is singular, clear, and in the expected position. The presence of diffuse, tailed, or redundant bands indicates template degradation or impurities. The overall integrity of the plasmid can be determined by observing the proportion of supercoiled plasmids. Capillary electrophoresis (e.g., LabChip, Fragment Analyzer) provides digital quantitative results, accurately calculating the target peak proportion, degradation level, dimerization, and average fragment size; this method is more sensitive and objective than gel electrophoresis. Fragment analysis software focuses on target fragment proportion (Purity) and degradation index, allowing the setting of thresholds (e.g., target proportion ≥90%, degradation index ≥0.8) to determine template suitability.

[0004] However, these existing technologies have the following drawbacks: plasmid templates used for IVT generally contain polyA sequences, the presence of which makes it easy for plasmids to generate multimers, and the proportion of supercoiled molecules is not easy to observe; incomplete IVT DNA templates are manifested as gaps, and such DNA molecules do not exhibit tailing, so electrophoresis-based quality control techniques cannot detect them; capillary electrophoresis and gel electrophoresis also have several consistent limitations, including: sensitivity to molecular conformation, sensitivity to sample purity, and limited resolution upper limit; none of these methods can accurately quantify the integrity of IVT DNA templates.

[0005] Therefore, there is an urgent need in this field for a method that can accurately quantify the integrity of IVT DNA templates. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a method for detecting the integrity of a DNA template.

[0007] This invention provides a method for detecting the integrity of a DNA template, the method comprising the following steps: 1) Provide a DNA template to be tested, and perform self-circularization ligation on the DNA template to obtain a first circularized molecule; 2) Provide a control DNA molecule, wherein the control DNA molecule has no gap; the control DNA molecule is circular, serving as a second circularized molecule; or the control DNA molecule is a linear molecule, and the linear molecule is self-circularized to obtain a second circularized molecule; 3) Take equal amounts of the first cyclized molecule and the second cyclized molecule and perform rolling ring replication under the same conditions to obtain the first rolling ring replication product and the second rolling ring replication product. 4) Take the first and second rolling circle replication products obtained in step 3) and perform gel electrophoresis analysis on them respectively, and detect the signal intensity of the first and second rolling circle replication products. 5) Calculate the ratio of the signal intensity of the first rolling circle replication product to the signal intensity of the second rolling circle replication product to determine the integrity of the DNA template to be tested.

[0008] This invention provides a method for detecting DNA template integrity. The method can compare template integrity under different conditions, establish batch-to-batch consistency standards, and has high sensitivity. This method does not rely on visual judgment of whether the main band is clear, has tailing, or is diffuse to detect template integrity. Compared with existing agarose gel electrophoresis methods, this method effectively avoids the problems of high subjectivity and lack of quantification. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the specific embodiments of the present invention, the accompanying drawings in the specific embodiments will be briefly described below.

[0010] Figure 1 This demonstrates that the mRNA product of IVT is affected by the gaps in the IVT DNA template.

[0011] Figure 2 A method for circularizing IVT DNA sequences in plasmids is shown.

[0012] Figure 3 The effects of different IVT DNA templates on rolling circle replication products are shown.

[0013] Figure 4 The gel electrophoresis results of different plasmids extracted from the same batch are shown. In the left image, the white arrow points to the supercoiled ordinary plasmid; in the right image, the white arrow points to the aggregates of plasmids containing the polyA sequence.

[0014] Figure 5 Electrophoresis results of plasmids and linear PCR products before and after nicking with restriction enzymes are shown.

[0015] Figure 6 The amount of RCA product of the cyclic molecule is shown without the use of nicking endonuclease and with different concentrations of nicking endonuclease.

[0016] Figure 7 The amounts of standard products and RCA products of plasmids 1-5 are shown.

[0017] Figure 8 The integrity of mRNA prepared from plasmids with more and fewer defects is shown. Detailed Implementation

[0018] The present invention will be described in detail below. It should be understood that the following description is merely illustrative and is not intended to limit the scope of the invention; the scope of protection of the invention is defined by the appended claims. Furthermore, those skilled in the art will understand that modifications can be made to the technical solutions of the present invention without departing from its spirit and intent. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0020] In the context of this invention, many embodiments use the expressions "comprising," "including," or "basically / mainly composed of." The expressions "comprising," "including," or "basically / mainly composed of" should be understood as open-ended expressions, indicating that they include not only the elements, components, parts, and method steps specifically listed after the expression, but also other elements, components, parts, and method steps. Additionally, in this document, the expressions "comprising," "including," or "basically / mainly composed of" may also be understood as closed-ended expressions in certain circumstances, indicating that they only include the elements, components, parts, and method steps specifically listed after the expression, and do not include any other elements, components, parts, or method steps. In this case, the expression is equivalent to the expression "composed of."

[0021] In this paper, ordinal numbers such as "first," "second," and "third" are sometimes used to modify or limit elements, components, parts, method steps, solutions, solvents, temperatures, systems, etc. It should be noted that in this paper, these expressions are only used to distinguish the elements, components, parts, method steps, solutions, solvents, temperatures, systems, etc. that they modify or limit, and are not intended to limit their order or importance.

[0022] In this paper, the term "in vitro transcription (IVT)" refers to the technique of synthesizing RNA using DNA as a template by using RNA polymerase to simulate the in vivo transcription process in vitro.

[0023] In this article, the term "Rolling Circle Amplification (RCA)" refers to a nucleic acid detection method that mimics the rolling circle replication process of circular DNA in natural microorganisms. Its principle is to use circular DNA / RNA as a template, and through a short primer, using dNTPs / NTPs as raw materials, amplify to produce a long repeating single-stranded DNA / RNA under the action of DNA / RNA polymerase. Alternatively, a nicking endonuclease can be used to specifically cleave one strand of the DNA double helix, utilizing the nick to perform the RCA reaction without primers.

[0024] Figure 1 This demonstrates that the IVT mRNA product is affected by gaps in the IVT DNA template. For example... Figure 1As shown, when the IVT DNA template has no nicks, it can be transcribed into a complete mRNA product (top image); when the IVT DNA template has nicks, transcription terminates prematurely, resulting in an incomplete mRNA product (bottom image). Because nicks in the IVT DNA template are random, they lead to interrupted products of varying lengths, which are difficult to detect using electrophoresis-based analytical methods.

[0025] Therefore, the present invention proposes a method for detecting the integrity of a DNA template, the method comprising the following steps: 1) Provide a DNA template to be tested and perform self-circularization ligation on the DNA template to obtain a first circularized molecule, wherein the DNA template to be tested may or may not have a gap; 2) Provide a control DNA molecule, wherein the control DNA molecule has no gap; the control DNA molecule is circular, serving as a second circularized molecule; or the control DNA molecule is a linear molecule, and the linear molecule is self-circularized to obtain a second circularized molecule; 3) Take equal amounts of the first cyclized molecule and the second cyclized molecule and perform rolling ring replication under the same conditions to obtain the first rolling ring replication product and the second rolling ring replication product. 4) Take the first and second rolling circle replication products obtained in step 3) and perform gel electrophoresis analysis on them respectively, and detect the signal intensity of the first and second rolling circle replication products. 5) Calculate the ratio of the signal intensity of the first rolling circle replication product to the signal intensity of the second rolling circle replication product to determine the integrity of the DNA template to be tested.

[0026] In one embodiment, the method provided by the present invention can be used to detect the integrity of in vitro transcribed DNA templates, but the application scenarios of the above method are not limited to this. For example, linear DNA expression cassettes used for treatment or immunization must be highly intact; gaps may lead to interruption of therapeutic gene expression and the production of truncated proteins (which may induce immunogenicity). The above method can be used to quantitatively detect the integrity of gene therapy DNA molecules.

[0027] In this paper, the term "gap" refers to a break in the sugar-phosphate backbone of a double-stranded DNA molecule where two adjacent nucleotides lack a phosphodiester bond. The formation of a gap does not involve the loss of nucleotides; the bases on either side of the break still bind to the other strand through complementary pairing, thus maintaining the overall continuity of the DNA double helix and preventing it from dissociating into two fragments.

[0028] Regarding step 1), in practice, the DNA template to be tested can be obtained directly, and then circularization can be performed directly using the DNA template to be tested. The DNA template to be tested refers to a linear double-stranded DNA molecule containing a specific bacteriophage promoter sequence and a target gene sequence.

[0029] In one optional implementation, the experimenter needs to cut the test DNA template from the plasmid DNA. The test DNA template is present in the plasmid and has a first restriction enzyme site and a second restriction enzyme site upstream and downstream thereon, thereby allowing the test DNA template to be obtained by digesting the first restriction enzyme site and the second restriction enzyme site.

[0030] Regarding step 2), in practice, a no-gap linear control DNA molecule (also referred to as a standard molecule or standard product in this paper) can be directly obtained, and self-circularization ligation can be performed to obtain a second circularized molecule.

[0031] In an optional embodiment, the experimenter can use a test DNA template from plasmid DNA amplified by PCR as a control DNA molecule. In a further embodiment, the control DNA molecule has a third restriction enzyme site and a fourth restriction enzyme site upstream and downstream, thereby allowing the control DNA molecule to be obtained by digesting the third and fourth restriction enzyme sites.

[0032] In this paper, the term "self-circularization ligation" refers to the process by which a linear DNA molecule forms a covalently closed circular DNA molecule by directly ligating its two ends together. Experimentally, the DNA concentration in the reaction system can be controlled at a low level to promote intramolecular ligation rather than intermolecular ligation at the ends of the same molecule. Furthermore, additional restriction endonuclease sites can be selected at sites outside the target DNA template of the plasmid, and the corresponding enzymes can be used for cleavage, resulting in the DNA in the non-target DNA template region being cut into small fragments (e.g., less than 100 bp). Since intramolecular ligation efficiency is related to fragment length (small fragments have extremely low self-circularization efficiency and may not form a stable circular structure), this prevents them from self-circulating. Further, the small fragments cut from the non-target DNA template region can be designed with different sticky ends to avoid self-circularization of these small fragments. Ultimately, only the fragment containing the target DNA template region can efficiently form a circular product by ligating its two ends together, reducing background interference and improving detection specificity. "Self-circularization ligation" can be achieved through blunt ends or sticky ends of the DNA molecule.

[0033] In this paper, sticky ends refer to a 2-4 nucleotide single-stranded protrusion that appears at the nick after DNA double-strands are cleaved by restriction endonucleases. This protruding sequence can transiently anneal to its complementary end through base pairing, forming a stable local double-stranded structure. Since one or two nicks still exist after annealing, ligation can be completed simply by DNA ligases (such as T4 DNA ligase and T7 DNA ligase) catalyzing the formation of phosphodiester bonds.

[0034] In this article, blunt ends refer to the cleavage of two strands by restriction endonucleases at the same base position without producing any single-stranded protrusions. There is no pre-annealed complementary structure between two blunt-end fragments, and the formation of a phosphodiester bond between the two adjacent ends can only be achieved by direct catalysis of DNA ligases (such as T4 DNA ligase).

[0035] The cyclization methods in steps 1) and 2) of this method are as follows: Figure 2 As shown in the figure. The top figure shows how to prepare cyclized molecules containing the template sequence to be detected, while the bottom figure shows how to prepare standard unnotched cyclized molecules.

[0036] The figure above shows the cutting of the DNA template to be tested from plasmid DNA. IVT DNA (sequence a) can be linearized through the restriction sites at both ends, and then circularized to obtain the first circularized molecule.

[0037] The figure below illustrates how IVT DNA (sequence a) can be amplified by PCR to produce a gap-free control DNA molecule using the test DNA template on the plasmid DNA. Enzyme restriction sites are introduced upstream and downstream of the control DNA molecule, and the molecule is digested with the corresponding enzymes. The digested fragments are then isolated, purified, and ligated into a second circularized molecule.

[0038] In step 2), there are no special requirements regarding the length and sequence of the control DNA molecule. In one embodiment, the length difference between the control DNA molecule and the test DNA template may not exceed one-fold. In an exemplary embodiment, the length of the test DNA template is 1000 bp, and the length of the control DNA molecule can be 500-1500 bp, for example, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, or 1500 bp. In this invention, the role of the control DNA molecule is to provide a reference signal when there is no gap. Even when the lengths of the control DNA molecule and the test DNA template are slightly different, the RCA products generated by both when there is no gap, although differing in total length, can both enter the high molecular weight region. Therefore, the difference in length does not affect the final calculation of the integrity of the test DNA template.

[0039] In a preferred embodiment, the control DNA molecule is the same length as the test DNA template. In a further preferred embodiment, the control DNA molecule has the same sequence as the test DNA template.

[0040] In one embodiment, the first restriction enzyme site is located 0-100 bp upstream of the 5' end of the DNA template to be tested, and the second restriction enzyme site is located 0-100 bp downstream of the 3' end of the DNA template to be tested. In practice, the closer the restriction enzyme site is to the end of the DNA template to be tested, the better. In a preferred embodiment, the first restriction enzyme site is located at the 5' end of the DNA template to be tested, and the second restriction enzyme site is located at the 3' end of the DNA template to be tested, so as not to affect the promoter region at the 5' end of the DNA template to be tested, nor to affect the polyA sequence at the 3' end.

[0041] In one embodiment, when cutting the test DNA template from plasmid DNA, in addition to the first and second restriction enzyme sites, multiple additional restriction endonuclease sites are selected at sites outside the test DNA template of the plasmid, and the corresponding enzymes are used for cutting. This results in the DNA in the non-test DNA template region being cut into small fragments (e.g., less than 100 bp). Since intramolecular ligation efficiency is related to fragment length (small fragments have extremely low self-circularization efficiency and may not form stable circular structures), self-circularization of these fragments is prevented. Furthermore, the small fragments cut from the non-test DNA template region can be designed with different sticky ends to avoid self-circularization of these small fragments. Ultimately, only the fragment containing the test DNA template region can efficiently form a circular product through ligation at its two ends, reducing background interference and improving the specificity of the detection. When selecting multiple additional restriction endonuclease sites in a non-test DNA template region, it is necessary to avoid the small fragments generated after enzyme digestion of the non-test DNA template region from joining the fragment containing the test DNA template region. For example, if the fragment containing the test DNA template region has blunt ends, the small fragments generated after enzyme digestion of the non-test DNA template region can have sticky ends, thus preventing the two from joining.

[0042] In another embodiment, the third restriction enzyme site is located 0-100 bp upstream of the 5' end of the control DNA molecule, and the fourth restriction enzyme site is located 0-100 bp downstream of the 3' end of the control DNA molecule. In a preferred embodiment, the third restriction enzyme site is located at the 5' end of the control DNA molecule, and the fourth restriction enzyme site is located at the 3' end of the control DNA molecule.

[0043] In one embodiment, the first and second cleavage sites are isoschizoid cleavage sites or isoschizoid cleavage sites. In another embodiment, the third and fourth cleavage sites are isoschizoid cleavage sites or isoschizoid cleavage sites.

[0044] Isocaudomers are a group of restriction endonucleases from different sources that recognize different DNA sequences but produce the same sticky ends after cleavage. For example, the restriction endonucleases BamHI (recognizing the 5'-G^GATCC-3' sequence) and BglII (recognizing the 5'-A^GATCT-3' sequence) both produce a 5'-GATC protruding sticky end after cleavage. Using isocaudomers in this invention has the following advantages: because they produce the same sticky ends at both ends, the experimental procedure is greatly simplified; different isocaudomer pairs can be selected according to experimental conditions without changing the detection protocol, thus improving the adaptability and robustness of the method.

[0045] Isoschizomers are restriction endonucleases from different strains that recognize the same nucleotide sequence. Based on whether they cleave in the same way, isoschizomers can be divided into two categories: Isolytic enzymes that produce the same ends after cleavage: For example, both SmaI and XmaI recognize the sequence 5'-CCCGGG-3', but SmaI produces blunt ends (5'-CCC^GGG-3', which is blunt after cleavage), while XmaI produces sticky ends (5'-C^CCGGG-3', which produces 5'-CCGG protrusions after cleavage); as another example, both MspI and HpaII recognize 5'-CCGG-3', but MspI is not sensitive to methylation, while HpaII is sensitive to cytosine methylation; Isolytic enzymes that produce identical ends can be cleaved: for example, AscI (from different strains) all recognize 5'-GG^CGCGCC-3' and produce the same sticky ends; different isolytic enzymes of NotI also produce the same ends.

[0046] Introducing isoschizase recognition sites into the IVT DNA template allows for the selection of different enzymes for linearization as needed: SmaI can be chosen to obtain blunt ends (avoiding multimer formation), while XmaI can be chosen to obtain sticky ends. This provides a flexible tool for end modification of IVT templates.

[0047] When methylation is present in the IVT DNA template, it may not be cleaved by a single isoschizase. In this case, an isoschizase insensitive to the methylation can be chosen for linearization (e.g., using MspI instead of HpaII). Therefore, introducing an isoschizase site ensures the robustness of the linearization reaction and avoids cleavage failure due to methylation.

[0048] In one embodiment, the first and second restriction enzyme sites may be the same or different restriction enzyme sites. In another embodiment, the third and fourth restriction enzyme sites may be the same or different restriction enzyme sites.

[0049] In one specific embodiment, the four restriction enzyme sites in the test DNA template and the control DNA molecule are the same. In another specific embodiment, the two restriction enzyme sites in the test DNA template are the same, and the two restriction enzyme sites in the control DNA molecule are the same, but the restriction enzyme sites in the test DNA template are different from those in the control DNA molecule. In yet another specific embodiment, the four restriction enzyme sites in the test DNA template and the control DNA molecule are all different.

[0050] In one embodiment, the first and second restriction enzyme sites in the DNA template to be tested are different restriction enzyme sites, thereby enabling the DNA template to generate complementary sticky ends after restriction enzyme digestion for self-circularization ligation.

[0051] In one embodiment, in steps 1) and 2), the non-circularized linear molecules are removed. In a specific embodiment, exonuclease V is used to remove the non-circularized linear molecules.

[0052] After the cyclization reaction, a large number of uncyclized linear DNA molecules remain in the reaction system. This invention allows for selective enzymatic digestion of the reaction product using exonuclease V (ExoV) after the cyclization reaction is complete. ExoV can degrade linear DNA bidirectionally from both ends, but it is inactive against all circular DNA molecules. This one-step enzymatic treatment efficiently removes uncyclized linear molecules, yielding high-purity circular molecules.

[0053] In another implementation, a combination of λ exonuclease and RecJf exonuclease can be used to remove uncircularized linear molecules. The λ exonuclease first degrades one strand of the linear double-stranded DNA, producing single-stranded DNA, and then RecJf degrades the remaining single strand into mononucleotides. Because the λ exonuclease cannot initiate digestion at the gap in the circular molecule, the gapped first circularized molecule can be retained.

[0054] In another embodiment, the products of steps 1) and 2) can be subjected to electrophoresis and then the uncyclized molecules can be removed by gel cutting.

[0055] In another implementation, since the uncirculated molecules are unlikely to produce the high molecular weight DNA (length > 20 kb) described herein and will not affect subsequent quantification results, the uncirculated molecules do not need to be removed.

[0056] In one embodiment, in step 2), a gap-free control DNA molecule is prepared using a method based on the principle of DNA polymerase isothermal amplification. In a specific embodiment, in step 2), the gap-free control DNA molecule is prepared using PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), etc.

[0057] In one implementation, in step 3), a nicking endonuclease is used to create a nick for rolling circle replication. A nicking endonuclease (also written as "nicking enzyme" herein) is a special type of Type II restriction enzyme that, unlike ordinary restriction enzymes, does not cleave double-stranded DNA. Instead, it cleaves only one strand of the DNA double helix based on a recognition site, creating a "nick"-type nick. Depending on the strand being cleaved, nicking endonucleases are classified into two types: Nt. (cleaving the upper strand) and Nb. (cleaving the lower strand). In a specific implementation, the nicking endonuclease may be Nt.BstNBI, Nt.BspQI, or Nt.BbvCI, but is not limited to these.

[0058] In one implementation, in step 3), rolling circle replication is performed using random primers. The random primers are typically a mixture of random sequences (random hexamers) of 6 nucleotides in length, theoretically containing 4... 6 =4096 different sequence combinations, sufficient for non-specific binding at multiple sites on a circular DNA template. In this case, since either strand of the circular molecule is randomly used as a template for both the first and second circularized molecules, it does not affect subsequent quantification results.

[0059] In one embodiment, in step 3), rolling circle replication is performed using a DNA polymerase. In a specific embodiment, the DNA polymerase is phi29 DNA polymerase, Bst DNA polymerase, or Vent DNA polymerase.

[0060] Rolling circle amplification (RCA) is a linear isothermal amplification technique in which a long single-stranded DNA product composed of numerous tandem repetitive sequences is formed by the continuous extension of a circular template, DNA polymerase, and primers along the circular template. However, when a nicking endonuclease is introduced into the system, RCA can be performed without exogenous primers. Its core principle is a "polymerization-nicking" cycle: the nicking endonuclease introduces a single-stranded nick at a specific position in the double-stranded DNA product, exposing the 3'-OH end; the DNA polymerase with strand displacement activity initiates polymerization and extension from this 3'-OH end, synthesizing a new strand using the unnicked complementary strand as a template, while simultaneously displacing the downstream double-stranded DNA. The displaced single strand can expose a new nicking enzyme recognition site (or serve as a new primer), which is then recognized and cleaved by the nicking endonuclease, thus initiating a new round of polymerization-displacement-nicking cycle.

[0061] In step 3), "same conditions" includes identical reaction system components, identical reaction environment conditions, identical template treatment and starting steps, etc. In this invention, "same conditions" means that, except for the cyclization molecule itself (either the first or second cyclization molecule), all other components and reaction parameters in the two rolling circle replication reaction systems are kept consistent. Specifically, the two reactions use DNA polymerase from the same source and with the same activity units, primers of the same concentration (or random primers from the same batch), dNTPs of the same concentration, and reaction buffer with the same formulation, added to the same final volume; the two reactions are set with the same reaction temperature and the same reaction time. Furthermore, the number of moles of cyclization molecules added in the two reactions is the same. Under these conditions, the difference in the yield of the high molecular weight product obtained from the two reactions directly reflects the difference in template integrity.

[0062] Those skilled in the art will understand that "same conditions" does not refer to absolute indifference in a mathematical sense, but rather to controlling the reaction system, environment, and operating procedures as consistently as possible under standard experimental precision, so that any minor differences may have a negligible impact on the total amount of rolling ring replication products. Generally, relative errors in parameters such as volume, temperature, and incubation time controlled within 5% are considered to meet "same conditions."

[0063] Figure 3 The schematic diagram illustrates the gap in the IVT DNA template detected by rolling circle replication. (See diagram for example.) Figure 3 As shown, for IVT DNA templates without gaps (top figure), RCA will produce high molecular weight tandem polymers, typically >20 kb in length; for IVT DNA templates with gaps (bottom figure), RCA cannot proceed effectively, resulting in lower product yields and the inability to produce large molecular weight tandem polymers.

[0064] In this paper, the term "rolling circle replication product" refers to a high-molecular-weight linear DNA molecule produced by continuous tandem extension of multiple intact template sequences around a circular DNA molecule as a template, catalyzed by a DNA polymerase with strand displacement activity, starting from the 3' end of the primer. Rolling circle replication lacks a natural termination signal; it does not automatically stop after one loop, nor does it have a predetermined product length threshold. In fact, as long as sufficient active polymerase and substrate (dNTPs) remain in the reaction system, the extended strand will continue to be synthesized, causing the product length to increase continuously with the extension of reaction time. Therefore, the final product length mainly depends on the reaction duration; the longer the reaction time, the more tandem repetitions, and the larger the product molecular weight, potentially reaching tens or even hundreds of kb.

[0065] In this invention, the length of the rolling circle replication product is the product of the length of the circular molecule and the number of replication cycles. In one embodiment, the length of the circular molecule (test DNA template / control DNA molecule) can be 100 bp-10 kb, for example, 100 bp, 500 bp, 2 kb, 5 kb, or 8 kb. In one embodiment, the number of replication cycles can be 100-10000, for example, 100, 500, 1000, 3000, 5000, or 8000 cycles.

[0066] In step 4), the first rolling circle replication product and the second rolling circle replication product obtained in step 3) are respectively subjected to gel electrophoresis analysis, and the signal intensity of the first rolling circle replication product and the second rolling circle replication product is detected.

[0067] In this invention, the length of the rolling circle replication product is related to the length of the circular molecule and the number of replication cycles. The signal intensities of the first and second rolling circle replication products detected in step 4) refer to the signal intensities of products with a length greater than 10 kb for the test DNA template / control DNA molecule. The length of the control DNA molecule depends on the continuous synthetic capacity (processivity) of the polymerase used for rolling circle replication. Based on the inventors' numerous experimental observations, the gel electrophoresis bands of the control DNA molecule rarely fall below 10 kb. Therefore, this invention uses the signal intensity of products with a length greater than 10 kb for integrity detection.

[0068] In one specific implementation, an unexposed TIFF image is acquired using a high dynamic range CCD camera. After subtracting the background of the rolling sphere using software such as ImageJ, a grayscale peak map of each lane is plotted. The peak area of ​​the target band is integrated (IntDen). The signal intensity can be obtained by calculating the percentage of the peak area of ​​the high molecular weight rolling ring replication product band to the sum of the peak areas of all bands in that lane.

[0069] Finally, the ratio of the signal intensity of the first rolling circle replication product to the signal intensity of the second rolling circle replication product is calculated to determine the integrity of the DNA template to be tested.

[0070] Under the same starting molecules and reaction conditions, the band signal intensity (e.g., brightness or peak area in gel electrophoresis) of the rolling circle replication product directly reflects the number of intact circular molecules that can serve as effective templates. The integrity of the DNA template to be tested ranges from 0% to 100%. The closer the signal of the DNA template sample to the control signal, and the closer the ratio is to 1, the more intact the DNA template to be tested; the lower the ratio, the more missing molecules are present in the template. By simultaneously measuring the control sample (in its ideal intact state) and taking the ratio, systematic errors caused by batch-to-batch differences (e.g., enzyme activity, reaction temperature, imaging conditions, etc.) can be eliminated, achieving quantification.

[0071] Example The present invention and its technical effects will be clearly and completely described below with reference to embodiments and accompanying drawings, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0072] Example 1 This embodiment uses agarose gel electrophoresis to detect the integrity of the IVT DNA template.

[0073] The plasmid used in this embodiment is pUC57-eGFP-A121 (GenScript SD1171), approximately 3.5 kb in length, containing a T7 promoter, an enhanced green fluorescent protein (eGFP) coding sequence (720 bp), and a 121 bp polyA sequence. The plasmid contains BamHI and BglII restriction enzyme sites (isosuppressive enzymes) flanking the eGFP sequence. The T7 promoter is located 24 bp upstream of the BamHI site, and the polyA sequence is immediately downstream of the BglII site. The plasmid contains a kanamycin resistance gene, and the host bacterium is *Escherichia coli* DH5α.

[0074] Gel electrophoresis steps: 1. Prepare 1% agarose gel: Weigh 1.0 g agarose powder and add it to 100 mL of 1× TAE electrophoresis buffer. Heat in a microwave oven until completely dissolved (the solution is clear and free of particles). Cool to about 50-55℃, add an appropriate amount of nucleic acid dye GelRed, mix well, pour into a horizontal gel casting mold, insert a comb, and let stand at room temperature for 20-30 minutes until the gel is completely solidified.

[0075] 2. Preparation of circularized molecules: Take 1 μg of plasmid DNA and digest it with BamHI and BglII at 37℃ for 2 hours. After purification of the digestion product using a PCR purification kit (Novizan, catalog number DC201-01), ligate it overnight at 16℃ using T4 DNA ligase (NEB, catalog number M0202S). Add exonuclease V (Exo V, NEB, catalog number M0345S) to the ligation product and treat it at 37℃ for 30 minutes to remove uncircularized linear molecules. Then inactivate it at 70℃ for 10 minutes to obtain the first circularized molecule.

[0076] 3. Load 15 μL of plasmid solution into each well, containing 150 ng of plasmid sample.

[0077] 4. After electrophoresis, carefully remove the gel and use the GelDoc XR+ imaging system to perform gel imaging, and use Image Lab software to acquire images.

[0078] The results are as follows Figure 4 As shown, the white arrow in the left image points to plasmid supercoiling. Generally, the molecular mobility of a supercoiled conformation is 80% of the expected value. That is, the supercoiled band of a 1 kb plasmid molecule is around 800 bp. The white arrow in the right image points to plasmid aggregates. Because the plasmid in the right image contains a polyA sequence, it easily forms aggregates, and the specific molecules are often larger than the expected molecular weight. After linearization, they revert to the expected size. For plasmids containing polyA sequences, it is difficult to use the supercoiling ratio to detect integrity.

[0079] Example 2 In this embodiment, the plasmid and linear PCR product were treated with a nicking endonuclease to create a nick, and then agarose gel electrophoresis was performed.

[0080] The experimental steps in this embodiment are as follows: This example uses the same plasmid (pUC57-eGFP-A121) as in Example 1.

[0081] 1. PCR amplification of plasmid DNA: 50 ng of plasmid pUC57-eGFP-A121 (same as in Example 1) was used as a template, and 25 μL of 2× PCR premix (Novizan, catalog number P222-01) was added, with ddH2O added to a total volume of 50 μL. PCR conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; final extension at 72℃ for 5 min. The primers used in PCR included: forward primer 5'-GGGATGTGCTGCAAGGCGATTAA-3' (SEQ ID NO:1) and reverse primer 5'-CAAGCGCGCAATTAACCCTCACTAA-3' (SEQ ID NO:2). After identification by 1% agarose gel electrophoresis, the PCR products were purified using a PCR purification kit (Novizan, catalog number DC201-01) to obtain a nick-free linear control DNA molecule.

[0082] 2. Nicking endonuclease treatment (for creating a nicked DNA template): The nicking enzyme used in this example is Nt.BbvCI (Yugong, catalog number EG25520). In a 50 μL reaction system, add 1 μg of DNA template (the plasmid digestion product obtained in Example 1 or the PCR product obtained in step 1 of this example), 5 μL of 10× CutSmart Buffer (NEB, catalog number B7204S), and 1 μL of nicking enzyme. After reacting at 37°C for 60 minutes, inactivate at 80°C for 20 minutes.

[0083] The agarose gel electrophoresis results of the complete plasmid digestion product, the complete PCR product, and the two nicking enzyme treatment products obtained in the above steps are as follows: Figure 5 As shown, the linearized plasmid and PCR product did not show significant differences in electrophoresis before and after nicking endonuclease treatment.

[0084] Example 3 1. Nucleation endonuclease concentration gradient experiment ( Figure 6 ) In a 50 μL reaction system, 100 ng of circular DNA template (prepared according to the method in Example 1), 5 μL of 10×CutSmart Buffer (NEB, catalog number B7204S), 10 U of phi29 DNA polymerase (NEB, catalog number M0269S), and 10 nmoldNTPs (NEB, catalog number N0447S) were added. Four experimental groups were set up, including a control group without nicking enzyme and experimental groups with 0.1 Unit, 0.01 Unit, and 0.001 Unit of nicking enzyme Nt.BbvCI (Yugong, catalog number EG25520), respectively. The reaction was carried out at 37℃ for 60 minutes to perform rolling circle replication. 5 μL of the reaction product was analyzed by 1% agarose gel electrophoresis.

[0085] The results are as follows Figure 6 As shown, the amount of RCA product in the cyclic molecule varies after treatment with different concentrations of nicking enzyme. The higher the concentration of nicking enzyme used, the more nicks are created, and the less RCA product is produced.

[0086] 2. Determination of plasmid integrity in different batches ( Figure 7 ) In this step, the integrity of plasmids 1-5 was determined using the method provided by this invention. All plasmids 1-5 used were from the same construct, pUC57-eGFP-A121, and were derived from five independent extraction batches. Slight differences existed in the fermentation, lysis, and purification conditions among the batches, resulting in varying nick proportions in the final plasmid products.

[0087] Take 1 μg of each of plasmids 1-5 and circularize them according to the method described in Example 1 to obtain circular molecules. Use these circular molecules and the no-gap linear control DNA molecule obtained in Example 2 to perform the RCA step according to step 1 of this example to detect the integrity of the DNA.

[0088] The gel electrophoresis results of the RCA products of the cyclized molecules obtained from plasmids 1-5 are as follows: Figure 7 As shown, the integrity of plasmids 1-5, calculated using the method of the present invention, is 70%, 47%, 30%, 52%, and 36%, respectively.

[0089] Each batch of plasmid pUC57-eGFP-A121 was linearized and purified using restriction endonucleases, then transcribed in vitro using T7 RNA polymerase, followed by digestion with DNase I (Yugong Biotechnology, catalog number EG24207) and purification by LiCl precipitation to obtain the corresponding batch of mRNA.

[0090] The integrity of another plasmid (pUC57-eGFP-A121) was calculated using the same method as above. The integrity of plasmid 6 was calculated to be 85%, and mRNA was obtained by in vitro transcription using plasmid 6.

[0091] Capillary electrophoresis was performed using these mRNAs: Following the instructions for use of the Agilent 2100 Bioanalyzer, the Gel-Dye Mix was prepared using the RNA 6000 Nano kit and injected into the RNA Nano chip. 5 μL of RNA Marker and 1 μL of the mRNA sample to be tested (concentration adjusted to 50-100 ng / μL) were added to each chip. The mRNA Smear Assay program was run using the accompanying 2100 Expert software to record the electrophoresis pattern.

[0092] Figure 8 The results of chromatography and capillary electrophoresis of mRNA prepared from plasmids with more defects (top image, template integrity 47%, i.e., plasmid 2) and plasmids with fewer defects (bottom image, template integrity 85%, i.e., plasmid 6) are shown. The plasmid with more defects shows obvious tailing.

[0093] The present invention has provided a detailed description of a method for detecting DNA template integrity. Specific embodiments have been used to illustrate the principle and implementation of the present invention. The description of the embodiments above is only for the purpose of helping to understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the idea of ​​the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for detecting the integrity of a DNA template, characterized in that, The method includes the following steps: 1) Provide a DNA template to be tested, and perform self-circularization ligation on the DNA template to obtain a first circularized molecule; Optionally, the DNA template to be tested is present in a plasmid and has a first restriction enzyme site and a second restriction enzyme site upstream and downstream thereon; 2) Provide a control DNA molecule, wherein the control DNA molecule has no gap; the control DNA molecule is circular, serving as a second circularized molecule; or the control DNA molecule is a linear molecule, and the linear molecule is self-circularized to obtain a second circularized molecule; 3) Take equal amounts of the first cyclized molecule and the second cyclized molecule and perform rolling ring replication under the same conditions to obtain the first rolling ring replication product and the second rolling ring replication product. 4) Take the first and second rolling circle replication products obtained in step 3) and perform gel electrophoresis analysis on them respectively, and detect the signal intensity of the first and second rolling circle replication products. 5) Calculate the ratio of the signal intensity of the first rolling circle replication product to the signal intensity of the second rolling circle replication product to determine the integrity of the DNA template to be tested.

2. The method according to claim 1, characterized in that, The first restriction enzyme site is located 0-100 bp upstream of the 5' end of the DNA template to be tested, and the second restriction enzyme site is located 0-100 bp downstream of the 3' end of the DNA template to be tested.

3. The method according to claim 2, characterized in that, The first restriction enzyme site is located at the 5' end of the DNA template to be tested, and the second restriction enzyme site is located at the 3' end of the DNA template to be tested.

4. The method according to any one of claims 1-3, characterized in that, The first and second restriction sites are either isoschizoid restriction sites or isoschizoid restriction sites.

5. The method according to any one of claims 1-4, characterized in that, The first restriction site and the second restriction site may be the same restriction site or different restriction sites.

6. The method according to any one of claims 1-4, characterized in that, In the DNA template to be tested, the first restriction enzyme site and the second restriction enzyme site are different restriction enzyme sites, so that the DNA template to be tested can generate complementary sticky ends after restriction enzyme digestion, so as to perform self-circularization ligation.

7. The method according to any one of claims 1-6, characterized in that, In steps 1) and 2), the non-circularized linear molecules are removed, for example, by using exonuclease V to remove the non-circularized linear molecules.

8. The method according to any one of claims 1-7, characterized in that, In step 3), nicks are generated using nick endonucleases (e.g., Nt.BstNBI, Nt.BspQI, Nt.BbvCI) to induce rolling circle replication.

9. The method according to any one of claims 1-7, characterized in that, In step 3), rolling circle replication is performed using random primers.

10. The method according to any one of claims 1-9, characterized in that, In step 3), rolling circle replication is performed using a DNA polymerase (e.g., phi29 DNA polymerase, Bst DNA polymerase, or Vent DNA polymerase).

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