Single-stranded DNA molecular weight marker and preparation method thereof

Abstract

The invention relates to the technical field of molecular biology, in particular to a single-stranded DNA molecular weight marker and a preparation method thereof. The method comprises the following steps: 1) mixing a sample containing single-stranded DNA with single-stranded DNA binding protein, and incubating for a period of time to obtain a conjugate containing the single-stranded DNA and the single-stranded DNA binding protein; (2) separating and purifying the conjugate obtained in the step (1), and removing impurities which are not combined with the single-chain binding protein to obtain a high-purity conjugate of the single-chain DNA and the single-chain DNA binding protein; and 3) purifying the high-purity conjugate of the single-stranded DNA and the single-stranded DNA binding protein obtained in the step 2) to at least remove the single-stranded DNA binding protein to obtain a target product, and (4) repeating the steps (1)-(3) for m times (m is greater than or equal to 1) to obtain target products of different single-stranded DNA, and mixing the target products of the different single-stranded DNA, so that the mixed mixture at least contains two kinds of single-stranded DNA with different lengths.

Description

Single-stranded DNA molecular weight marker and preparation method thereof

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 202310075047.3 filed on January 19, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of molecular biology, and in particular to a single-stranded DNA molecular weight marker and a preparation method thereof. Background Art

[0004] In the field of gene therapy, recombinant adeno-associated virus is the most commonly used vector in this field. The genome of this virus is a linear DNA with ITRs at both ends, and its longest packaging length is 4500 bases in length. During the process of viral packaging, due to the replication mechanism of the viral genome itself, the packaged viral genome may have fragment deletions, resulting in incomplete or even ineffective drug efficacy. It may also contain redundant sequences due to the integration of the plasmid vector backbone sequence of the target gene. When using non-viral CRISPR gene editing strategies, single-stranded DNA (ssDNA) as an HDR (Homology directed repair, homology-mediated double-stranded DNA repair) template has many advantages in CRISPR gene knock-in experiments, such as improving editing efficiency, editing accuracy, and reducing off-target effects and cytotoxicity. Therefore, single-stranded DNA templates are very suitable for the insertion, replacement and correction of genes based on CRISPR technology, and are an ideal technical solution for primary cell and stem cell gene editing and the establishment of genetically modified animal models.

[0005] In the quality control of drug-encoding DNA vectors for gene therapy, whether adeno-associated virus vectors or non-viral single-stranded DNA templates, after completing GMP production, the purified single-stranded DNA must be size-determined and purity-identified to ensure the integrity and effective purity of the active pharmaceutical ingredient. However, due to limitations in their preparation processes, currently available single-stranded DNA molecular weight markers (ssDNA ladders) cannot clearly mark single-stranded DNA over 3000 nt, and undigested DNA polymers are often present on gel images (reference: Gu, H. and Breaker, RR Production of single-stranded DNAs by self-cleavage of rolling-circle amplification products. BioTechniques 54:337-343 (June 2013) doi 10.2144 / 000114009). Therefore, double-stranded DNA markers are the only alternative for size calibration. Double-stranded DNA molecules generally exist in a relatively stretched and relaxed double helix form in solution, while single-stranded DNA molecules generally exist in a relatively compact coil structure in solution. There are obvious differences between the two in terms of physical properties such as molecular weight and molecular fluid mechanics, resulting in different migration rates in agarose gel electrophoresis, making it impossible to achieve the purpose of accurate size calibration.

[0006] Summary of the Invention

[0007] The first object of the present invention is to provide a method for preparing a single-stranded DNA molecular weight marker, comprising:

[0008] 1) mixing a sample containing single-stranded DNA with a single-stranded DNA binding protein and incubating the mixture for a period of time to obtain a complex containing the single-stranded DNA and the single-stranded DNA binding protein;

[0009] 2) isolating and purifying the conjugate obtained in step 1) to remove impurities that are not bound to the single-stranded DNA binding protein, thereby obtaining a high-purity conjugate of the single-stranded DNA and the single-stranded DNA binding protein;

[0010] 3) purifying the high-purity conjugate of the single-stranded DNA and the single-stranded DNA binding protein obtained in step 2) to at least remove the single-stranded DNA binding protein to obtain the target product;

[0011] 4) Repeat steps 1) to 3) m (m ≥ 1) times to obtain different target products, and mix the different target products so that the mixture contains at least two single-stranded DNAs of different lengths. A second object of the present invention is to provide a single-stranded DNA molecular weight marker prepared by the above method.

[0012] A third object of the present invention is to provide a method for determining the length range of single-stranded DNA, comprising the following steps:

[0013] a) allowing a sample containing single-stranded DNA to be measured, which has been pre-contacted with a dye, and a detectably labeled single-stranded DNA molecular weight marker as described above to migrate under mobility-dependent separation conditions, and allowing the single-stranded DNA molecular weight marker to form a single-stranded size determination ladder;

[0014] b) determining the relative position of the ladder according to the size of the single-stranded DNA to determine the length range of the single-stranded DNA in the sample.

[0015] The present invention also relates to a kit comprising the single-stranded DNA molecular weight marker described above.

[0016] The preparation method of the single-stranded DNA molecular weight standard product developed by the present invention can effectively remove the residues of non-specific amplification products, especially double-stranded DNA impurities, during the preparation process of single-stranded DNA. Based on the separation and purification method of single-stranded DNA binding protein, the test materials can all be conventional protein purification tools, which will not cause adverse effects such as degradation and digestion on the target product, and the system is easy to amplify, and mg-level target products can be obtained in a relatively short time. The selected reagents may not contain carcinogenic agents, which improves the safety of operation and application and provides great convenience for clinical experiments. The method may not involve amplification or denaturation in the purification process, and will not cause sequence errors, losses or mutations due to the presence of difficult sequences in the target DNA. The difficult sequences refer to sequences containing but not limited to hairpin structures caused by reverse repeats, high and low GC sequences or poly structures, which are difficult to amplify by PCR and result in inefficient synthesis, thereby ensuring the sequence integrity of the purified single-stranded DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] FIG1 shows the amplification results of 2k-DNA PCR products provided by one embodiment of the present invention; 12 lanes other than the first lane marker contain PCR products of completely identical samples;

[0019] FIG2 shows the results of restriction enzyme digestion of a 2k-RCA product and the results of purification of a single-stranded binding protein provided by one embodiment of the present invention; Lane M: Marker (DL3000 DNA ladder); Lane 1: 2k-RCA product restriction enzyme digestion product; Lane 2: Purification of a single-stranded binding protein;

[0020] Figure 3 shows the final product of 1k-5k single-stranded DNA purification provided by one embodiment of the present invention; Lane M: Marker (DL3000 DNA ladder); Lane 1: Final product after 1k-ssDNA purification; Lane 2: Final product after 2k-ssDNA purification; Lane 3: Final product after 3k-ssDNA purification; Lane 4: Final product after 4k-ssDNA purification; Lane 5: Final product after 5k-ssDNA purification;

[0021] FIG4 is an agarose gel electrophoresis result of a mixture of 1k-5k single-stranded DNAs provided in one embodiment of the present invention; Lane M: Marker (DL3000 DNA ladder); Lane S: ssDNA Ladder (single-stranded DNA molecular weight marker provided by the present invention);

[0022] FIG5 is an agarose gel electrophoresis comparison result of a 1k-5k single DNA sequence and ssDNA Ladder (single-stranded DNA molecular weight marker provided by the present invention) provided by one embodiment of the present invention; Lane M: Marker (DL3000 DNA ladder); Lane 1: 1k-final product after single-stranded DNA purification; Lane 2: 2k-final product after single-stranded DNA purification; Lane 3: 3k-final product after single-stranded DNA purification; Lane 4: 4k-final product after single-stranded DNA purification; Lane 5: 5k-final product after single-stranded DNA purification; Lane S: ssDNA Ladder (single-stranded DNA molecular weight marker provided by the present invention);

[0023] FIG6 is a ssDNA Ladder Agilent TapeStation RNA detection result provided by one embodiment of the present invention;

[0024] FIG7 is the result of agarose gel electrophoresis comparison of five single-stranded DNA sequences of different lengths provided by the present invention and ssDNA Ladder (single-stranded DNA molecular weight marker provided by the present invention). DETAILED DESCRIPTION

[0025] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0026] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0028] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0029] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0030] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for 2%, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within ±10% are permitted.

[0031] In the present invention, "substantially" means that the defined component / production varies within a range of less than about 5%, such as ±5%, ±4%, ±3%, ±2% or ±1% (mass percentage).

[0032] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0033] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0034] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0035] Reference is made herein to various patents, patent applications, and other publications, all of which are incorporated herein by reference in their entirety. In addition, the following standard reference works are incorporated herein by reference: Current Protocols in Molecular Biology, John Wiley & Sons, New York, editions as of October 2007; Sambrook, Russell & Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. In the event of a conflict between this specification and any document incorporated by reference, this specification shall control, with the understanding that the determination of whether a conflict or inconsistency exists is within the discretion of the inventors and may be made at any time.

[0036] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0037] The present invention relates to a method for preparing a single-stranded DNA molecular weight marker, comprising:

[0038] 1) mixing a sample containing single-stranded DNA with a single-stranded DNA binding protein and incubating the mixture for a period of time to obtain a complex containing the single-stranded DNA and the single-stranded DNA binding protein;

[0039] 2) isolating and purifying the conjugate obtained in step 1) to remove impurities that are not bound to the single-stranded DNA binding protein, thereby obtaining a high-purity conjugate of the single-stranded DNA and the single-stranded DNA binding protein;

[0040] 3) purifying the high-purity conjugate of the single-stranded DNA and the single-stranded DNA binding protein obtained in step 2) to at least remove the single-stranded DNA binding protein to obtain the target product;

[0041] 4) Repeat step 1) - step 3) m (m ≥ 1) times to obtain different target products, and mix the different target products so that the mixed mixture contains at least two single-stranded DNAs of different lengths. The main function of single-stranded DNA binding protein (SSB) is to stabilize the unwound single strands of DNA during DNA replication, prevent renaturation and protect the single-stranded portion from being degraded by nucleases. This specification proposes that the binding ability of single-stranded DNA binding protein to single-stranded DNA can be utilized to purify single-stranded DNA on a large scale and efficiently to obtain a higher yield.

[0042] In some embodiments, the sample may be a product obtained from a large-scale production process of single-stranded DNA, and therefore the sample may contain non-specific amplification products, such as double-stranded DNA. In order to remove double-stranded DNA impurities, the traditional method is to use gel electrophoresis to recover single-stranded DNA in the sample, but this method has a low yield and greatly increases the time and labor costs of the downstream purification process, hindering the process of large-scale preparation. By adopting the purification method based on single-stranded DNA binding protein proposed in this specification, the single-stranded DNA binding protein that binds to single-stranded DNA can be purified on a large scale and efficiently by utilizing the binding ability of the single-stranded DNA binding protein to single-stranded DNA.

[0043] In some embodiments, the sample can refer to any sample containing single-stranded DNA of a certain purity. The proportion of single-stranded DNA in the sample can be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc., and this specification is not limited thereto. The single-stranded DNA is also referred to as the target sequence.

[0044] In some embodiments, when the sample is mixed with the single-stranded DNA binding protein, the final concentration of the single-stranded DNA binding protein can be 0.1 ng / μL-1000 ng / μL. In some embodiments, when the sample is mixed with the single-stranded DNA binding protein, the final concentration of the single-stranded DNA binding protein can be 0.5 ng / μL-500 ng / μL. In some embodiments, when the sample is mixed with the single-stranded DNA binding protein, the final concentration of the single-stranded DNA binding protein can be 1 ng / μL-200 ng / μL, for example, 5 ng / μL, 10 ng / μL, 50 ng / μL, 100 ng / μL, or 150 ng / μL.

[0045] In some embodiments, the first time of incubation after the sample is mixed with the single-stranded DNA binding protein can be at least 30 minutes, for example, 30 minutes to 24 hours. In some embodiments, the first time of incubation after the sample is mixed with the single-stranded DNA binding protein can be 30 minutes to 15 hours. In some embodiments, the first time of incubation after the sample is mixed with the single-stranded DNA binding protein can be 30 minutes to 12 hours, for example, 2 hours or 4 hours. In some embodiments, the first time of incubation after the sample is mixed with the single-stranded DNA binding protein can be 30 minutes to 6 hours. In some embodiments, the first time of incubation after the sample is mixed with the single-stranded DNA binding protein can be 2-4 hours.

[0046] In some embodiments, the sample is mixed with the single-stranded DNA binding protein and then incubated at 30-37° C. For example, the incubation temperature can be 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., etc.

[0047] In some embodiments, in step 2), the conjugate obtained in step 1) is further separated and purified to wash away impurities that are not bound to the single-stranded binding protein to obtain a high-purity conjugate of single-stranded DNA and single-stranded DNA binding protein. This step can be achieved by various protein separation and purification methods. For example, the purification method can be affinity chromatography, ion exchange chromatography or gel filtration chromatography (molecular sieve) method, etc., which is not limited in this specification. In some embodiments, the separation and purification method described in step 2) is affinity chromatography. In some embodiments, the affinity chromatography purification can adopt various affinity chromatography methods for separating and purifying proteins, which is not limited in this specification.

[0048] In some embodiments, the single-stranded DNA binding protein may further include a purification tag for affinity chromatography purification, such as a polyhistidine tag, a glutathione-S transferase tag, a FLAG tag, or a Strep-tag II tag.

[0049] As an example only, the single-stranded DNA binding protein may have a purification tag and may be purified using magnetic beads corresponding to the purification tag. For example, the single-stranded DNA binding protein may include a polyhistidine tag, and the corresponding magnetic beads may include nickel; the single-stranded DNA binding protein may include a glutathione-S transferase (GST) tag, and the corresponding magnetic beads may be GST fusion protein purification magnetic beads; the single-stranded DNA binding protein may also have a Strep-tag II tag, and the corresponding magnetic beads may be Strep-tag II protein purification magnetic beads. For another example, the single-stranded DNA binding protein may have a FLAG tag, and may be purified using a FLAG tag fusion protein purification kit.

[0050] For the convenience of description, the sample containing single-stranded DNA is mixed with the single-stranded DNA binding protein and incubated for a period of time, and the product obtained is defined as the first product; it is defined that in step 2), the conjugate obtained in step 1) is further separated and purified, and impurities that are not bound to the single-stranded binding protein are washed away to obtain the second product.

[0051] In some embodiments, the first product can be incubated (or incubated) with an affinity chromatography purification medium at room temperature for 30 minutes to 1 hour, and the components that are not bound to the single-stranded DNA binding protein are eluted by buffers with different concentration gradients (e.g., buffers containing different concentrations of imidazole) or buffers with different pH gradients to obtain a second product containing a conjugate of single-stranded DNA and single-stranded DNA binding protein. This method is suitable for large-scale purification of single-stranded DNA, with high yield, high speed, and lower cost. The entire purification process does not involve DNA dyes and carcinogenic agents, and the product has good biosafety, which provides great convenience for clinical trials and other applications; and, since the final product purification process does not involve amplification or denaturation, sequence errors, losses or mutations will not be caused by the presence of difficult sequences in the target sequence. Therefore, the method for purifying single-stranded DNA provided in this specification is applicable to the purification of single-stranded DNA of various lengths and structures, and has a wide range of applications. The reagents and materials required for the entire process are all conventional protein purification reagents and tools, and the system is easy to amplify and the cost is low. The term "high-difficulty sequence" refers to a sequence containing, but not limited to, hairpin structures caused by inverted repeats, high and low GC sequences, or poly structures, which are difficult to amplify by PCR and result in low synthesis efficiency.

[0052] In some embodiments, during the affinity chromatography purification process, optionally, after the affinity adsorption step is completed, the remaining liquid can be collected and analyzed to determine whether the single-stranded DNA has been well adsorbed to the affinity adsorption medium. For example, if affinity chromatography is performed using a magnetic bead method, after the first product and the magnetic beads are incubated for 30-60 minutes, the magnetic beads can be collected using a magnetic separation rack, and the supernatant can be obtained for analysis. By way of example only, a certain amount of the supernatant can be taken and analyzed by gel electrophoresis. If the results show that there is no single-stranded DNA to be purified in the supernatant, it means that the DNA in the sample has been substantially completely adsorbed to the affinity adsorption medium. If the results show that a large amount of single-stranded DNA is still in the supernatant, it means that the single-stranded DNA binding protein has not completely bound to the single-stranded DNA in the sample, or the selected affinity chromatography medium is not effective. In this case, the purification yield can be improved by checking whether the single-stranded DNA binding protein used can properly bind to the single-stranded DNA, increasing the concentration of the single-stranded DNA binding protein used, increasing the incubation time after mixing the sample with the single-stranded DNA binding protein, or replacing the affinity chromatography medium.

[0053] In some embodiments, after completing the affinity chromatography purification step, the second product can be optionally sampled and analyzed to determine the effect of the affinity chromatography purification. For example, a certain amount of the second product can be taken and analyzed by gel electrophoresis. If the result shows that there is only single-stranded DNA in the second product, it means that the double-stranded DNA impurity has been removed; if the result shows that there are both single-stranded DNA and double-stranded DNA in the second product, it means that the double-stranded DNA impurity has not been completely removed, and the selected affinity chromatography process is not suitable. For example, it may be due to an excess of single-stranded DNA binding protein, resulting in non-specific adsorption of double-stranded DNA. This can be improved by reducing the concentration of the single-stranded DNA binding protein used, reducing the incubation time after mixing the sample with the single-stranded DNA binding protein, or performing a secondary incubation purification of the sample with a small amount of single-stranded DNA binding protein, so as to avoid the situation where the double-stranded DNA impurity is not completely removed.

[0054] In some embodiments, the second product is purified to remove unwanted impurities such as single-stranded DNA binding proteins, imidazole, and salts. This purification step can be selected from any one or more of the following methods: phenol-chloroform extraction, DNA adsorption centrifugal column recovery, plasmid extraction and ion exchange column recovery, high performance liquid chromatography, isopropanol precipitation, ethanol precipitation, and molecular sieve chromatography (or molecular sieve chromatography supported by high performance liquid chromatography), and this specification does not impose any restrictions thereon.

[0055] In some embodiments, the single-stranded DNA binding protein comprises a single-stranded DNA binding protein derived from Escherichia coli, phi29 phage (Bacillus phage Nf), Salmonella phage Vi II-E1, Thermus thermophilus HB8, Klebsiella Phi K02 phage, Yersinia phage Py54 phage or Synechococcus phage S-CBS2, its core functional fragment, or its homologous sequence or mutant sequence. Here, the term "core functional fragment" refers to a fragment in the protein structure that enables the protein to normally perform its specific function. "Homologous sequence" refers to a sequence that originates from the same ancestor but gradually develops partial differences during evolution. "Mutant sequence" refers to a sequence after a natural mutation or artificially induced mutation occurs in the wild-type sequence. The core functional fragment, homologous sequence or mutant sequence of the single-stranded DNA binding protein derived from the above-mentioned microorganisms mentioned here still has the ability to bind to single-stranded DNA. It should be noted that the single-stranded DNA binding protein used in this specification can be a protein from various sources that can bind to single-stranded DNA, such as from other microorganisms not mentioned above, from artificial synthesis, etc., and this specification does not impose any restrictions on this.

[0056] In some embodiments, the single-stranded DNA binding protein is a mutant of the single-stranded DNA binding protein of the above-mentioned microorganism; the single-stranded DNA binding protein mutant comprises a sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the single-stranded DNA binding protein of the above-mentioned microorganism and has the function of binding to single-stranded DNA.

[0057] As an example only, the single-stranded DNA binding protein from Thermomyces HB8 has an NCBI accession number of WP_011227803.1 (SEQ ID NO: 16); the amino acid sequence of the mutant of the single-stranded DNA binding protein from Thermomyces HB8 is SEQ ID NO: 15, and the nucleotide sequence is SEQ ID NO: 19. The single-stranded DNA binding protein from Escherichia coli K-12 has an NCBI accession number of AKK14256.2 (SEQ ID NO: 17); and the single-stranded DNA binding protein from phi29 phage has an NCBI accession number of YP_009910721.1 (SEQ ID NO: 18). In some embodiments, the single-stranded DNA binding protein comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 15-18 and has the function of binding to single-stranded DNA. In some specific embodiments, the single-stranded DNA binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 15-18. In a specific embodiment, the amino acid sequence of the single-stranded DNA binding protein is as shown in any one of SEQ ID NOs: 15-18.

[0058] In some embodiments, the single-stranded DNA binding protein has greater than or equal to 80% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 85% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 90% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 92% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 95% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 97% identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the single-stranded DNA binding protein has greater than or equal to 99% identity to the amino acid sequence of SEQ ID NO: 15. In one embodiment, the amino acid sequence of the single-stranded DNA binding protein is as set forth in SEQ ID NO: 15.

[0059] As described herein, sequence consistency (also referred to as "sequence identity") refers to the amount of consistency between two nucleotide / amino acid sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of consistency between two nucleotide / amino acid sequences, a sequence alignment is performed and gaps (if any) are introduced. If, at a certain alignment position, the bases or amino acids in the two sequences are the same, the two sequences are considered to be consistent or matched at that position; if the bases / amino acids in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence consistency. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purposes of this specification, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence consistency between the two nucleotide / amino acid sequences using the default settings.

[0060] In some embodiments, after obtaining the target product, the target product can be optionally subjected to PCR amplification and DNA sequencing to verify whether the target product contains the complete sequence of the single-stranded DNA. If the sequencing result is consistent with the sequence of the single-stranded DNA, it means that the single-stranded DNA in the target product is intact and can be used for the next step, including but not limited to being used for CRISPR / Cas9 technology to achieve specific gene knockout, knock-in and site-directed modification functions. If the sequencing result is inconsistent with the sequence of the single-stranded DNA, it means that the sample may contain multiple single-stranded DNAs or other impurities may be mixed in during the purification process, and further purification is required.

[0061] The sample containing single-stranded DNA can be prepared by any known method. In some embodiments, the sample containing single-stranded DNA is prepared by biotin-modified magnetic bead separation, asymmetric PCR, reverse transcription first-strand synthesis, exonuclease digestion, or rolling circle amplification.

[0062] As an example, the biotin-modified magnetic bead separation method can be the method described in Yuji Wakimoto, et al. Current Protocols in Molecular Biology, 2014; the asymmetric PCR can be the method described in Citartan Marimuthu. Analyst, 2012, 137, 1307-1315.

[0063] Rolling circle amplification is preferably a method of automatic double-stranded DNA circularization mediated by uracil-specific subtractase (USER) and rolling circle replication (RCA) of precise cutting and isothermal amplification of single-stranded DNA by type II restriction endonucleases to achieve the preparation of single-stranded DNA of different lengths. A typical example is the method described in WO / 2020 / 135651. According to the method described in this patent, 5 single-stranded DNAs of different lengths (1000nt / 2000nt / 3000nt / 4000nt / 5000nt) can be designed and prepared. Specifically including: primer design process, template amplification preparation-automatic circularization, rolling circle replication, annealing-automatic folding, restriction endonuclease cutting-release of long single-stranded monomers of target fragments.

[0064] In some embodiments, the length of the longest single-stranded DNA in the single-stranded DNA molecular weight marker is 3000 nt to 7000 nt, for example, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 5500 nt, 6000 nt, or 6500 nt.

[0065] Another effect that can be achieved by this embodiment is that it can make up for the deficiency of single-stranded DNA molecular weight markers on the market that cannot identify larger single-stranded DNA sequences, and can achieve stable large-scale production. The raw materials used are mainly conventional molecular biology experimental preparations, with high yield, low cost, simple operation, and high mass production feasibility.

[0066] In some embodiments, the length difference between adjacent sized single-stranded DNAs is 50 nt to 3000 nt, such as 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 1000 nt, 1500 nt, 2000 nt, 2500 nt. The length difference between adjacent sized single-stranded DNAs is preferably substantially uniform.

[0067] Ideally, the amount of single-stranded DNA of each length is identical, resulting in nearly uniform brightness across the resulting single-stranded size ladder. However, a certain degree of variation should be tolerated, as long as it does not significantly affect molecular weight determination. In some embodiments, the difference in the amount of single-stranded DNA of each length is ≤10%, ≤5%, or ≤1%.

[0068] In some embodiments, single-stranded DNAs of different lengths are mixed in an equimolar ratio, that is, the amount of single-stranded DNA of different lengths is the same.

[0069] In some embodiments, the single-stranded DNA marker contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more single-stranded DNAs of different lengths.

[0070] In some embodiments, the single-stranded DNA molecular weight markers include at least five lengths of single-stranded DNA, including 1000 nt, 2000 nt, 3000 nt, 4000 nt and 5000 nt.

[0071] The present invention also relates to a single-stranded DNA molecular weight marker prepared by the above method.

[0072] In some embodiments, the single-stranded DNA molecular weight marker has a detectable label.

[0073] In some embodiments, the detectable label is selected from pyrene, naphthalene, aminopyridine, xanthene, cyanine, coumarin, boropolyazaindole, benzofuran, or indole dyes.

[0074] The present invention also relates to a method for determining the length range of single-stranded DNA, comprising the following steps:

[0075] a) allowing a sample containing single-stranded DNA to be measured, which has been pre-contacted with a dye, and a detectably labeled single-stranded DNA molecular weight marker as described above to migrate under mobility-dependent separation conditions, and allowing the single-stranded DNA molecular weight marker to form a single-stranded size determination ladder;

[0076] b) determining the relative position of the ladder according to the size of the single-stranded DNA to determine the length range of the single-stranded DNA in the sample.

[0077] Typically, to facilitate observation of the results, the sample containing the single-stranded DNA to be measured and the single-stranded DNA molecular weight marker are assigned to different channels. As used herein, "channel" and its derivatives refer to different lanes in the electrophoresis process, or a groove, tube, capillary, or other structure capable of supporting a certain volume of separation medium for electrophoresis. The geometry of the channel can vary greatly and include (but are not limited to) grooves, capillaries, or tubes, grooves, etc. with annular, rectangular, or square cross-sections, and can be manufactured using a variety of techniques.

[0078] As used herein, the term "mobility-dependent separation" refers to the separation of single-stranded DNA to be measured and / or single-stranded DNA molecular weight markers under the influence of factors such as nucleic acid charge and / or nucleic acid size.

[0079] Obviously, the sample may contain one or more single-stranded DNAs of different lengths to be measured. When there are multiple single-stranded DNAs, they can also be separated under mobility-dependent separation conditions and then measured separately.

[0080] In some embodiments, the mobility-dependent separation conditions may or may not include a denaturing additive.

[0081] In some embodiments, the dye is configured to not substantially label double-stranded nucleic acids.

[0082] In some embodiments, the sample containing single-stranded DNA to be assayed does not substantially contain double-stranded nucleic acid (eg, double-stranded DNA).

[0083] In some embodiments, the detectable label and the dye are independently selected from pyrene, naphthalene, aminopyridine, xanthene, cyanine, coumarin, boropolyazaindole, benzofuran, or indole dyes.

[0084] In some embodiments, the mobility-dependent separation condition is electrophoresis, preferably agarose gel electrophoresis.

[0085] The detectable label and the dye may be pre-dissolved in the electrophoresis gel.

[0086] It is easy to understand that the sample containing the single-stranded DNA to be measured and the single-stranded DNA molecular weight marker are preferably carried out under the same mobility-dependent separation conditions (e.g., in different lanes of the same agarose gel). In this case, the detectable label and the dye (in this case, the two are usually the same substance) can be added to the matrix (e.g., agarose gel) used to form the mobility-dependent separation conditions. Of course, the single-stranded DNA to be measured and the molecular weight marker can also be pre-labeled with the detectable label and the dye (in this case, the two are the same or different substances), and no additional detectable label and dye are added to the matrix.

[0087] The present invention also relates to a kit comprising the single-stranded DNA molecular weight marker described above.

[0088] In some embodiments, the kit further comprises a loading buffer; the loading buffer and the single-stranded DNA molecular weight marker are packaged separately, or the two are premixed.

[0089] In some embodiments, the kit further comprises the dye and / or the detectable label; the detectable label is packaged separately from the single-stranded DNA molecular weight marker, or is pre-labeled on the single-stranded DNA molecular weight marker.

[0090] In some embodiments, the detectable label and the dye are independently selected from pyrene, naphthalene, aminopyridine, xanthene, cyanine, coumarin, boropolyazaindole, benzofuran, or indole dyes.

[0091] The method for purifying single-stranded DNA described in step 1), and its additional technical solutions, can achieve at least one of the following technical effects: (1) It is suitable for large-scale purification of single-stranded DNA with high yield; (2) Compared with traditional methods (such as gel purification), the method for purifying single-stranded DNA proposed in this specification adopts an affinity chromatography process, which is fast and efficient; (3) The entire purification process does not involve DNA dyes and carcinogenic agents, and the product has good biosafety, which provides great convenience for clinical trials and other applications; (4) Since the purification process of the final product does not involve amplification or denaturation, sequence errors, losses or mutations will not be caused by the presence of difficult sequences in the target sequence. Therefore, the method for purifying single-stranded DNA provided in this specification is applicable to the purification of single-stranded DNA of various lengths and structures, and has a wide range of applications; (5) The reagents and materials required for the entire process are all conventional protein purification reagents and tools, and the system is easy to scale up and the cost is low.

[0092] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0093] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0094] Example

[0095] The single-stranded DNA ladder marker is a mixture of single-stranded DNA of varying lengths, with five sequences in 1000-base increments: 1000nt, 2000nt, 3000nt, 4000nt, and 5000nt. This ssDNA ladder clearly and accurately indicates the positional distribution of single-stranded DNA during agarose gel electrophoresis, allowing for the relative size determination of other sequences to be determined.

[0096] Experimental steps:

[0097] 1. Sequence and Primer Design

[0098] 1k-DNA sequence (SEQ ID: No. 1)

[0099] PCR amplification primer is forward amplification primer

[0100] Pf:5'-AACTATACUACGTCAATCGGCTCTTCAGGACACCAGGATTTATTTAT-3'

[0101] (SEQ ID: No.6)

[0102] Reverse amplification primer

[0103] Pr: 5'-TATAGTUACGTCAATCGGCTCTTCAGGACGTAATCTCTTGCTCTGAAAAC-3' (SEQ ID: No. 7).

[0104] 2k-DNA sequence (SEQ ID: No. 2)

[0105] PCR amplification primer is forward amplification primer

[0106] Pf:5'-AACTATACUACGTCAATCGGCTCTTCAGGACACCAGGATTTATTTAT-3'

[0107] (SEQ ID: No.6)

[0108] Reverse amplification primer

[0109] Pr:5'-TATAGTUACGTCAATCGGCTCTTCAGGAATTCGCCAGTGG CACAAAGAG-3'(SEQ ID:No.8)

[0110] 3k-DNA sequence (SEQ ID: No. 3)

[0111] PCR amplification primer is forward amplification primer

[0112] Pf:5'-AACTATACUACGTCAATCGGCTCTTCACTGAAGCGCCATAACATCG-3'

[0113] (SEQ ID: No. 9)

[0114] Reverse amplification primer

[0115] Pr:5'-TATAGTUACGTCAATCGGCTCTTCACTGCCAGCGGGTAGCCAATCT-3'

[0116] (SEQ ID: No. 10)

[0117] 4k-DNA sequence (SEQ ID: No. 4)

[0118] PCR amplification primer is forward amplification primer

[0119] Pf:5'-AACTATACUACGTCAATCGGCTCTTCATTCATCGACTCATGCCTTTC-3'

[0120] (SEQ ID: No.11)

[0121] Reverse amplification primer

[0122] Pr:5'-TATAGTUACGTCAATCGGCTCTTCATTCTTCCAGACGCACCAGTTC-3'

[0123] (SEQ ID: No. 12)

[0124] 5k-DNA sequence (SEQ ID:No.5)

[0125] PCR amplification primer is forward amplification primer

[0126] Pf:5'-AACTATACUACGTCAATCGGCTCTTCACGTGGTAACAACCTTCAAAT-3'

[0127] (SEQ ID: No. 13)

[0128] Reverse amplification primer

[0129] Pr:5'-TATAGTUACGTCAATCGGCTCTTCACGTTTCCATCGAGTGGGTAAGA-3'

[0130] (SEQ ID: No. 14)

[0131] The above primers were synthesized.

[0132] 2. Template Amplification Preparation - Automatic Circularization

[0133] The target fragment was amplified using primers (Pf, Pr) modified with uracil. The PCR reaction system was as follows:

[0134] Table 1. PCR reaction system

[0135] The PCR reaction program is:

[0136] Table 2. PCR reaction program

[0137] After 30 cycles, the electrophoresis pattern of the PCR product is shown in Figure 1. The PCR product is then recovered and subjected to a self-circularization reaction. The self-circularization reaction system for preparing a nicked circular double-stranded DNA using USER enzyme and T4 DNA ligase is as follows:

[0138] Table 3. Reaction system for double-stranded DNA self-circularization

[0139] The reaction conditions were: 37°C, 30 min, 20°C, 30 min, and then cooled to 4°C for storage.

[0140] 3. Rolling Circle Replication

[0141] In a 200 μL PCR tube, add 100 ng of the nicked circular DNA sample prepared in step 2 to a rolling circle replication reaction using the following system. Incubate at 30°C for 4-8 hours, then inactivate the enzyme at 80°C for 20 minutes. The rolling circle replication reaction system (using 100 μL as an example) is:

[0142] Table 4. Rolling circle replication reaction system

[0143] 4. Annealing-Automatic Folding

[0144] The rolling circle replication product was annealed to generate the designed hairpin structure. The annealing program was as follows: 80°C, 5 min, 0.1°C / s to 65°C, 65°C, 5 min, 0.1°C / s to 42°C, 42°C, 5 min, 37°C, 5 min, 0.1°C / s to 4°C, and then stored at 4°C.

[0145] 5. Restriction enzyme cleavage - release of target fragment long single-chain monomer

[0146] To the above reaction product, add 12 μL of 10× restriction enzyme buffer and 8 μL of the previously selected type II restriction enzyme. Incubate at the optimal reaction temperature for the restriction enzyme for 60 minutes, then heat denature and inactivate. The electrophoresis pattern of the crude reaction product is shown in lane 1 of Figure 2.

[0147] 6. Binding of Single-stranded Binding Protein to Crude Single-stranded DNA

[0148] The single-stranded DNA purification method established by utilizing the binding ability of single-stranded binding protein to single-stranded DNA and the purification and screening function of polyhistidine tag protein is used for subsequent single-stranded DNA purification operations.

[0149] 6.1 Preparation of single-chain synaptic proteins

[0150] 1) Gene synthesis and recombinant vector construction:

[0151] The mutant sequence of the SSB gene from Thermomyces HB8 selected in this specification (SEQ ID NO: 19) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0152] The pET28a kanamycin-resistant vector was used as an expression vector. The SSB gene (SEQ ID NO: 19) was inserted between the restriction enzyme sites NdeI and HindIII, i.e., between bases 5130 and 5923, to generate a protein expression vector with a polyhistidine tag at the N-terminus. Homologous recombination was used to generate a recombinant plasmid, as shown in Figure 1. The amino acid sequence of the SSB protein encoded by the SSB gene mutant sequence is shown in SEQ ID NO: 15.

[0153] 2) Protein expression:

[0154] Transform E. coli BL21(DE3) with the recombinant plasmid. Inoculate a single colony into a large-scale TB medium containing kanamycin resistance and culture at 37°C. When the OD600 reaches approximately 1.2, induce expression of the target protein with 0.5 mM IPTG at 15°C for 16 hours. Harvest the cells by centrifugation.

[0155] 3) Purification and analysis:

[0156] The cell pellet was resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 5% glycerol), then sonicated and centrifuged to collect the supernatant. The supernatant was further purified using a nickel-containing affinity chromatography column to obtain the target protein. The target protein was sterilized by filtration using a 0.22 μm filter and aliquoted for storage.

[0157] Protein concentration was determined by Bradford protein assay using BSA as a standard. The concentration of the purified single-stranded DNA-binding protein was 2.62 mg / ml. Protein purity and molecular weight were confirmed by reducing SDS-PAGE and Western blot (the primary antibody involved was mouse anti-His antibody, purchased from Nanjing GenScript, Cat. No. A00186). See Figure 2. Lane M1 is a protein marker (Bio-rad, Cat. No. 1610374S); lane M2 is a protein marker (Genscript, Cat. No. M00521); lane BSA is a standard; lane R is the purified target single-stranded DNA-binding protein. The band of the purified single-stranded DNA-binding protein is consistent with the theoretical molecular weight (32.05 kDa), indicating a purity of 90%.

[0158] The gene synthesis and recombinant vector construction, protein expression, and purification analysis involved in the above methods were all completed by the gene synthesis service and protein purification service of Nanjing GenScript Biotechnology Co., Ltd. (https: / / www.genscript.com.cn / guaranteed-bacterial-protein-expression-services.html?src=pullmenu).

[0159] 6.2 Binding of ssDNA-binding proteins to crude ssDNA products

[0160] Dilute the purified ssDNA-binding protein obtained in 6.1 5-fold (to a concentration of 10 ng / μL to 50 ng / μL). Add the diluted ssDNA-binding protein to 10 mL of crude ssDNA and mix thoroughly. The volume ratio of the mixture is shown in Table 5. Incubate at 37°C for 2 h to ensure that the ssDNA-binding protein and the target ssDNA in the crude product are fully bound to obtain an incubation product.

[0161] Table 5

[0162] 7. Purification of target single-stranded DNA by affinity chromatography

[0163] Ni-Charged MagBeads (Ni-filled magnetic beads, Nanjing GenScript Biotechnology Co., Ltd., Cat. No. L00295) were used to purify the target DNA bound to the single-stranded binding protein to obtain the affinity chromatography purification product. The specific steps are as follows:

[0164] 1) Completely resuspend the Ni-Charged MagBeads by shaking or vortexing the vial.

[0165] 2) Transfer 40uL of magnetic beads to a clean EP tube.

[0166] 3) Place the tube on a magnetic separation rack to collect the beads. Remove and discard the supernatant.

[0167] 4) Add 1 mL of LE Buffer (LE buffer: 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) to the EP tube and invert the tube several times to mix. Collect the beads using a magnetic separation rack and discard the supernatant. Repeat this step twice.

[0168] 5) Add an equal volume of the incubation product obtained in step 1 to the EP tube and gently invert the tube to mix.

[0169] 6) Incubate with mixing (on a shaker) at 20°C for 30-60 minutes.

[0170] 7) Collect the beads using a magnetic separation rack and discard the supernatant. If necessary, retain the supernatant for analysis.

[0171] 8) Add 2 ml of Wash Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) to the tube and mix thoroughly. Collect the beads using a magnetic separation rack and discard the supernatant. Repeat this wash step twice.

[0172] 9) Add 40 μL of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) to the tube, mix well, and incubate at 20°C (on a shaker) for 5 minutes.

[0173] 10) Collect the magnetic beads using a magnetic separation rack and transfer the supernatant containing the eluted protein into a clean EP tube.

[0174] 11) If necessary, repeat steps 9 and 10 twice to prepare the sample for subsequent purification.

[0175] 12) The supernatant (purified product) collected in step 11 is subjected to agarose gel electrophoresis, and the bands before and after purification are compared and analyzed to preliminarily determine whether the purification is complete.

[0176] 8. Removal and Purification of Single-Stranded Bound Proteins

[0177] The affinity chromatography purification product obtained above contains single-stranded binding proteins, imidazole, salts and other byproducts. In this example, the Qiagen-DNA plasmid extraction kit was used to remove byproducts such as proteins, imidazole and salts in the reaction system, and the purified product was finally obtained. See Figure 2, where lane 1 is the crude single-stranded DNA product and lane 2 is the purified single-stranded DNA product. The electrophoresis results show that the single-stranded DNA has been completely purified. This step can also be performed by phenol-chloroform extraction, DNA adsorption spin column recovery, isopropanol / ethanol precipitation, or molecular sieve chromatography supported by high-performance liquid chromatography to remove proteases and salts in the reaction system. According to the above steps, the other four single-stranded DNAs in the ssDNA ladder were also prepared, including 2000nt / 3000nt / 4000nt / 5000nt. The final product was detected by agarose gel electrophoresis in Figure 3.

[0178] 9. Mixing single-stranded DNA of different lengths and verifying their actual function

[0179] The obtained single-stranded DNAs of different lengths are mixed. After testing, it was found that when single-stranded DNA sequences of different lengths are mixed in equimolar proportions, the proportions of each band during agarose gel electrophoresis are relatively consistent. The specific operation is: first, the obtained single-stranded DNAs of different lengths are diluted to the same concentration c, and then the software is used to calculate the molecular weight M of the single-stranded DNAs of different lengths. The amount of substance n = m / M, then m = n×M, when equimolar, n is fixed, and the volume v = n×M / c of ​​single chains of different lengths that need to be mixed, so the added volume ratio is the ratio of molecular weights. After continuously adjusting the mixing ratio, it was determined that after mixing in an equimolar ratio, the brightness of each band was beautiful during agarose gel electrophoresis detection (see Figure 4). At the same time, the 5 original single-stranded DNAs of different lengths before mixing were electrophoresed together with the mixed ssDNA Ladder to verify the indication accuracy of the ssDNA Ladder. The comparison results are shown in Figure 5. After the initial verification was successful, 5 single-stranded DNAs with lengths different from the original sequence were randomly selected and subjected to agarose gel electrophoresis together with the prepared ssDNA Ladder to measure its actual use effect. The results are shown in Figure 7, and the size calibration effect of single-stranded DNAs of different sizes on the gel map is good. At the same time, the prepared ssDNA Ladder was subjected to Agilent TapeStation RNA detection, and the results are shown in Figure 6. In the TapeStation RNA detection, it can be found that the prepared ssDNA Ladder can be clearly separated when detected by the instrument. On the one hand, it proves that the purity of the ladder product is high and can achieve the purpose of accurate calibration. On the other hand, since there is no dedicated single-stranded DNA TapeStation detection kit on the market, single-stranded DNA can only be roughly detected using an RNA kit when performing purity detection on this instrument. This result provides the possibility of developing a kit specifically suitable for single-stranded DNA purity detection in the future.

[0180] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A method for preparing a single-stranded DNA molecular weight marker, comprising: 1) mixing a sample containing single-stranded DNA with a single-stranded DNA binding protein and incubating them for a period of time to obtain a complex containing the single-stranded DNA and the single-stranded DNA binding protein; 2) separating and purifying the conjugate obtained in step 1) to remove impurities that are not bound to the single-stranded binding protein, and obtaining a high-purity conjugate of the single-stranded DNA and the single-stranded DNA binding protein; 3) purifying the high-purity conjugate of the single-stranded DNA and the single-stranded DNA binding protein obtained in step 2) to at least remove the single-stranded DNA binding protein to obtain the target product; 4) Repeat step 1) to step 3) m (m≥1) times to obtain different target products, and mix the different target products so that the mixed mixture contains at least two single-stranded DNAs of different lengths. 2 . The method according to claim 1 , wherein when the sample is mixed with the single-stranded DNA binding protein, the final concentration of the single-stranded DNA binding protein is 0.5 ng / μL-500 ng / μL. 3 . The method according to claim 1 , wherein when the sample is mixed with the single-stranded DNA binding protein, the final concentration of the single-stranded DNA binding protein is 1 ng / μL-200 ng / μL.

4. The method of claim 1, wherein the sample is mixed with the single-stranded DNA binding protein and incubated for at least 30 minutes, preferably 2-4 hours.

5. The method according to claim 1, wherein the sample is mixed with the single-stranded DNA binding protein and then incubated at 30-37°C.

6. The method according to claim 1, wherein the separation and purification method in step 2) is selected from at least one of the following methods: affinity chromatography, ion exchange chromatography or gel filtration chromatography, preferably affinity chromatography.

7. The method according to claim 6, wherein the single-stranded DNA binding protein carries a purification tag, and the purification tag is a polyhistidine tag, a glutathione-S transferase tag, a FLAG tag or a Strep-tagII tag.

8. The method according to any one of claims 1 to 7, wherein the method for purifying the conjugate is selected from at least one of the following methods: phenol-chloroform extraction, DNA adsorption centrifugal column recovery, plasmid extraction ion exchange column recovery, high performance liquid chromatography, isopropanol precipitation, ethanol precipitation and molecular sieve chromatography.

9. The method according to claim 1, further comprising performing PCR amplification and DNA sequencing on the target product to verify whether the target product contains the complete sequence of the single-stranded DNA.

10. The method of claim 1, wherein the single-stranded DNA binding protein comprises a single-stranded DNA binding protein derived from Escherichia coli, phi29 phage, Salmonella phage Vi II-E1, Thermomyces HB8, Klebsiella Phi K02 phage, Yersinia Py54 phage or Synechococcus phage S-CBS2, a core functional fragment thereof, a homologous sequence thereof or a mutant sequence thereof.

11. The method according to claim 1, wherein the single-stranded DNA binding protein comprises a sequence having at least 80% sequence identity with any one of the amino acid sequences shown in SEQ ID NOs: 15-18 and having a function of binding to single-stranded DNA.

12. The method according to claim 1, wherein the single-stranded DNA binding protein comprises any one of the amino acid sequences shown in SEQ ID NOs: 15-18 and has a sequence that can bind to single-stranded DNA.

13. The method according to any one of claims 1 to 12, wherein the sample containing single-stranded DNA is prepared by biotin-modified magnetic bead separation, asymmetric PCR, reverse transcription first-strand synthesis, exonuclease digestion or rolling circle amplification.

14. The method according to any one of claims 1 to 13, wherein the length of the longest single-stranded DNA in the single-stranded DNA molecular weight marker is 3000 nt to 7000 nt.

15. The method according to claim 14, wherein the length difference between adjacent single-stranded DNAs is 50 nt to 3000 nt.

16. The method according to claim 15, wherein the difference in the amount of single-stranded DNA of different lengths is ≤10%, or ≤5%, or ≤1%.

17. The method according to any one of claims 1 to 16, wherein the single-stranded DNAs of different lengths are mixed in an equimolar ratio.

18. The method according to any one of claims 15 to 17, wherein the single-stranded DNA molecular weight marker comprises at least five types of single-stranded DNA lengths, including 1000 nt, 2000 nt, 3000 nt, 4000 nt and 5000 nt.

19. The single-stranded DNA molecular weight marker prepared by the method according to any one of claims 1 to 18.

20. A method for determining the length range of single-stranded DNA, comprising the following steps: a) allowing a sample containing single-stranded DNA to be determined that has been pre-contacted with a dye and the detectably labeled single-stranded DNA molecular weight marker of claim 19 to migrate under mobility-dependent separation conditions, and allowing the single-stranded DNA molecular weight marker to form a single-stranded size determination ladder; b) determining the relative position of the ladder according to the size of the single-stranded DNA, and determining the length range of the single-stranded DNA in the sample.

21. The method of claim 20, wherein the detectable label and the dye are independently selected from pyrene, naphthalene, aminopyridine, xanthene, cyanine, coumarin, boropolyazindecine, benzofuran or indole dyes.

22. The method of claim 20, wherein the mobility-dependent separation condition is agarose gel electrophoresis.

23. A kit comprising the single-stranded DNA molecular weight marker according to claim 19.

24. The kit according to claim 23, further comprising a loading buffer; the loading buffer and the single-stranded DNA molecular weight marker are packaged separately, or the two are premixed.

25. The kit according to claim 23 or 24, further comprising the dye and / or the detectable label; the detectable label is packaged separately from the single-stranded DNA molecular weight marker, or is pre-labeled on the single-stranded DNA molecular weight marker.

26. The kit of claim 25, wherein the detectable label and the dye are independently selected from pyrene, naphthalene, aminopyridine, xanthene, cyanine, coumarin, boropolyazindecine, benzofuran or indole dyes.

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