A method for preparing a DNA ladder of customizable size without restriction enzymes

Abstract

The application discloses a method for preparing a DNA ladder with a self-defined size without a restriction endonuclease, and belongs to the technical field of biology. The method is characterized in that: a template is designed with a linker chain at the head and tail, so that the template can be self-circulated and amplified without a restriction endonuclease and plasmid construction; and the non-standard molecular weight of the DNA ladder can be customized by directly editing and adjusting the intermediate sequence in the template sequence and the base length of the linker chain. The gradient size of the DNA ladder is the sum of the sizes of the intermediate sequence and the linker chain, and the DNA ladder is suitable for precise indication of the molecular size in agarose electrophoresis. The preparation period of the application can be shortened to 6 hours, and the application provides a more simple and fast way for the preparation of the DNA ladder and a new strategy for the preparation of the DNA ladder with a non-standard molecular weight.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing a DNA ladder of custom size without the need for restriction endonucleases. Background Technology

[0002] In the field of molecular biology, DNA molecular weight standards (DNA ladders) are key reagents for indicating the size of DNA molecules in agarose gel electrophoresis. Their preparation methods include restriction endonuclease digestion and PCR amplification, but existing technologies have significant shortcomings. For example, the method disclosed in CN103667264A (publication date: March 26, 2014) constructs plasmids containing specific sequences and obtains DNA markers by digestion with restriction endonucleases such as XmnⅠ and BamHI. Although this method is highly stable and can be mass-produced, it requires cumbersome steps such as vector construction and host transformation. Furthermore, the fragment size is limited by the design of the restriction sites, making it difficult to flexibly customize non-standard fragment sizes. Additionally, the digestion reaction conditions can easily affect product efficiency. CN106011133A (publication date: October 12, 2016) constructs small fragments ranging from 100bp to 700bp into a single plasmid, and obtains a DNA marker with uniform bands by digestion with EcoRI and other enzymes. Although this method solves some problems in the preparation of small fragments, it still relies on a complex plasmid construction process, and the enzyme digestion step increases the operational complexity, making it difficult to quickly respond to diverse fragment requirements. PCR amplification methods, such as those proposed in CN104988134A (publication date: October 21, 2015), clone the target fragment into the pMD18-T vector, amplify it using universal primers M13F / M13R, and then prepare a DNA ladder by mixing them. This reduces the cost by 90% compared to the enzyme digestion method, but still requires a vector cloning step. For small fragments such as 100bp, there are problems such as low amplification efficiency and easy formation of primer dimers. Moreover, each new fragment size requires the reconstruction of a new vector, resulting in high customization costs. Although CN110714053A (publication date: 2020.01.21) achieves efficient amplification of 100bp DNA fragments by combining bridging primers with high-fidelity enzymes (such as Pfu enzymes) without the need for vector construction, this method relies on specific primer design and is only applicable to small molecular weight fragments. It lacks universality for customizing a wider range of fragments. At the same time, conventional PCR has large differences in amplification efficiency for fragments of different sizes, which can easily lead to uneven band brightness after mixing and affect the electrophoresis indication effect.

[0003] In existing technologies, enzyme digestion methods rely on restriction endonucleases and plasmid construction, which are cumbersome and offer limited freedom in fragment design. While PCR methods partially avoid enzyme digestion, the complexity of vector cloning or primer design still limits customization efficiency, especially for the preparation of non-standard molecular weight fragments (such as 350bp and 680bp), where it is difficult to balance ease of operation with size flexibility. Therefore, there is an urgent need for a method that does not require restriction endonucleases and can directly achieve customized preparation of DNA ladders of any size through sequence design and PCR amplification, in order to overcome the bottlenecks in efficiency, cost, and flexibility of existing technologies. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing custom-sized DNA ladders without the need for restriction endonucleases, thereby addressing the problems existing in the prior art. This invention achieves template self-circulating amplification through the design of the template's head and tail linker strands, eliminating the need for restriction endonucleases and plasmid construction. It allows for the direct customization of non-standard molecular weight DNA ladders by editing and adjusting the base length of the template sequence, making it suitable for precise molecular size indication in agarose gel electrophoresis. This invention shortens the preparation cycle to 6 hours, providing a simpler and faster method for DNA ladder preparation and offering a new strategy for preparing DNA ladders of non-standard molecular weights.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing a custom-sized DNA ladder without restriction endonucleases, comprising the following steps:

[0007] (1) Design and synthesize a template sequence; the template sequence consists of an intermediate sequence of customizable size and a linker chain connecting the beginning and end of the intermediate sequence;

[0008] (2) Design primers that are complementary to the intermediate sequence;

[0009] (3) Using the template sequence and the primers, amplification is performed using nucleic acid amplification technology to obtain a DNA ladder of customizable size;

[0010] The gradient size of the DNA ladder is the sum of the size of the intermediate sequence and the size of the linker chain.

[0011] This invention does not have special requirements for the type and sequence of bases in the intermediate sequence. It only needs to consider the molecular weight, that is, the number of bases contained in the fragment, and has no special limitations on the sequence itself.

[0012] Optionally, the linker chain size is 10-15 bp.

[0013] Optionally, the nucleotide sequence of the linker chain is shown in SEQ ID NO.2.

[0014] Optionally, the nucleotide sequence of the intermediate sequence is as shown in SEQ ID NO.1 or SEQ ID NO.6.

[0015] Furthermore, the nucleic acid amplification technology includes isothermal amplification technology or Taq enzyme temperature-variable amplification technology.

[0016] The present invention also provides a DNA ladder prepared using the above-described preparation method.

[0017] The present invention also provides a template sequence for preparing a DNA ladder, the template sequence comprising an intermediate sequence of customizable size and a linker strand connected to both ends of the intermediate sequence; the nucleotide sequence of the linker strand is shown in SEQ ID NO.2.

[0018] The present invention also provides a primer for preparing a DNA ladder, wherein the primer is complementary to the above-mentioned intermediate sequence.

[0019] The present invention also provides a kit for preparing a DNA ladder, the kit comprising the template sequence and the primers described above.

[0020] The present invention discloses the following technical effects:

[0021] (1) Linker chain self-circulation mechanism: This invention adopts the template head and tail linker chain repeat design to realize template "self-bridging" amplification, without the need for additional primers, which can simplify the primer design process and the preparation process.

[0022] (2) Enzyme-free design: The DNA ladder preparation method of the present invention completely eliminates the use of restriction endonucleases. The target fragment can be directly synthesized through nucleic acid amplification technology, and the cost is reduced by 70% compared with the enzyme digestion method.

[0023] (3) Size customization: This invention can directly control the product size by adjusting the template sequence length, breaking through the size limitations of vector preparation methods and fixed fragment preparation methods, and can quickly prepare fragments that are not conventionally covered, such as 800bp, 900bp, etc.

[0024] This invention achieves template self-circulating amplification through the design of linker strands at both ends of the template, eliminating the need for restriction endonucleases and plasmid construction. It allows for the direct customization of DNA ladders with non-standard molecular weights by editing and adjusting the base length of the template sequence, making it suitable for precise molecular size indication in agarose gel electrophoresis. The preparation cycle of this invention is shortened to 6 hours, providing a simpler and faster method for DNA ladder preparation and offering a new strategy for preparing DNA ladders with non-standard molecular weights. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The DNA ladder products obtained by RPA isothermal amplification in Example 1 are shown below. Lane 1 is an M2000 DNA ladder with DNA lengths of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom. Lanes 2 to 9 are the product bands amplified at 26.3℃, 29℃, 32℃, 35℃, 38℃, 41℃, 44℃, and 46.7℃, respectively.

[0027] Figure 2 The DNA ladder products obtained by temperature-controlled amplification in Example 2 are shown below. Lane 1 is an M2000 DNA ladder with DNA lengths of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom. Lanes 2 to 6 are the product bands amplified at 41℃, 45.5℃, 50℃, 54.5℃, and 59℃, respectively.

[0028] Figure 3 The DNA ladder products obtained by temperature-controlled amplification in Example 3 are shown below. Lane 1 is an M700 DNA ladder with DNA lengths of 700bp, 600bp, 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, and 50bp from top to bottom. Lanes 2 to 9 are the product bands amplified at 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, and 66℃, respectively.

[0029] Figure 4The bands are the product bands obtained by temperature-controlled amplification in Comparative Example 1. Lane 1 is the M2000 DNA Ladder, with DNA lengths of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom. Lanes 2 to 5 are the product bands amplified at 45.5℃, 50℃, 54.5℃, and 59℃ with a template concentration of 10nM, respectively. Lanes 6 to 9 are the product bands amplified at 45.5℃, 50℃, 54.5℃, and 59℃ with a template concentration of 10pM, respectively. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] The technical principle of this invention is as follows:

[0036] This invention achieves DNA ladder preparation without restriction endonucleases by designing template sequences containing identical repeating linker strands at both ends, utilizing specific primers complementary to the template, and combining high-fidelity PCR amplification technology. Specifically, the middle sequence of the template sequence serves as the primer binding site, enabling self-circulating amplification of the target fragment through PCR cycles, avoiding the steps of plasmid construction and restriction endonucleases required by traditional enzyme digestion methods. The editable sequence in the middle region directly determines the fragment size of the DNA ladder, allowing for customization of molecular weights from 50bp to 1000bp by adjusting the base length in this region. The 3'-5' repair capability of the high-fidelity DNA polymerase ensures the accuracy of the amplified products. Finally, after detection and purification, a homogeneous DNA molecular weight standard is obtained for molecular size indication by agarose gel electrophoresis.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0038] Example 1

[0039] In this embodiment, a 53bp DNA ladder (i.e., fragment sizes of 65bp, 118bp, 171bp, 224bp, 277bp, 330bp, 383bp, ...) was prepared using the RPA isothermal amplification method. The specific process is as follows:

[0040] 1. Template sequence design and synthesis

[0041] Design intermediate sequences:

[0042] 5'-TCTAACCGTACAGTATTTTCCCGGCGGCGCAGCAGTTAGAT-3' (SEQ ID NO. 1);

[0043] Design a linker chain connecting the beginning and end of the intermediate sequence:

[0044] 5'-GGTGGTGGTGGT-3' (SEQ ID NO. 2);

[0045] The final template sequence is synthesized artificially:

[0046] 5'-GGTGGTGGTGGTTCTAACCGTACAGTATTTTCCCGGCGGCGCAGCAGTTAGATGGTGGTGGTGGT-3' (SEQ ID NO. 3).

[0047] 2. Primer design

[0048] Specific primers were designed using Primer 5 software. The 5' end of the forward primer was complementary to the beginning of the middle sequence, and the 5' end of the reverse primer was complementary to the end of the middle sequence.

[0049] Forward primer: 5'-TCTAACCGTACAGTATTTTCC-3' (SEQ ID NO.4);

[0050] Reverse primer: 5'-ATCTAACTGCTGCGC-3' (SEQ ID NO.5).

[0051] 3. PCR amplification

[0052] PCR amplification was performed using RPA isothermal amplification, and the reaction system was as follows (50 μL):

[0053] Template sequence: 2 μL (5 ng / μL); forward primer: 2 μL (10 μM); reverse primer: 2 μL (10 μM); solubilizer: 20 μL; activator: 2 μL; ddH2O: 22 μL. The solubilizer contains potassium acetate, dithiothreitol, polyethylene glycol (molecular weight 1450-20000), ATP, creatine phosphate, creatine kinase, cryophage uvsX, cryophage gp32, cryophage uvsY, Staphylococcus aureus polymerase I large fragment (exo-), Bacillus subtilis polymerase I large fragment (exo-), dNTPs, and other components required for the RPA reaction. The activator contains magnesium acetate.

[0054] The reaction conditions were as follows: constant temperature incubation for 20 minutes at 26.3℃, 29℃, 32℃, 35℃, 38℃, 41℃, 44℃ and 46.7℃ respectively.

[0055] 4. Product testing

[0056] Take 5 μL of PCR amplification product, add 1 μL of 6× Loading Buffer, mix thoroughly, and then perform 2% agarose gel electrophoresis for detection.

[0057] The results are as follows Figure 1As shown, lane 1 is the M2000 DNA Ladder, and lanes 2 to 9 contain product bands amplified at isothermal temperatures of 26.3℃, 29℃, 32℃, 35℃, 38℃, 41℃, 44℃, and 46.7℃, respectively. It can be seen that the amplified products contain bands of various sizes: 65bp, 118bp, 171bp, 224bp, 277bp, 330bp, and 383bp. The number of amplified product bands varies at different temperatures, but the band fragment sizes are consistent. Notably, the product amplified at 26.3℃ contains only one band; due to the lower temperature and lower amplification efficiency, the product quantity is small, and the band clarity is low.

[0058] Example 2

[0059] In this embodiment, a 53bp DNA ladder (i.e., fragment sizes of 65bp, 118bp, 171bp, 224bp, ...) was prepared using the Taq enzyme temperature-controlled amplification method. The specific process is as follows:

[0060] 1. Template sequence design and synthesis

[0061] Design intermediate sequences:

[0062] 5'-TCTAACCGTACAGTATTTTCCCGGCGGCGCAGCAGTTAGAT-3' (SEQ ID NO. 1);

[0063] Design a linker chain connecting the beginning and end of the intermediate sequence:

[0064] 5'-GGTGGTGGTGGT-3' (SEQ ID NO. 2);

[0065] The final template sequence is synthesized artificially:

[0066] 5'-GGTGGTGGTGGTTCTAACCGTACAGTATTTTCCCGGCGGCGCAGCAGTTAGATGGTGGTGGTGGT-3' (SEQ ID NO. 3).

[0067] 2. Primer design

[0068] Specific primers were designed using Primer 5 software. The 5' end of the forward primer was complementary to the beginning of the middle sequence, and the 5' end of the reverse primer was complementary to the end of the middle sequence.

[0069] Forward primer: 5'-TCTAACCGTACAGTATTTTCC-3' (SEQ ID NO.4);

[0070] Reverse primer: 5'-ATCTAACTGCTGCGC-3' (SEQ ID NO.5).

[0071] 3. PCR amplification

[0072] PCR amplification was performed using Taq DNA polymerase at varying temperatures. The reaction volume was as follows (20 μL):

[0073] Template sequence: 0.8 μL (5 ng / μL); Forward primer: 0.8 μL (10 μM); Reverse primer: 0.8 μL (10 μM); 2× reaction premix (containing dNTPs, Taq DNA polymerase, Mg...) 2+ (Components required for PCR reaction): 10 μL; ddH2O: 7.6 μL.

[0074] The reaction conditions are as follows: Step 1: Pre-denaturation at 95℃ for 30s; Step 2: Denaturation at 95℃ for 5s; Step 3: Annealing and extension at 41℃, 45.5℃, 50℃, 54.5℃ or 59℃ for 30s. Skip to Step 2 and repeat the cycle for a total of 30 times.

[0075] 4. Product testing

[0076] Take 5 μL of PCR amplification product, add 1 μL of 6× Loading Buffer, mix thoroughly, and then perform 2% agarose gel electrophoresis for detection.

[0077] The results are as follows Figure 2 As shown, lane 1 is the M2000 DNA ladder, with DNA lengths from top to bottom of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. Lanes 2 to 6 show the product bands at extension temperatures of 41℃, 45.5℃, 50℃, 54.5℃, and 59℃, respectively. It can be seen that the amplified products contain bands of varying sizes: 65bp, 118bp, 171bp, and 224bp. The number of bands varies at different temperatures, but the band fragment sizes are consistent.

[0078] Example 3

[0079] In this embodiment, a 40bp DNA ladder (i.e., fragment sizes of 52bp, 92bp, 132bp, 172bp, ...) was prepared using the Taq enzyme temperature-dependent amplification method. The specific process is as follows:

[0080] 1. Template sequence design and synthesis

[0081] Design intermediate sequences:

[0082] 5'-CCGGATGCGGAGTAATCAAACGCGCACT-3' (SEQ ID NO. 6);

[0083] Design a linker chain connecting the beginning and end of the intermediate sequence:

[0084] 5'-GGTGGTGGTGGT-3'(SEQ ID NO.2)

[0085] The final template sequence is synthesized artificially:

[0086] 5'-GGTGGTGGTGGTCCGGATGCGGAGTAATCAAACGCGCACTGGTGGTGGTGGT-3' (SEQ ID NO. 7).

[0087] 2. Primer design

[0088] Specific primers were designed using Primer 5 software. The 5' end of the forward primer was complementary to the beginning of the middle sequence, and the 5' end of the reverse primer was complementary to the end of the middle sequence.

[0089] Forward primer: 5'-CCGGATGCGGAGT-3' (SEQ ID NO.8);

[0090] Reverse primer: 5'-AGTGCGCGTTTGATT-3' (SEQ ID NO.9).

[0091] 3. PCR amplification

[0092] PCR amplification was performed using Taq DNA polymerase at varying temperatures. The reaction volume was as follows (20 μL):

[0093] Template sequence: 0.8 μL (5 ng / μL); Forward primer: 0.8 μL (10 μM); Reverse primer: 0.8 μL (10 μM); 2× reaction premix (containing dNTPs, Taq DNA polymerase, Mg...) 2+ (Components required for PCR reaction): 10 μL; ddH2O: 7.6 μL.

[0094] The reaction conditions are as follows: Step 1: Pre-denaturation at 95℃ for 30s; Step 2: Denaturation at 95℃ for 5s; Step 3: Annealing and extension at 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃ or 66℃ for 30s. Skip to Step 2 and repeat the cycle for a total of 20 times.

[0095] 4. Product testing

[0096] Take 5 μL of PCR amplification product, add 1 μL of 6× Loading Buffer, mix thoroughly, and then perform 2% agarose gel electrophoresis for detection.

[0097] The results are as follows Figure 3 As shown, lane 1 is the M700 DNA Ladder, and lanes 2 to 9 contain product bands at extension temperatures of 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, and 66℃, respectively. It can be seen that the amplified products contain bands of various sizes, including 52bp, 92bp, 132bp, and 172bp. The number of product bands varies at different temperatures, but the band fragment sizes are consistent.

[0098] Comparative Example 1

[0099] The difference between this comparative example and Example 1 is that it does not use the preparation method of linker chain repeat sequence design. The specific process is as follows:

[0100] 1. Template sequence design and synthesis

[0101] The template sequence was designed as 5'-TCTAACCGTACAGTATTTTCCCGGCGGCGCAGCAGTTAGAT-3' (SEQ ID NO.1) and synthesized artificially.

[0102] 2. Primer design

[0103] Specific primers were designed using Primer 5 software. The 5' end of the forward primer was complementary to the head end of the template, and the 5' end of the reverse primer was complementary to the tail end of the template.

[0104] Forward primer: 5'-TCTAACCGTACAGTATTTTCC-3' (SEQ ID NO.4);

[0105] Reverse primer: 5'-ATCTAACTGCTGCGC-3' (SEQ ID NO.5).

[0106] 3. PCR amplification

[0107] PCR amplification was performed using Taq DNA polymerase at varying temperatures. The reaction volume was as follows (20 μL):

[0108] Template sequence: 0.8 μL (10 nM / 10 pM); Forward primer: 0.8 μL (10 μM); Reverse primer: 0.8 μL (10 μM); 2× reaction premix (containing dNTPs, Taq DNA polymerase, Mg...) 2+ (Components required for PCR reaction): 10 μL; ddH2O: 7.6 μL.

[0109] The reaction conditions are as follows: Step 1: Pre-denaturation at 95℃ for 30s; Step 2: Denaturation at 95℃ for 5s; Step 3: Annealing and extension at 45.5℃, 50℃, 54.5℃ or 59℃ for 30s. Skip to Step 2 and repeat the cycle for a total of 30 times.

[0110] 4. Product testing

[0111] Take 5 μL of PCR amplification product, add 1 μL of 6× Loading Buffer, mix thoroughly, and then perform 2% agarose gel electrophoresis for detection.

[0112] The results are as follows Figure 4 As shown, lane 1 is the M2000 DNA Ladder; lanes 2 to 5 represent the product bands amplified at extension temperatures of 45.5℃, 50℃, 54.5℃, and 59℃ when the template concentration is 10 nM; and lanes 6 to 9 represent the product bands amplified at the same temperatures when the template concentration is 10 pM. It is evident that only a 41 bp band exists in the amplified product.

[0113] The results of Examples 1-3 demonstrate that the linker repeat design method of this invention is crucial for preparing high-purity DNA ladders. Examples 1-3 also show that the intermediate sequence of the template has customizable characteristics, allowing for the amplification of DNA sequences of any size to obtain a DNA ladder with a target gradient, which can then be used as an indicator for gel electrophoresis.

[0114] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a custom-sized DNA ladder without restriction endonucleases, characterized in that, Includes the following steps: (1) Design and synthesize a template sequence; the template sequence consists of an intermediate sequence of customizable size and a linker chain connected to both ends of the intermediate sequence; the nucleotide sequence of the linker chain is shown in SEQ ID NO.2, and the nucleotide sequence of the intermediate sequence is shown in SEQ ID NO.1 or SEQ ID NO.6; (2) Design primers that are complementary to the intermediate sequence; (3) Using the template sequence and the primers, amplification is performed using nucleic acid amplification technology to obtain a DNA ladder of customizable size; The gradient size of the DNA ladder is the sum of the size of the intermediate sequence and the size of the linker chain; The nucleic acid amplification technology includes isothermal amplification technology or Taq enzyme temperature-variable amplification technology.

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