A method for preparing a DNA molecular weight standard based on self-assembly

Abstract

The application discloses a preparation method of DNA molecular weight standards based on self-assembly, comprising the following steps: S1, two single-stranded DNAs with equal length but not complete complementarity are chemically synthesized, and each single-stranded DNA is composed of two sequences, one of which is a complementary sequence, and the other is a self-assembly complementary sequence; S2, directional self-assembly is carried out in a reaction buffer solution, and a ligase is added; S3, reaction is carried out at room temperature for 0.5-10 min, the number of self-assembly is controlled by controlling the reaction time, the number of bases added by each connection is equal to the number of bases of the single-stranded DNA, and a plurality of DNA molecular weight standards with arithmetic progression of sequence length are formed; S4, heat inactivation treatment is carried out; S5, an electrophoresis loading buffer solution is added, and the DNA molecular weight standards are prepared, and the DNA molecular weight standards are stored at low temperature. The application has the advantages of simple process, low manufacturing cost, strong expandability and the like, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a preparation method of a DNA molecular weight standard based on self-assembly. BACKGROUND

[0002] The DNA molecular weight standard is a series of mixed DNA fragment standard reagents with different molecular weight sizes. In the field of molecular biology, the DNA molecular weight standard is used to calibrate the molecular weight of a DNA fragment to be measured. A specific calibration method is to perform electrophoresis experiments on the same agarose gel or polyacrylamide for the DNA molecular weight standard and the DNA fragment to be measured. The DNA fragments with known lengths in the DNA molecular weight standard form a gradient distribution band, indicating the size and distribution of the DNA fragment to be measured with unknown size.

[0003] In molecular biology experiments, since the DNA molecular weight standard is a necessary reagent with high frequency of use and large consumption, there is a great demand for its preparation, and it is required to have low production cost, high indication accuracy and high diversity.

[0004] In the prior art, there are two methods for preparing the DNA molecular weight standard, namely PCR amplification and enzyme digestion of plasmids. The main principle of PCR amplification is to design primers, amplify according to the required length by using a template DNA, purify after amplification, and finally mix the obtained different DNA fragments. The main principle of enzyme digestion of plasmids is to design a pair of primers, amplify by using a template, and obtain DNA fragments. However, for special modified DNA molecular weight standards, such as fluorescent labeling or single-stranded DNA molecular weight standards, there is a lack of simple and efficient preparation methods. Especially for small-fragment single-stranded or double-stranded DNA molecular weight standards, such as 20 nt DNA molecular weight standards, due to the small fragment length, the cost of the PCR amplification method is high, and the enzyme digestion of plasmids method often produces redundant bands, which is not conducive to subsequent purification and limits large-scale production. The currently available small-fragment single-stranded DNA molecular weight standards or fluorescent-modified DNA molecular weight standards have high purchase cost, and there is no fluorescent-modified single-stranded DNA molecular weight standard. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a DNA molecular weight standard based on self-assembly, so as to solve the technical bottleneck of high preparation cost and difficult modification of single-stranded small-fragment DNA molecular weight standards in the prior art.

[0006] In order to solve the above problems, the technical scheme adopted by the present application is as follows:

[0007] According to the first aspect of the present application, a preparation method of DNA molecular weight standard based on self-assembly is provided, comprising the following steps: S1: chemically synthesizing two single-stranded DNAs with equal length but not completely complementary, and each of the two single-stranded DNAs is composed of two sequences: one is a complementary sequence, and the other is a self-assembly complementary sequence; S2: mixing the two single-stranded DNAs in a reaction buffer, and adding a ligase after the two single-stranded DNAs are directionally self-assembled through base complementary pairing; S3: reacting at room temperature for 0.5-10 min, and controlling the self-assembly times by controlling the reaction time, and each ligation increases the number of bases equal to the number of bases of the single-stranded DNA, so as to form a plurality of DNA molecular weight standards with equal-difference sequence length; S4: placing the system after the reaction at high temperature to heat inactivate the ligase; S5: adding an electrophoresis loading buffer with a corresponding concentration in the system to prepare a DNA molecular weight standard, and storing at low temperature.

[0008] According to the present application, the two single-stranded DNAs are equal in the number of bases, and the number of bases determines the sequence length difference of the DNA molecular weight standard prepared therefrom.

[0009] Preferably, the length of the two single-stranded DNAs is between 20-100 nt. More preferably, 20 nt of DNA1-P and DNA2 are used as raw materials for preparing the single-stranded DNA molecular weight standard. It should be understood that the sequence is only used as a preferred example for illustration, and is not used for limitation. The sequence length is preferably between 20-100 nt, which can ensure a high efficiency of self-assembly, and at the same time ensure a high accuracy and low cost during synthesis.

[0010] According to the present application, if the single-stranded DNA molecular weight standard is prepared, the 5' end of one of the DNA chains is modified with a phosphate group; and if the double-stranded DNA molecular weight standard is prepared, the 5' ends of the two DNA chains are both modified with a phosphate group.

[0011] According to the present application, the ligase is an enzyme with DNA ligation function, and is selected from: T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, 9°N DNA Ligase, E. coli DNA ligase, SplintR ligase, 5´ App DNA / RNA thermostable ligase, T4 RNA ligase, T4 RNA ligase 2, or RtcB ligase.

[0012] According to an embodiment of the present application, the final concentration of the T4 DNA ligase is 20 U / μL.

[0013] According to the method of the present application, the concentration of the DNA ligase and the reaction time jointly determine the self-assembly times, but the above-mentioned concentration and reaction time are preferred conditions used in the experiment, and are not used for limitation.

[0014] It should also be understood that according to the method of the present application, the concentration of single-stranded DNA is not limited, and the mixing ratio is not limited. Preferably, in step S2, the concentration of the two single-stranded DNAs is 0.01-100 μM, and the mixing ratio is adjusted according to the target product.

[0015] Preferably, in step S4, three-step inactivation is performed in the order of heating at 65°C for 10 min, ice bath quenching, and EDTA chelation to ensure complete elimination of enzyme activity. The present application first uses such a three-step inactivation method to ensure complete inactivation and product stability. Among them, heating at 65°C for 10 min is to make the protein structure of DNA ligase denatured by heat, ice bath quenching can quickly fix the inactivated state of the enzyme to prevent its renaturation, and EDTA chelation is to further ensure complete elimination of enzyme activity by combining with metal ions to remove metal ions required for the active center of DNA ligase.

[0016] According to the present application, different functional groups or different numbers of functional groups can also be modified during synthesis, and the functional groups are selected from cy3, cy5, azide, methylation, digoxin, or methylene blue, etc. to prepare different modified DNA molecular weight standards. For example, cy3, cy5, etc. fluorescent group modification can make the DNA molecular weight standard easy to be detected and tracked in fluorescence detection experiments; methylation modification can affect the biological activity of DNA and has different application values in specific experimental requirements.

[0017] The reaction buffer includes but is not limited to 6.6 mM MgCl2, 1 mM ATP, 10 mM DTT, 66 mM Tris-HCl buffer with pH 7.6. It should be understood that other concentrations and pH buffers can be used in the present application as long as they can ensure the activity of the enzyme, such as PBS, D-PBS, potassium phosphate buffer, etc., and different concentrations of ATP such as 0.1-10 mM. It should be understood that the reaction buffer is the preferred condition in the experiment and is not used for limitation, and any ligase buffer can be used in the present application.

[0018] According to the second aspect of the present application, a DNA molecular weight standard prepared by the preparation method is provided, which includes a plurality of DNA molecular weight standards with equal-difference incremental sequence lengths.

[0019] According to one preferred embodiment of the present application, a method for preparing a DNA molecular weight standard based on controllable self-assembly of DNA is provided, which includes the following steps:

[0020] Directional self-assembly control: 0.01-100 μM 5-end phosphorylated modified DNA1-P is mixed with complementary strand DNA2 at 1:1 molar concentration to form a self-assembly structure in 66 mM Tris-HCl buffer (pH 7.6) containing 6.6 mM MgCl2, 1 mM ATP, 10 mM DTT;

[0021] Molecular weight gradient generation: T4 DNA ligase is added to a final concentration of 20 U / μL, and the number of self-assembly times is accurately controlled by controlling the reaction time (0.5-10 min), and each ligation increases the number of fixed bases to form an arithmetic gradient;

[0022] Product stabilization treatment: a three-step inactivation method (65°C heating for 10 min → ice bath quenching → EDTA chelation) is used to ensure complete elimination of enzyme activity.

[0023] Product storage: dilute the product to the corresponding concentration, and add the corresponding volume of electrophoresis loading buffer to the system to prepare a DNA molecular weight standard, which is stored at 4°C.

[0024] According to the preparation method of the DNA molecular weight standard provided by the present application, combined with Figure 1 The working principle is as follows: two single-stranded DNAs with equal length but not completely complementary, and each composed of two sequences: one is a complementary sequence, and the other is a self-assembly complementary sequence. Such two single-stranded DNAs can be self-assembled through base complementary pairing, and the 5-end phosphorylated modified DNA1 is connected by DNA ligase, and each ligation assembly increases the same number of bases, forming a gradient-increasing DNA fragment, i.e. a single-stranded DNA molecular weight standard. Similarly, when preparing double-stranded DNA molecular weight, two 5-end phosphorylated modified single-stranded DNAs are used as raw materials for directional self-assembly, and DNA ligase is used to connect DNA1 and DNA2. When preparing a DNA molecular weight standard with modification, single-stranded DNA raw materials with corresponding modification are used.

[0025] In the present application, self-assembly refers to the process of two single-stranded DNAs spontaneously forming a specific spatial structure through base complementary pairing. Specifically, each of the two single-stranded DNAs contains a complementary sequence and a self-assembly complementary sequence. The two parts recognize and bind to each other through base pairing (such as A-T, C-G pairing), so that the two single-stranded DNAs are directionally assembled together to form a stable complex structure. The complementary sequence is used to preliminarily orient the two single-stranded DNAs close to each other during self-assembly, and the self-assembly complementary sequence determines the specificity and stability of the final self-assembly structure.

[0026] This self-assembly is the basis for the subsequent DNA ligase to work, and the assembled structure provides a recognizable connection site for the ligase, allowing the enzyme to connect adjacent DNA chains, and by controlling the reaction time and other conditions, DNA molecular weight standards with equal differences in length increments (such as 20 nt, 40 nt, 60 nt, etc.) are generated.

[0027] According to the present application, the lengths of the complementary sequences of the two single-stranded DNAs and the self-assembly complementary sequences are not fixed, and the number of bases of the two single-stranded DNAs is the same (which determines the sequence length difference of the molecular weight standard), which provides the basis for the flexibility and gradient control of self-assembly.

[0028] Although self-assembly and enzyme ligation are prior art, the innovation of the present application is mainly in the gradient precise control and raw material versatility. The present application method overcomes the high cost and high complexity of the preparation process of traditional single-stranded DNA molecular weight standards, and a plurality of DNA molecular weight standards with equal differences in sequence length increments can be prepared through a simple reaction process.

[0029] Compared with the prior art, the superiority of the technical scheme of the present application mainly lies in the following aspects:

[0030] Directional self-assembly control: using the self-assembly characteristics of 5' phosphorylated DNA and complementary strands, directional connection is achieved through base complementary pairing, which is different from the random fragment generation of traditional enzyme cutting or PCR;

[0031] Gradient precise control: by controlling the reaction time of T4 ligase, the equal difference gradient of "increasing a fixed number of bases each time" is realized, solving the problem of irregular gradient intervals in the prior art;

[0032] Three-step inactivation method: through the combination of heating, ice bath, and EDTA chelation, the enzyme activity is completely eliminated, and the stability of the product is improved, while the prior art may only use single heating inactivation, which has relatively limited effect;

[0033] Raw material versatility: using short-chain DNA (such as 20 nt) as raw material, which can be extended to long fragments through self-assembly, reducing the synthesis cost, while the traditional chemical synthesis of long fragments has high cost.

[0034] In summary, the preparation method of the DNA molecular weight standard based on self-assembly provided by the application can prepare DNA molecular weight standards of different gradients, and the process is simple and the preparation cost is low. The method can also prepare DNA molecular weight standards with different modifications, such as fluorescent molecule modification and cholesterol modification. The method is particularly suitable for preparing DNA molecular weight standards with a chemical modification function in the range of 20-100 nt, and is not limited to single-stranded or double-stranded DNA molecular weight standards, solving the technical bottleneck of high preparation cost and modification difficulty of single-stranded small fragment DNA molecular weight standards in the prior art. The application adopts a one-pot method to quickly prepare DNA molecular weight standards, has the advantages of simple process, low manufacturing cost, strong expandability and the like, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A schematic diagram of controllable self-assembly of two single-stranded DNAs to prepare a DNA molecular weight standard according to the method of the application;

[0036] Figure 2 A comparison diagram of the DNA molecular weight standard prepared by using 20 nt single-stranded DNA as a raw material and a commercial DNA molecular weight standard of a certain company; wherein, band 1 is the commercial DNA molecular weight standard of the certain company, and band 2 is the DNA molecular weight standard prepared by the method;

[0037] Figure 3 A gel diagram of the DNA molecular weight standard prepared by using 20 nt single-stranded DNA as a raw material and a single-stranded DNA with a known length of 42 nt; wherein, band 1 is the DNA molecular weight standard prepared by using 20 nt single-stranded DNA as a raw material, and band 2 is the 42 nt single-stranded DNA chain;

[0038] Figure 4 A gel diagram of the DNA molecular weight standard prepared by using 25 nt single-stranded DNA as a raw material and a DNA with a known length of 42 nt; wherein, band 1 is the DNA molecular weight standard prepared by using 25 nt single-stranded DNA as a raw material, and band 2 is the 42 nt single-stranded DNA chain;

[0039] Figure 5 An assembly effect of the DNA molecular weight standard prepared by using DNA ligase to connect different times by using 20 nt single-stranded DNA as a raw material; wherein, band 1 is the product obtained by connecting for 0.5 min, band 2 is the product obtained by connecting for 1 min, band 3 is the product obtained by connecting for 2 min, band 4 is the product obtained by connecting for 3 min, band 5 is the product obtained by connecting for 4 min, band 6 is the product obtained by connecting for 5 min, band 7 is the product obtained by connecting for 6 min, band 8 is the product obtained by connecting for 7 min, band 9 is the product obtained by connecting for 8 min, band 10 is the product obtained by connecting for 9 min, and band 11 is the product obtained by connecting for 10 min.

[0040] Figure 6 Assemble effect of preparing DNA molecular weight standard at different concentrations using 20 nt single-stranded DNA as raw material; wherein, band 1 is commercialized DNA molecular weight standard, band 2 is 0.1 μM product, band 3 is 0.5 μM product, band 4 is 1 μM product, band 5 is 2 μM product, band 6 is 4 μM product, band 7 is 6 μM product, band 8 is 8 μM product, and band 9 is 10 μM product;

[0041] Figure 7 Assemble effect of preparing DNA molecular weight standard using different concentrations and different types of DNA ligase to connect 5 min using 20 nt single-stranded DNA as raw material; wherein, band 1 is 15 U / μL T3 DNA ligase product, band 2 is 75 U / μL T3 DNA ligase product, band 3 is 150 U / μL T3 DNA ligase product, band 4 is 225 U / μL T3 DNA ligase product, band 5 is 300 U / μL T3 DNA ligase product, band 6 is 1.75 U / μL T4 DNA ligase product, band 7 is 8.75 U / μL T4 DNA ligase product, band 8 is 17.5 U / μL T4 DNA ligase product, band 9 is 26.25 U / μL T4 DNA ligase product, band 10 is 35 U / μL T4 DNA ligase product, band 11 is 15 U / μL T7 DNA ligase product, band 12 is 75 U / μL T7 DNA ligase product, band 13 is 150 U / μL T7 DNA ligase product, band 14 is 225 U / μL T7 DNA ligase product, and band 15 is 300 U / μL T7 DNA ligase product. DETAILED DESCRIPTION

[0042] The present application is further described in conjunction with the following specific examples. It should be understood that the following examples are intended to illustrate the application and are not intended to limit the scope of the application. Unless otherwise indicated, the techniques utilized in the examples are routine procedures available to those skilled in the art, or as suggested by the manufacturer of the kits and instruments. The reagents and materials used in the examples are commercially available unless otherwise specified.

[0043] Table 1 DNA sequences used in the present specification

[0044]

[0045] Example 1: Preparation of DNA ladder using 20 nt single-stranded DNA as raw material

[0046] After mixing 5 μM of 20 nt DNA1-P and 20 nt DNA2, T4 DNA ligase was added and reacted for 5 min; the reaction system was placed at 65 ℃ for 10 min, after heat inactivation of the ligase, the DNA chain was diluted to 2 μM, the same volume of 2× denaturation loading buffer was added to the system, and it was stored at 4 ℃.

[0047] As shown in the results Figure 2 , band 1 is a commercial DNA ladder of a certain company, and band 2 is the DNA ladder prepared in this example. It can be seen that 11 DNA ladders are successfully prepared according to the method of this example within 5 min.

[0048] As shown in the results Figure 3 , band 1 is a DNA ladder prepared using 20 nt single-stranded DNA as raw material, and band 2 is a 42 nt single-stranded DNA chain. It can be seen that the DNA ladder prepared according to the method of this example within 5 min meets the expectation.

[0049] Example 2: Preparation of DNA ladder using 25 nt single-stranded DNA as raw material

[0050] After mixing 5 μM of 25 nt DNA1-P and 25 nt DNA2, T4 DNA ligase was added and reacted for 5 min; the reaction system was placed at 65 ℃ for 10 min, after heat inactivation of the ligase, the DNA chain was diluted to 2 μM, the same volume of 2× denaturation loading buffer was added to the system, and it was stored at 4 ℃.

[0051] As shown in the results Figure 4 , band 1 is a DNA ladder prepared using 25 nt single-stranded DNA as raw material, and band 2 is a 42 nt single-stranded DNA chain. It can be seen that the DNA ladder prepared according to the method of this example within 5 min meets the expectation.

[0052] Example 3: Preparation of 20 nt single-stranded DNA ladder with different ligation times

[0053] After mixing 5 μM of 20 nt DNA1-P and 20 nt DNA2, T4 DNA ligase was added, and different reaction times were used: 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min; after the reaction, the system was placed at 65 ℃ for 10 min, after the heat inactivation of the ligase, the DNA chain was diluted to 2 μM, the same volume of 2 × denaturation loading buffer was added to the system, and it was stored at 4 ℃.

[0054] The results are shown in Figure 5 As shown in the table, different concentrations of 20 nt single-stranded DNA were used as raw materials to prepare DNA molecular weight standards, and the assembly effects were different, and with the increase of the reaction time, the molecular weight standards with higher molecular weight (such as 220 nt, 200 nt) were also more.

[0055] Example 4: Preparation of DNA molecular weight standards using different concentrations of 20 nt single-stranded DNA as raw materials

[0056] After mixing different concentrations (0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM) of 20 nt DNA1-P and 20 nt DNA2, T4 DNA ligase was added, and the reaction was carried out for 5 min; after the reaction, the system was placed at 65 ℃ for 10 min, after the heat inactivation of the ligase, the DNA chain was diluted to 2 μM, the same volume of 2 × denaturation loading buffer was added to the system, and it was stored at 4 ℃.

[0057] The results are shown in Figure 6 As shown in the table, band 1 is a commercial DNA molecular weight standard of a certain company, and bands 2-9 are the products obtained in this example, and thus it can be seen that with the increase of the concentration of single-stranded DNA, the molecular weight standards with lower molecular weight (such as 40 nt, 60 nt) are also more.

[0058] Example 5: Preparation of DNA molecular weight standards using different DNA ligases and different concentrations of 20 nt single-stranded DNA as raw materials

[0059] After mixing 5 μM of 20 nt DNA1-P and 20 nt DNA2, different concentrations (0.1 × - 2 ×) of DNA ligase (T3 DNA ligase, T4 DNA ligase, T7 DNA ligase) were added, and the reaction was carried out for 5 min; after the reaction, the system was placed at 65 ℃ for 10 min, after the heat inactivation of the ligase, the DNA chain was diluted to 2 μM, the same volume of 2 × denaturation loading buffer was added to the system, and it was stored at 4 ℃.

[0060] The results are as follows Figure 7 As shown, different DNA ligases have similar ligation effects, and as the concentration of the ligase increases, more molecular weight standards with higher molecular weights are produced.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A method for preparing a self-assembly based DNA molecular weight standard, characterized by, The method comprises the following steps: S1: chemically synthesizing two single-stranded DNAs with equal length but not completely complementary, the length of the two single-stranded DNAs is between 20-100 nt, and each single-stranded DNA is composed of two sequences: one is a complementary sequence, and the other is a self-assembling complementary sequence, the two sequences are recognized and combined by base pairing, so that the two single-stranded DNAs are directionally assembled together to form a stable complex structure; S2: mixing the two single-stranded DNAs in a reaction buffer, the concentration of the two single-stranded DNAs is 0.01-100 μM, and after the directional self-assembly of the two single-stranded DNAs through base complementary pairing, a ligase is added; S3: reacting at room temperature for 0.5-10 min, and controlling the self-assembly times by controlling the reaction time, each ligation increases the number of bases equal to the number of bases of the single-stranded DNA, to form a plurality of DNA molecular weight standards with equidifferent incremental sequence lengths; S4: placing the reaction system at 65°C to heat inactivate the ligase; S5: adding an electrophoresis loading buffer with a corresponding concentration in the system to prepare a DNA molecular weight standard, and storing at 4°C.

2. The production method according to claim 1, characterized by, The number of bases of the two single-stranded DNAs is equal, and the number of bases determines the sequence length difference of the DNA molecular weight standard prepared therefrom.

3. The preparation method according to claim 1, characterized in that, The sequence lengths of the two parts of the two single-stranded DNAs are not fixed.

4. The method of claim 1, wherein, If a single-stranded DNA molecular weight standard is prepared, the 5' end of one DNA strand is modified with a phosphate group; if a double-stranded DNA molecular weight standard is prepared, the 5' ends of the two DNA strands are both modified with phosphate groups.

5. The preparation method according to claim 1, characterized in that, The ligase is an enzyme with DNA ligation function, and is selected from: T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, 9°N DNA Ligase, E. coli DNA ligase, SplintR ligase, 5´ App DNA / RNA thermostable ligase, T4 RNA ligase, T4 RNA ligase 2, or RtcB ligase.

6. The method of claim 1, wherein, In step S4, three steps of heating at 65°C for 10 min, ice bath quenching, and EDTA chelation are sequentially performed to ensure that the enzyme activity is completely eliminated.

7. The preparation method according to claim 1, characterized in that, During the synthesis process, modification of different functional groups or different numbers of functional groups is performed, and the functional groups are selected from: cy3, cy5, azide, methylation, digoxin, or methylene blue, to prepare DNA molecular weight standards with different modifications.

8. A DNA molecular weight standard prepared by the method according to any one of claims 1-7, characterized in that, The method comprises a plurality of DNA molecular weight standards with equidifferent incremental sequence lengths. The method comprises a plurality of DNA molecular weight standards with equidifferent incremental sequence lengths.

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