High-resolution capillary electrophoresis screening medium and application

Abstract

The invention discloses a high-resolution capillary electrophoresis screening medium. The high-resolution capillary electrophoresis screening medium is prepared by mixing a screening polymer and a self-coating viscosity adjusting polymer, wherein the screening polymer is linear polyacrylamide, the molecular weight Mn of the linear polyacrylamide is 1.4 to 1.5 MDa, and the PDI of the linear polyacrylamide is 1.5 to 2.0; the self-coating viscosity adjusting polymer is a mixture of a plurality of linear poly N, N-dimethylacrylamide with different molecular weights, the molecular weight Mn of the self-coating viscosity adjusting polymer is 340 kDa to 360 kDa, and the PDI is 3.8 to 4.2. The prepared screening medium is high in resolution ratio, good in stability and accurate in typing.

Description

Technical Field

[0001] The invention relates to a capillary electrophoresis screening medium, in particular to a high-resolution capillary electrophoresis screening medium for forensic DNA detection, and also to a preparation method and use of the capillary electrophoresis screening medium, belonging to the technical field of biological detection. Background Art

[0002] Capillary electrophoresis (CE) is an efficient biological sample separation technology. It is widely used in many fields such as biochemistry, molecular biology and clinical medicine due to its rapidity, high efficiency, high resolution, good repeatability, high sensitivity and easy automation. Especially in the field of forensic DNA detection, the DNA detection technology based on the STR-PCR-capillary electrophoresis fluorescence detection platform has developed rapidly, and the unique advantages of capillary electrophoresis technology have been fully demonstrated, becoming one of the most important technical means in the field of modern forensic science.

[0003] Capillary electrophoresis sieving media are key consumables in capillary electrophoresis. They are polymer solutions with specific molecular weight and viscosity characteristics. As separation media, they can be analyzed and separated according to the size of DNA molecules. The type, composition, structure and corresponding properties of the sieving media determine the migration characteristics, separation degree, read length, reproducibility, injection time, injection pressure and capillary service life of biomolecules. They have a significant impact on capillary electrophoresis separation technology and are the core key to ensuring the accuracy, detection resolution and detection throughput of DNA testing.

[0004] Initially, the sieving medium used in capillary electrophoresis for DNA detection was cross-linked polyacrylamide gel. Although this gel has high resolution, it is difficult to prepare, the process is complicated, the service life is short, and it cannot be automated. The polymerization reaction is affected by many factors, resulting in poor batch stability, and it does not have the ability of capillary self-coating, which cannot meet the use requirements of the existing technical system. Gel-free sieving medium, that is, non-cross-linked linear polymer solution, has been widely used in the field of capillary electrophoresis due to its advantages such as low viscosity, simple preparation, good repeatability, high separation efficiency, wide molecular weight application range and automated operation. In the late 1990s, gene sequencing centers generally abandoned gel media and switched to gel-free sieving media based on automated capillary arrays (CAE) for DNA screening, which greatly promoted the early completion of the human genome project.

[0005] In Chinese invention patent ZL 201510477236.9, a sieving medium used for capillary electrophoresis is disclosed. The quasi-interpenetrating polymer network of long-chain linear poly(N,N dimethylacrylamide) and short-chain linear poly(N,N dimethylacrylamide) and random copolymer (acrylamide-N,N dimethylacrylamide) is prepared by redox free radical polymerization, which solves the mutual solubility and reproducibility problems of linear polyacrylamide (LPA) and linear poly-N,N dimethylacrylamide (PDMA). However, due to limited resolution, it is difficult for copolymers to achieve long-chain polymerization and the molecular weight is small, which has great limitations in forensic DNA detection with higher requirements. In addition, U.S. Patent US008221607B2 describes the preparation of non-cross-linked linear polyacrylamide polymers by solution polymerization and reverse emulsion polymerization, and mixing them with PDMA and a specific sol buffer to form a sieving medium for capillary electrophoresis. PDMA in the sieving medium acts as a surface interaction component, which helps to improve the interaction between the inner wall of the capillary and the separation medium, reduce the nonspecific adsorption of the analyte to the tube wall, and enable the sieving medium to have the function of capillary self-coating and can be used for uncoated capillaries. However, in the process of mixing the two polymers, PDMA needs to be adjusted to a lower concentration to achieve mutual solubility. Too high a PDMA concentration will lead to poor mutual solubility of the sieving medium, making it difficult to ensure that PDMA plays an effective role in the capillary electrophoresis process while maintaining the fluidity, operability and sieving performance of the composition.

[0006] Although the existing mainstream technologies have many advantages in preparing capillary electrophoresis screening media for DNA fragment analysis and DNA sequencing, they also have some disadvantages:

[0007] 1. Resolution limitation:

[0008] Sieving media with poly (N,N-dimethylacrylamide) (PDMA) as the main component have good capillary self-coating ability, but in the field of forensic DNA testing, especially when separating larger DNA fragments or two DNA fragments with a difference of 1 bp, their sieving ability is low, resulting in reduced separation efficiency and typing errors.

[0009] 2. Viscosity problem:

[0010] Studies have shown that increasing the concentration of polymers in the sieving medium can improve DNA resolution, but this also leads to an increase in the viscosity of the sieving medium. The increase in the viscosity of the sieving medium will make capillary injection difficult and cannot meet the requirements of existing automated capillary electrophoresis equipment.

[0011] 3. Phase separation problem:

[0012] Linear polyacrylamide (LPA) and poly (N,N-dimethylacrylamide) (PDMA) are two commonly used polymers that are mixed in appropriate proportions to form blends for capillary electrophoresis sieving media. The compatibility of these two polymers is crucial to achieve efficient electrophoretic separations. The mixing ratio of LPA and PDMA affects the viscosity of the solution and the separation efficiency. Increasing the LPA ratio will improve the resolution, but the self-coating ability will deteriorate; increasing the PDMA concentration will reduce the stability of the sieving medium and increase the risk of polymer precipitation and phase separation. Therefore, it is necessary to determine the optimal ratio of LPA and PDMA through experiments to ensure the uniformity, stability and separation efficiency of the electrophoretic medium.

[0013] 4. Stability issues:

[0014] Stability is an important performance indicator of sieving media, including batch preparation stability and storage stability. The polymer molecules in the current mainstream sieving media are prone to degrade or lose stability under room temperature conditions, affecting the separation effect and repeatability. This may be due to chemical degradation (such as oxidation, hydrolysis or photodegradation) or physical degradation (such as repeated mechanical stress). Changes in the pH value of the polymer solution, solvent evaporation, and interactions between different polymers may affect the stability of the sieving media. For the control of batch stability, the batch difference of the sieving media may be caused by a variety of factors, among which the molecular weight control of the polymer is a critical and challenging process.

[0015] In summary, these shortcomings have prompted researchers to continuously explore and develop new screening media. The ideal screening media preparation method should, on the basis of ensuring screening performance, reduce the viscosity of the screening media as much as possible, avoid phase separation, improve the fluidity of the screening, ensure the self-coating ability of the capillary, realize automatic glue filling, and enhance the stability of the screening media at room temperature, while controlling the batch difference. Summary of the invention

[0016] The primary technical problem to be solved by the present invention is to provide a method for preparing a capillary electrophoresis screening medium with high resolution and high stability.

[0017] Another technical problem to be solved by the present invention is to provide a high-resolution capillary electrophoresis screening medium obtained by the above preparation method.

[0018] Another technical problem to be solved by the present invention is to provide a use of the above-mentioned high-resolution capillary electrophoresis screening medium.

[0019] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0020] According to a first aspect of an embodiment of the present invention, a high-resolution capillary electrophoresis sieving medium is provided, which is prepared by mixing a sieving polymer and a self-coating viscosity regulating polymer, wherein the sieving polymer is a linear polyacrylamide, and the molecular weight Mn of the linear polyacrylamide is 1.4 to 1.5 MDa, and the PDI is 1.5 to 2.0; the self-coating viscosity regulating polymer is a mixture of multiple linear poly-N,N-dimethylacrylamides of different molecular weights, and the molecular weight Mn of the self-coating viscosity regulating polymer is 340 kDa to 360 kDa, and the PDI is 3.8 to 4.2.

[0021] Among them, the self-coating viscosity regulating polymer is prepared by mixing a plurality of different polymers, and the mixing ratio is not limited. It is only necessary to ensure that the number average molecular weight Mn of the mixed polymer is between 340 and 360 kDa and the PDI is between 3.8 and 4.2 to achieve the technical effect of the present invention;

[0022] Preferably, the self-coating viscosity regulating polymer is PDMA-M, and the PDMA-M is prepared by mixing PDMA-1, PDMA-2 and PDMA-3;

[0023] The number average molecular weight Mn of the PDMA-1 polymer is less than 350 kDa, and the PDI is 2.5 to 3.5; the molecular weight Mn of the PDMA-2 polymer is greater than 350 kDa, and the PDI is 2.5 to 3.5; the molecular weight Mn of the PDMA-3 polymer is greater than 350 kDa, and the PDI is 2.5 to 3.5; the mass ratio of the above three polymers is not limited, and the PDMA-M prepared by blending polymers within the three molecular weight and PDI ranges can meet the requirements that the molecular weight and distribution range can reach about 350,000 Daltons and the distribution range PDI is 4.0.

[0024] Preferably, the weight ratio of PDMA-1, PDMA-2 and PDMA-3 is 1.5-2.5:2.5-3.5:4-6.

[0025] According to a second aspect of an embodiment of the present invention, there is provided a method for preparing a high-resolution capillary electrophoresis sieving medium, comprising the following steps:

[0026] (1) preparing a sieving polymer, wherein the sieving polymer is a linear polyacrylamide, wherein the molecular weight Mn of the linear polyacrylamide is 1.4 to 1.5 MDa, and the PDI is between 1.5 and 2.0;

[0027] (2) preparing a self-coating viscosity regulating polymer, wherein the self-coating viscosity regulating polymer is a mixture of multiple linear poly-N,N-dimethylacrylamides of different molecular weights, wherein the molecular weight Mn of the self-coating viscosity regulating polymer is 340 kDa to 360 kDa, and the PDI is between 3.8 and 4.2;

[0028] (3) The products prepared in step (1) and step (2) are mixed under the action of a DNA denaturant and a stabilizer to obtain the product.

[0029] Preferably, the preparation method of the sieving polymer is as follows: nitrogen is introduced into acrylamide monomer and isopropanol under magnetic stirring to deoxygenate the solution; ammonium persulfate is added to carry out monomer polymerization reaction and then the reaction is stopped by cooling; and freeze-drying is performed after dialysis.

[0030] Preferably, the self-coating viscosity regulating polymer is PDMA-M, which is prepared by mixing PDMA-1, PDMA-2 and PDMA-3;

[0031] The molecular weight Mn of the PDMA-1 polymer is less than 350 kDa, and the PDI is 2.5-3.5; the molecular weight Mn of the PDMA-2 polymer is greater than 350 kDa, and the PDI is 2.5-3.5; the molecular weight Mn of the PDMA-3 polymer is greater than 350 kDa, and the PDI is 2.5-3.5.

[0032] Preferably, the preparation method of PDMA-1 is as follows: polymer monomer, chain transfer agent and first initiator are mixed uniformly, a stirring magnet is added, and then deoxygenated by freeze-thaw-degassing method, and then reacted at a high temperature of 70-85°C to obtain a viscous transparent fluid; dialyzed; and freeze-dried; the polymer monomer is N,N-dimethylacrylamide, the first initiator is benzoyl peroxide, and the chain transfer agent is β-mercaptoethanol.

[0033] Preferably, the preparation method of PDMA-2 is: uniformly mix the polymer monomer and the first initiator, add a stirring magnet, deoxygenate by freeze-thaw-degassing method, react at 70-85°C to obtain a viscous transparent fluid; dialyze; and freeze-dry. The polymer monomer is N,N-dimethylacrylamide, and the first initiator is benzoyl peroxide.

[0034] Preferably, the preparation method of PDMA-3 is as follows: the polymer monomer and water are mixed evenly, after deoxygenation with N2, a second initiator ammonium persulfate is added, a stirring magnet is added, and the mixture is evenly mixed; a high temperature reaction is performed at 70 to 85° C. in a nitrogen environment to obtain a viscous transparent fluid; dialyzed; and freeze-dried to obtain the PDMA-3; the polymer monomer is N,N-dimethylacrylamide, and the second initiator is ammonium persulfate.

[0035] According to a third aspect of an embodiment of the present invention, there is provided a high-resolution capillary electrophoresis sieving medium (referred to as sieving medium for short), which is prepared by the above-mentioned preparation method.

[0036] The high-resolution capillary electrophoresis screening medium provided by the present invention can be used in the fields of biochemistry, molecular biology, clinical medicine, etc., and can also be used in the field of forensic DNA detection and analysis.

[0037] Compared with the prior art, the present invention has the following technical effects:

[0038] (1) The present invention can effectively improve the resolution of DNA detection. Improving the resolution of DNA detection is the focus and difficulty of current DNA testing. Related studies have shown that it is difficult for the screening medium prepared by the prior art to separate two DNA fragments with a difference of only 1 bp and amplified fragments of more than 350 bp. The screening medium prepared by the present invention can significantly improve the single base capacity of DNA in the range of 350 to 600 bp, and is particularly suitable for forensic DNA detection.

[0039] (2) The sieving medium prepared by the present invention has excellent storage stability and batch stability. It can be seen from the test results that the sieving medium prepared by the present invention can still achieve normal detection under room temperature storage conditions within 1 month. Under the same conditions, the commercial sieving medium produced a broad peak and a reduced signal. Among the sieving media prepared by the present invention, the sieving media of multiple different batches of products have stable test results for the same allele standard control, which shows that the batch stability of the present invention is also good.

[0040] (3) Good typing accuracy: The sieving medium prepared by the present invention was used to detect the product of STR composite amplification of standard DNA 9947A. After comparison with ladder, the GeneMapper IDX software was used for analysis, and the accuracy of the obtained detection result was 100%.

[0041] (4) The present invention solves the problems of viscosity, phase separation and fluidity of the screening medium in the prior art, improves product performance, makes its application range wider, and has a good market prospect.

[0042] The present invention is further described below in conjunction with specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the screening result diagram of the allele ladder of the Identifiler plus kit (five colors);

[0044] Figure 2 This is the screening result diagram of the allele ladder of the Verifiler plus kit (six colors);

[0045] Figure 3 It is the detection profile of 9947A STR;

[0046] Figure 4A This is a comparison diagram of the resolution between the screening medium prepared by the present invention and the commercial screening medium;

[0047] Figure 4B For the general Figure 4A The internal standard screening was magnified to show only the fragment separation after 350 bp;

[0048] Figure 5 The results of the test of the same internal standard sample with two different sieving media stored at room temperature for 30 days;

[0049] Fig. 6A A graph showing the overlapping detection results of the screening media prepared by the present invention for the D13S317 locus in the allele ladder prepared from multiple different batches;

[0050] Figure 6B A graph showing the results of allele overlap detection at 202 bp of the D13S317 locus in the allele ladder using the screening media prepared in the present invention from multiple different batches (SIZE value);

[0051] Figure 7 Phase separation occurs when LPA prepared by the prior art is mixed with conventional PDMA;

[0052] Figure 8 This is a product diagram of two polymers blended together by the preparation method provided by the present invention;

[0053] Fig. 9 The figure is the molecular weight internal standard diagram (self-coating ability) of the sieving medium detection standard prepared by the present invention. DETAILED DESCRIPTION

[0054] The technical content of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0055] The professional terms mentioned in the following embodiments are explained as follows:

[0056] CE: Capillary Electrophoresis is a highly efficient separation and analysis technology. It uses high-voltage electric field as the driving force and uses capillaries as separation channels to achieve separation based on the differences in mobility and distribution behavior between components in the sample.

[0057] Sieving media: usually refers to polymer solutions with specific molecular weight and viscosity characteristics used in capillary electrophoresis. These polymer solutions are used as separation media to analyze and separate DNA molecules according to their size.

[0058] STR: Short Tandem Repeats, also known as microsatellite DNA, is a type of DNA sequence formed by tandem repetitions of 2 to 6 base pairs as core units. Since the number of core unit repeats is highly variable among individuals and is abundant, it constitutes the genetic polymorphism of the STR locus.

[0059] PCR: Polymerase Chain Reaction, is a molecular biology technique used to rapidly replicate large numbers of specific DNA fragments in vitro. The core of PCR technology is to use DNA polymerase to catalyze the amplification of specific DNA sequences in vitro through a series of temperature cycles.

[0060] EOF: Electroosmotic Flow is a fluid movement phenomenon that occurs at the interface between a charged solid surface (such as glass or silicon-based materials) and an electrolyte solution. When an electric field is applied to this interface, since the solid surface usually carries a negative charge, it attracts positive ions in the solution to form a double electric layer. Under the action of the electric field, these positive ions and the water molecules around them will move toward the negative electrode, thereby driving the entire solution layer to flow along the charged surface. This flow is uniform and involves the entire liquid layer, unlike electrophoresis, which involves the migration of charged particles in an electric field.

[0061] The research and development idea of ​​the present invention is: to prepare the screening medium by adopting the step-by-step synthesis and mixed preparation method, that is, generally speaking, two different monomers are respectively initiated to polymerize according to a specific method, and then the screening medium is prepared by mixing them according to a certain proportion and technical means, while ensuring the self-coating ability of the capillary, the DNA fragment resolution ability and detection speed of the screening medium are greatly increased. First, a high-resolution linear polyacrylamide is prepared, and at the same time, a PDMA mixture with a specific molecular weight and distribution having self-coating ability and viscosity adjustment ability is prepared; then, by adding necessary ingredients such as stabilizers and denaturants, the preparation of a capillary electrophoresis screening medium with high resolution, high stability and low viscosity can be achieved. For a certain monomer polymer, the screening medium can be improved in terms of capillary self-coating, DNA screening ability (resolution), stability, etc., forming a complementary performance advantage.

[0062] Example 1 Study on Viscosity and Phase Separation of Screening Media

[0063] 1. Research on Viscosity of Screening Media:

[0064] The inventors discovered and confirmed three regular problems during the research process. First, the viscosity of the screening medium is positively correlated with the molecular weight of the polymer it contains; second, for a screening medium prepared with a high molecular weight polymer, the higher the polymer concentration it contains, the greater the viscosity; third, for forensic DNA detection, the higher the polymer molecular weight and concentration, the higher the resolution; based on the above three regular facts, many researchers have tried to increase the molecular weight of the polymer or the polymer concentration in the screening medium to improve the screening performance. However, the increase in the viscosity of the screening medium will bring about a series of problems such as the difficulty of capillary injection described in the background technology, which makes it more difficult to improve the resolution of the screening medium.

[0065] The preparation method provided by the present invention can effectively solve the above problems. During the research process, it was found that when the sieving medium prepared by LPA of a specific molecular weight and concentration has a fixed viscosity, when the special mixed PDMA (specific molecular weight and distribution) of the present invention is added, the viscosity of the sieving medium will decrease, so the above contradiction can be cleverly solved, that is, the viscosity of the sieving medium can be increased while the LPA concentration is increased; in addition, PDMA itself has the ability to self-coat, and can also effectively inhibit the electroosmotic flow. However, it should be noted that the special mixed PDMA mentioned above has also been studied to determine its special requirements: (1) a specific number average molecular weight (340kDa~360kDa) and its distribution range (PDI is 3.8~4.2); (2) the above standard PDMA cannot be directly synthesized in one go by free radical polymerization. The present invention has found through a large number of studies that a variety of PDMAs with different molecular weights and distributions can be innovatively mixed and formulated to finally prepare a mixture of PDMA polymers. At the same time, it has been tested that another advantage of this method is that the sieving medium prepared by this method has stable performance and good batch-to-batch repeatability. The goal is to increase resolution without increasing viscosity and to have low batch-to-batch variability.

[0066] 2. Study on phase separation problem:

[0067] When LPA is mixed with conventional PDMA, phase separation is very likely to occur. Figure 7 As shown in the figure, a clear dividing line can be seen at the intersection of the two arrows, indicating that the two polymers have poor compatibility. The mixed screening medium cannot be used because it does not have capillary self-coating ability.

[0068] The present invention solves the phase separation problem in two ways: (1) By matching the molecular weights of PDMA and LPA, the compatibility between polymers can be improved while reducing viscosity. The main reason may be that the balance of molecular weights can lead to a more uniform mixture and avoid the occurrence of phase separation. That is, the molecular weight Mn of LPA is between 1.4 and 1.5 MDa, and the PDI (Polydispersity Index) is between 1.5 and 2.0. The molecular weight Mn of the self-coating viscosity regulating polymer PDMA-M is 340 kDa to 360 kDa, and the PDI is 3.8 to 4.2, which can achieve a special balance and matching, and can effectively reduce the probability of phase separation. While ensuring compatibility, the screening capacity is maximized; (2) Adding a stabilizer (polyethylene glycol) to the screening medium (added in the sol buffer) can also reduce the interfacial tension and further improve the uniformity of the LPA / PDMA mixture.

[0069] like Figure 8 As shown, the preparation method provided by the present invention can ensure the compatibility of the two polymers and is transparent throughout.

[0070] 3. Research on fluidity of screening media:

[0071] If the fluidity of the screening medium, i.e., the self-coating ability, has problems, then electroosmotic flow will occur, which will lead to a broad peak phenomenon in the sample. The present invention utilizes a special PDMA-M mixture and limits its number average molecular weight to 340kDa to 360kDa. The PDI is between 3.8 and 4.2 to achieve excellent self-coating performance. If it is lower than the above range, the self-coating ability decreases and the performance requirements cannot be met. The molecular weight internal standard of the screening medium test standard prepared by the present invention is used, and the test results are as follows: Fig. 9 As shown, the mass number of the internal standard is 1, indicating that there is no peak width phenomenon (for example, for a broad peak, the mass number of the internal standard would be lower than 1.0).

[0072] The above studies show that the sieving medium prepared by the present invention effectively solves the problems of viscosity, phase separation and self-coating ability in the prior art.

[0073] Example 2 Preparation of Sieving Polymer

[0074] (1) Take 230 ml of ultrapure water and place it in a 500 ml three-necked round-bottom flask, add 18.3 g of acrylamide monomer, and then add 2 ml of isopropanol. Place it on a magnetic stirrer and pass high-purity nitrogen (99.99%) to deoxygenate the solution for 120 minutes.

[0075] (2) 0.8 ml of 10% (w / v) ammonium persulfate (APS) was added and reacted at 50 degrees Celsius for 60 minutes. The viscosity of the system increased, and ice water was added to lower the temperature of the system to stop the reaction.

[0076] (3) After one end of the pretreated dialysis bag (10000D) is clamped with a dialysis bag clamp, the polymerized viscous liquid is poured into the pretreated dialysis bag, and both ends are clamped with dialysis bag clamps. The bag is placed in a large glass beaker, and ultrapure water is added to cover the dialysis bag. The dialysis is performed for three days, during which the water is changed every 12 hours.

[0077] (4) After dialysis, the polymer solution was placed in a culture dish and placed in a freeze dryer for freeze drying. The white solid obtained was LPA. GPC characterization analysis showed that the molecular weight Mn of LPA was 1.44 MDa and the PDI was 1.7.

[0078] Example 3 Preparation of self-coating and viscosity-adjusting polymers

[0079] (1) Purification of monomer and initiator BPO

[0080] The DMA monomers used in the polymerization are purified to remove the inhibitors and some impurities in the monomers, reducing the impact of impurities on the polymerization process. Because the DMA monomer contains inhibitors such as HEMQ, it is purified by vacuum distillation. The temperature of the vacuum distillation is set to 80°C, and the dripping rate of the condensed monomer is maintained at 1 drop / s. The 5% at the beginning of the distillation and the remaining 10% of the monomers in the flask at the end need to be discarded, leaving only the middle section components, and the purified monomers are stored in a refrigerator at -20°C.

[0081] The initiator benzoyl peroxide BPO was purified by recrystallization: 5 g of BPO was dissolved in 20 mL of dichloromethane, reduced by half and filtered, and then dripped into 20 mL of ice methanol, filtered, dried under reduced pressure at 40° C. for 12 h, and stored in a brown bottle under refrigeration.

[0082] (2) Preparation of PDMA-1

[0083] Take 20g N,N-dimethylacrylamide (0.20mol), 1.93mg benzoyl peroxide BPO (8.0μmol), 14μL (200μmol) β-mercaptoethanol and mix them evenly in a reaction bottle, and add a stirring magnet. Oxygen will consume part of the initiator and form an inhibitory effect, so we use the freeze-pump-thaw method to deoxygenate. Freeze the entire system with liquid nitrogen, then use a vacuum pump to evacuate the air so that the bottle is kept under negative pressure for thawing. Do this three times, and finally fill the bottle with nitrogen to keep the reaction bottle sealed. At this time, most of the oxygen in the reaction system has been removed. Put the reaction bottle into an oil bath pot at 80℃ for 7h to obtain a viscous transparent fluid. Put the transparent fluid into a pre-treated dialysis bag (10000D), put it into a large glass beaker, put ultrapure water to cover the dialysis bag, and dialyze for three days, changing the water every 12 hours. After dialysis, the polymer solution was placed in a culture dish and freeze-dried in a freeze dryer to obtain a white solid, which was PDMA-1. Gel permeation chromatography was used to characterize and analyze it. The molecular weight Mn of the obtained polymer was 150 kDa and the PDI was 3.24. The reaction structure is as follows:

[0084]

[0085] (3) Preparation of PDMA-2

[0086] Take 12g N,N-dimethylacrylamide (0.12mol) and 1.2mg benzoyl peroxide BPO (4.95μmol) and mix them evenly in the reaction bottle, and add a stirring magnet. Oxygen will consume part of the initiator and form an inhibitory effect, so we use freeze-thaw-degassing method to remove oxygen. Freeze the entire system with liquid nitrogen, then use a vacuum pump to evacuate the air so that the bottle is kept under negative pressure for thawing. Repeat this cycle three times, and finally fill the bottle with nitrogen to keep the reaction bottle sealed. At this time, most of the oxygen in the reaction system has been removed. Put the reaction bottle into an oil bath pot at 80℃ for 7h to obtain a viscous transparent fluid. Put the transparent fluid into a pre-treated dialysis bag (10000D) and put it into a large glass beaker. Put ultrapure water to cover the dialysis bag and dialyze for three days, changing the water every 12 hours. After dialysis, the polymer solution is placed in a culture dish, and the white solid obtained after freeze drying in a freeze dryer is PDMA-2. The polymer was characterized and analyzed by gel permeation chromatography. The molecular weight Mn of the obtained polymer was 379 kDa and the PDI was 3.04.

[0087] (4) Preparation of PDMA-3

[0088] Take 20g N,N-dimethylacrylamide (0.20mol), add it to a three-necked flask containing 40ml pure water, deoxygenate with N2 for 60 minutes, add 200ul 10% (w / v) ammonium persulfate solution (APS), mix well in the reaction bottle, and add a stirring magnet. Under N2 environment, put the reaction bottle into an oil bath at 45℃ for 3h to obtain a viscous transparent fluid. Put the transparent fluid into a pre-treated dialysis bag (10000D) and put it into a large glass beaker. Put ultrapure water to cover the dialysis bag and dialyze for three days, changing the water every 12 hours. After dialysis, the polymer solution was placed in a culture dish, placed in a freeze dryer for freeze drying, and the white solid obtained was PDMA. It was characterized and analyzed by gel permeation chromatography. The molecular weight Mn of the obtained polymer was 526kDa and the PDI was 3.32.

[0089] Reasons for using ammonium persulfate APS as the second initiator:

[0090] (1) The synthesis of small molecular weight PDMA (e.g. 150 kDa) requires a higher reaction temperature;

[0091] (2) APS is not suitable for high reaction temperatures and degrades at 40-50 degrees Celsius, while BPO can adapt to high reaction temperatures;

[0092] (3) BPO is not suitable for synthesizing polymers with a molecular weight exceeding 500 kDa. Therefore, this section fully utilizes the characteristics of two different initiators to form complementary advantages.

[0093] Example 4 Mixing of PDMA-M

[0094] Weigh 1.58g PDMA-1 sample, 3.0g PDMA-2 sample, 5.42g PDMA-3 sample, add to a beaker containing 90mL ultrapure water, seal with plastic wrap, stir with a magnetic stirrer at 400rpm for 72h, and after the sample is fully dissolved and mixed evenly, draw 100ul sample for data characterization. The molecular weight Mn of PDMA is 349kDa, and the PDI is 4.0. The remaining samples are freeze-dried and used for the preparation of screening media. The present invention innovatively adopts a multi-polymer mixing method to achieve precise control of the molecular weight and PDI of high molecular polymers. The characterization parameters of each polymer are shown in Table 1.

[0095] Table 1

[0096] Wi(%) Mw(kDa) Mn(kDa) PDI PDMA-1 15.8 491 151 3.24 PDMA-2 30 1155 379 3.04 PDMA-3 54.2 1751 526 3.32 PDMA-M 100 348.6 4.03

[0097] In other embodiments of the present invention, PDMA-1, PDMA-2 and PDMA-3 have a variety of mixing methods to ensure that the number average molecular weight Mn of the final PDMA-M polymer is about 350kDa, where "about" means between 340kDa and 360kDa, and PDI is between 3.8 and 4.2. The screening medium obtained after preparing with the LPA obtained in Example 1 can achieve the technical effect of the present invention. The molecular weight Mn of the PDMA-1 polymer given in this embodiment is less than 350kDa, and the PDI is 2.5 to 3.5; the molecular weight Mn of the PDMA-2 polymer is greater than 350kDa, and the PDI is 2.5 to 3.5; the molecular weight Mn of the PDMA-3 polymer is greater than 350kDa, and the PDI is 2.5 to 3.5. After mixing PDMA-1, PDMA-2, and PDMA-3 within the above range, it can be ensured that the number average molecular weight Mn of the PDMA-M polymer is about 340kDa to 360kDa, and the PDI is between 3.8 and 4.2.

[0098] Effects of changes in the molecular weight and distribution of PDMA-M beyond the above range:

[0099] (1) When the number average molecular weight of PDMA-M is lower than the above range, the coating performance of the PDMA-M mixture on the inner wall of the capillary is reduced, and a higher concentration or blending with other polymers may be required to improve the stability and effectiveness of the coating;

[0100] (2) When the number average molecular weight of PDMA-M is higher than the above range, the higher PDMA mixture will intensify the phase separation with LPA. This phase separation is mainly due to the interaction and kinetic process between polymer chains, which may cause the polymer chains to aggregate to form different phases under certain conditions, breaking the balance between LPA and PDMA-M.

[0101] (3) The PDI range of PDMA-M becomes narrower. When the PDI is lower than 3.8, it will affect the viscosity of its solution. Too high a viscosity may lead to the appearance of bubbles, difficulty in perfusion and flushing, and long electrophoresis time in the application of capillary electrophoresis. The narrowing of PDI may also lead to the uneven dynamic pore structure of the sieving medium, affecting the sieving efficiency, resolution (R) and length-of-read (LOR).

[0102] (4) The molecular weight of PDMA-M is widened (PDI is higher than 4.2) on the basis of the number average molecular weight of 350 kDa, which itself makes the preparation process difficult, because the preparation requires a screening medium with a higher molecular weight. PDMA with a molecular weight that is too high is difficult to synthesize, and is prone to long chain breakage, which causes changes in molecular weight. In addition, the viscosity is relatively high, resulting in unstable self-coating ability and screening performance of the final prepared screening medium.

[0103] Example 5 Preparation of Screening Media

[0104] (1) Preparation of sol buffer

[0105] Use a balance to take 6.05g of tris-base, 12.16g of N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), and 0.74g of EDTA and place them in a 100ml reagent bottle. First add 80ml of ultrapure water, mix thoroughly until all reagents are dissolved, and then add ultrapure water to make up to 100ml.

[0106] (2) Preparation of blended screening media

[0107] A. Weigh 0.5 g of PDMA-M prepared in Example 4 and place it in a 200 ml reagent bottle;

[0108] B. Add 10 ml of sol buffer and 40 ml of pure water and mix thoroughly;

[0109] C. Weigh 36.036 g of urea (Sigma, U5378-500G) and mix thoroughly;

[0110] D. Weigh 3.8 g of thiourea (Sigma, T8656-500G) and mix thoroughly;

[0111] E. Add polyethylene glycol, specifically PEG200, to a final concentration of 20% (range 15-30%) (m / v);

[0112] F. Weigh 5.0 g of the sieved polymer prepared in Example 2 and adjust the volume to 100 ml.

[0113] G. Stir at low speed (≤300rmp) on a magnetic stirrer and swell for 30h. After the polymer is fully swollen, use a 0.45μm nylon filter membrane filtration device for vacuum filtration (the filtration device is directly connected to the vacuum pump), collect the filtrate into a clean reagent bottle, and then divide it into 8ml reagent bottles or 30ml reagent bottles, which can be used for experiments, and the others can be stored according to the conditions.

[0114] In the above preparation method, urea is a DNA denaturant, but it is unstable under alkaline conditions; thiourea is a stabilizer that can maintain the stability of urea under alkaline conditions; however, the addition of thiourea will affect the spatial correction after the injection of the screening medium, that is, the laser passability, resulting in the screening medium being unable to be used on the genetic analyzer. Polyethylene glycol can largely ensure the laser passability, while also improving the solubility of the polymer in the screening medium and maintaining the denatured state of DNA, thereby ensuring the stability and resolution of the screening medium.

[0115] Example 6 Application of high-resolution capillary electrophoresis screening media in forensic DNA detection

[0116] (1) Single-base resolution test

[0117] The screening medium prepared in the embodiment of the present invention was used to detect the allelic ladder in the commercial STR typing kit, and to observe whether the two alleles with a difference of 1 bp can be effectively screened under the default parameter setting conditions of the GeneMapper software. The specific test results are shown in FIG. Figure 1 and Figure 2 As shown, Figure 1 This is the screening result diagram of the allele ladder of the Identifiler plus kit (five colors). Figure 2 This is the screening result diagram of the allele ladder of the Verifiler plus kit (six colors).

[0118] The above test results show that the screening medium prepared by the present invention can fully achieve the separation requirements of the alleles "9.3" and "10" in the TH01 locus in the allele ladder of the Identifiler kit, and the separation requirements of the alleles "19.3" and "20" in the D12S391 locus in the Verifiler plus kit.

[0119] (2) Typing accuracy test

[0120] The screening medium prepared by the present invention was used to detect the product of STR composite amplification of standard DNA 9947A, and the obtained detection result was correct by comparing with the ladder and analyzing with GeneMapper IDX software. The specific typing and comparison results are as follows: Figure 3 As shown in Table 2. Figure 3 It is the detection spectrum of 9947A STR. It can be seen from the detection results that the detection results of the sieving medium prepared by the present invention are highly accurate.

[0121] Table 2 Comparison of 9947A typing results

[0122]

[0123] (3) Comparison experiment of the resolution of the screening medium prepared by the present invention and the commercial screening medium

[0124] Improving the resolution of DNA detection is the focus and difficulty of current DNA testing. Relevant studies have shown that it is difficult to separate two DNA fragments with a difference of only 1 bp when the amplified fragments reach more than 350 bp using the existing technology for preparing screening media. The screening media prepared by the method of the present invention can significantly improve the single base capacity of DNA in the range of 350 to 600 bp, and is suitable for forensic DNA detection.

[0125] In order to verify the advantage of resolution, the existing commercial capillary electrophoresis screening medium (POP4) was used as a control, and the specific resolution test internal standard (each node contained two fluorescent DNA fragments with a difference of 1 bp) was electrophoretically separated under the same electrophoresis parameters, such as Figure 4A As shown, the screening medium prepared by the present invention has obvious advantages in single base resolution of large fragments above 350 bp. Figure 4B As shown in the figure, Figure 4A The screening of some fragments in the internal standard was magnified, and only the separation of fragments after 350bp was shown. The results showed that when the commercial screening medium was used to detect the resolution test standard, the DNA fragments in the range of 350bp to 600bp could no longer be recognized and distinguished by single bases, while the screening medium prepared by the present invention could fully achieve single base distinction and recognition. The corresponding lengths were marked, 359 / 360, 379 / 380, 399 / 400, 419 / 420, 439 / 440,

[0126] 459 / 460、479 / 480、499 / 500、524 / 525、549 / 550、574 / 575、

[0127] 599 / 600.

[0128] The test results show that the commercial screening medium cannot achieve single-base resolution after 350 bp, while the screening medium prepared by the present invention can still maintain relatively excellent single-base resolution after 350 bp.

[0129] It can be seen from the above detection results that the present invention can effectively improve the resolution of DNA detection.

[0130] (4) Screening medium stability test

[0131] The stability of the screening medium is mainly reflected in two aspects, namely storage stability and batch stability. Regarding storage stability, LPA is easily degraded under alkaline conditions. The degradation of urea is the main reason for the instability or short shelf life of the screening medium. The hydrolysis, degradation or change of polymers in the DNA screening medium may also play a decisive role in the instability of the screening medium. The present invention adopts stabilizers and DNA denaturants, which can maintain the stability of the screening medium at room temperature for a long time, and will not cause problems such as hydrolysis and degradation that affect the screening effect of the screening medium. The test results show that the screening medium prepared by the present invention can still achieve normal detection under room temperature storage conditions within 1 month. However, the commercial screening medium produces a wide peak and a reduced signal. The specific results are as follows Figure 5 As shown, Figure 5 The figure above shows the test results of commercial sieving media. The figure shows the test results of two different sieving media stored for 30 days on the same internal standard sample.

[0132] Regarding batch-to-batch stability, the present invention adopts LPA and PDMA blending to achieve high stability of polymer preparation by precisely controlling the molecular weight and distribution range of the polymer. The same allele standard control was tested using multiple batches of different screening media and analyzed using GeneMapperIDX software. The test results are as follows Fig. 6A As shown, Fig. 6A This is a graph showing the overlapping detection results of the D13S317 locus in the allele ladder for screening media from multiple different batches. Figure 6B for Fig. 6A The allele overlap detection result diagram of the D13S317 locus 202bp. It can be seen from the figure that the standard deviation of the DNA fragment SIZE value is 0.1, indicating that the screening medium prepared by the present invention has good batch stability.

Claims

1. A high-resolution capillary electrophoresis screening medium, characterized in that The self-coating viscosity regulating polymer is prepared by mixing a sieving polymer and a self-coating viscosity regulating polymer. The sieving polymer is a linear polyacrylamide with a molecular weight Mn of 1.4-1.5 MDa and a PDI of 1.5-2.

0. The self-coating viscosity regulating polymer is a mixture of linear poly-N,N-dimethylacrylamides with different molecular weights, with a molecular weight Mn of 340-360 kDa and a PDI of 3.8-4.

2.

2. The high-resolution capillary electrophoresis screening medium according to claim 1, characterized in that: The self-coating viscosity regulating polymer is PDMA-M, which is obtained by mixing PDMA-1, PDMA-2 and PDMA-3; The molecular weight Mn of the PDMA-1 polymer is less than 350 kDa, and the PDI is 2.5-3.5; the molecular weight Mn of the PDMA-2 polymer is greater than 350 kDa, and the PDI is 2.5-3.5; the molecular weight Mn of the PDMA-3 polymer is greater than 350 kDa, and the PDI is 2.5-3.

5.

3. A method for preparing a high-resolution capillary electrophoresis screening medium, characterized in that The steps include: (1) preparing a sieving polymer, wherein the sieving polymer is a linear polyacrylamide, wherein the molecular weight Mn of the linear polyacrylamide is 1.4 to 1.5 MDa, and the PDI is 1.5 to 2.0; (2) preparing a self-coating viscosity regulating polymer, wherein the self-coating viscosity regulating polymer is a mixture of multiple linear poly-N,N-dimethylacrylamides of different molecular weights, wherein the molecular weight of the self-coating viscosity regulating polymer is between 340 and 360 kDa, and the PDI is between 3.8 and 4.2; (3) The products prepared in step (1) and step (2) are mixed in the presence of a DNA denaturant and a stabilizer to obtain the product.

4. The method for preparing a high-resolution capillary electrophoresis screening medium according to claim 3, characterized in that The preparation method of the sieving polymer is as follows: Nitrogen is introduced into the solution of acrylamide monomer and isopropanol under magnetic stirring to deoxygenate the solution; ammonium persulfate is added to carry out monomer polymerization reaction and then the reaction is stopped by cooling; and freeze drying is carried out after dialysis.

5. The method for preparing a high-resolution capillary electrophoresis screening medium according to claim 3, characterized in that: The self-coating viscosity regulating polymer is PDMA-M, which is obtained by mixing PDMA-1, PDMA-2 and PDMA-3; The molecular weight Mn of the PDMA-1 polymer is less than 350 kDa, and the PDI is 2.5-3.5; the molecular weight Mn of the PDMA-2 polymer is greater than 350 kDa, and the PDI is 2.5-3.5; the molecular weight Mn of the PDMA-3 polymer is greater than 350 kDa, and the PDI is 2.5-3.

5.

6. The method for preparing a high-resolution capillary electrophoresis screening medium according to claim 5, characterized in that The preparation method of PDMA-1 is: The polymer monomer, the chain transfer agent and the first initiator are mixed uniformly, a stirring magnet is added, and then the mixture is deoxygenated by freeze-thaw-degassing method, and then reacted at a high temperature of 70 to 85° C. to obtain a viscous transparent fluid; dialyzed; and freeze-dried to obtain the polymer monomer N,N-dimethylacrylamide, the first initiator is benzoyl peroxide, and the chain transfer agent is β-mercaptoethanol.

7. The method for preparing a high-resolution capillary electrophoresis screening medium according to claim 5, characterized in that The preparation method of PDMA-2 is: The polymer monomer and the first initiator are mixed evenly, a stirring magnet is added, and after deoxygenation by freeze-thaw-degassing method, a high temperature reaction is performed at 70-85° C. to obtain a viscous transparent fluid, which is dialyzed and freeze-dried to obtain the polymer monomer N,N-dimethylacrylamide, and the first initiator is benzoyl peroxide.

8. The method for preparing a high-resolution capillary electrophoresis screening medium according to claim 5, characterized in that The preparation method of PDMA-3 is: The polymer monomer and water are mixed evenly, and after deoxygenation with N2, a second initiator ammonium persulfate is added, and a stirring magnet is added to mix evenly; a high temperature reaction is performed at 70 to 85° C. in a nitrogen environment to obtain a viscous transparent fluid; dialyzed; and freeze-dried to obtain the polymer monomer N,N-dimethylacrylamide, and the second initiator is ammonium persulfate.

9. The method for preparing a high-resolution capillary electrophoresis screening medium according to claim 3, characterized in that: The DNA denaturant is urea, and the stabilizer is thiourea and polyethylene glycol.

10. A high-resolution capillary electrophoresis screening medium, characterized in that The compound is prepared by the preparation method described in any one of claims 3 to 9.

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