DNA synthetic matrix as well as preparation method and application thereof

Abstract

The invention relates to the field of DNA synthesis, in particular to a DNA synthetic matrix, a DNA synthetic matrix capable of avoiding cross contamination and a preparation method and application of the DNA synthetic matrix. Compared with a synthetic matrix of a carrier with DMT protection, the DNA synthetic matrix capable of avoiding cross contamination provided by the invention has the advantages that a deprotection reagent does not need to be added in the first-step synthetic reaction, the operation is convenient, and the dosage of the reagent is saved. Secondly, according to the DNA synthesis matrix capable of avoiding cross contamination, provided by the invention, high-strength DMT removal is performed on a sample adding site before synthesis, and by adopting the DNA synthesis matrix capable of avoiding cross contamination, the accuracy of DNA synthesis can also be improved, and the probability of N-1 and N-2 deletion of a synthesized gene sequence can be reduced.

Description

Technical Field

[0001] The present application relates to the field of DNA synthesis, and in particular to a DNA synthesis matrix and a DNA synthesis matrix avoiding cross contamination, and a preparation method and application thereof. Background Art

[0002] With the development of molecular biology technology, including drug development, diagnosis of genetic and infectious diseases, gene research, polymerase chain reaction (PCR) and hybridization experiments, a large number of DNA fragments are needed, and most of these DNA fragments need to be synthesized artificially. DNA synthesized fragments often have different expressions, such as oligonucleotides, PCR primers, adapters, probes, short-chain primers and long-chain primers.

[0003] At present, the DNA synthesis field is mainly based on the solid phase phosphoamidite trisester method. The synthesis of one monomer is completed by a cycle consisting of four reaction steps, that is, each additional cycle adds one base to the growing oligonucleotide chain on the solid support. The four reaction steps contained in each cycle are specifically as follows: in the first step, the nucleotide (usually contained in a synthesis column) connected to the solid phase support (generally Au, Pt, Si, SiNx, controlled pore glass beads (CPG) or polystyrene microspheres (PS), etc.) with the 5' terminal hydroxyl protected by dimethoxytrityl (DMT) is deprotected by adding trichloroacetic acid to obtain a free 5'-hydroxyl end for the next synthesis reaction, so that the DNA chain adds one monomer on the original basis. In the second step, the phosphoramidite monomer is mixed with a tetrazolium activator and enters the synthesis column to form a phosphoramidite tetrazolium active intermediate (its 5' end is still protected by DMT and the 3' end is activated). The third step is to couple the 5'-hydroxyl group of the nucleoside previously connected to the solid phase support in the sequence to form a phosphite triester, thereby extending the synthetic sequence chain forward by one base. The fourth step is to use an iodine tetrahydrofuran solution to convert the unstable, easily acidic and alkaline hydrolyzed phosphite bond into a stable pentavalent phosphate bond. After the same cycle steps as above, the desired DNA sample can be obtained.

[0004] After the above four steps, a deoxynucleotide is connected to the nucleotide of the solid phase carrier (usually CPG). Then the DMT protecting group on its 5'-hydroxyl group is removed with trichloroacetic acid, and the above steps are repeated until all the bases required to be synthesized are connected.

[0005] However, this synthesis method in the prior art requires the removal of DMT during the reaction, which is a cumbersome process. When CPG with DMT protection is used to prepare a DNA synthesis matrix, it is sintered together with the fixing material, and the surface of CPG will be largely shielded by the fixing material and plasticizer. Therefore, it is difficult to completely remove the DMT on CPG, thereby limiting the yield and accuracy of DNA synthesis. Summary of the invention

[0006] Based on this, it is necessary to provide a DNA synthesis matrix and a DNA synthesis matrix that avoids cross contamination and a preparation method thereof, so as to solve the technical problems of low synthesis yield and poor accuracy when preparing a DNA synthesis matrix from a vector carrying DMT.

[0007] On one hand, the present application provides a DNA synthesis matrix, comprising a carrier without DMT and a fixing material for fixing the carrier, wherein the carrier without DMT comprises an active group without DMT.

[0008] In one embodiment, the thickness of the matrix is ​​0.01 mm to 10 mm, the average pore size of the matrix is ​​1 μm to 500 μm, and the ratio of the thickness to the average pore size is (10:1) to (100:1).

[0009] In one embodiment, the active group is at least one of a phosporamidite group, a universal linkerphosphoramidite group and a modified phosporamidites group.

[0010] In one embodiment, the carrier has an average of 0.1 nmol / cm 2 ~10nmol / cm 2 Active groups.

[0011] In one embodiment, the hydrophobic contact angle of the substrate is 10 to 45 degrees.

[0012] In one embodiment, the steps include:

[0013] The carrier without DMT and the fixing material are heated to melt the carrier to form an intermediate, and the intermediate is subjected to calendaring and / or stretching treatment to obtain the DNA synthesis matrix without DMT protection.

[0014] On the other hand, the present application provides a DNA synthesis matrix that avoids cross-contamination, comprising at least two synthesis sites, wherein there is a gap between the synthesis sites; among the vectors in the synthesis sites, more than 95% are vectors without DMT; among the vectors between the synthesis sites, more than 95% are vectors with DMT.

[0015] In one embodiment, the steps include:

[0016] The DMT-carrying carrier and the fixing material are heated to melt the carrier to form an intermediate, and the intermediate is subjected to calendaring and / or stretching treatment to obtain the DMT-protected DNA synthesis matrix, and the synthesis site is subjected to de-DMT treatment, while the synthesis sites are not subjected to de-DMT treatment.

[0017] In one embodiment, the method further comprises ultrasonic cleaning of the synthesis site.

[0018] According to the above DNA synthesis substrate, the above DNA synthesis substrate avoiding cross contamination is used in DNA synthesis.

[0019] Compared with the traditional technology, the beneficial effects of this application include:

[0020] The DNA synthesis matrix and preparation method thereof provided by the present application on the one hand, compared with the synthetic matrix of the carrier with DMT protection, the first step of the synthesis reaction does not need to add a deprotection reagent, easy to operate, save the amount of reagents. Due to the lack of DMT protection, the hydroxyl group participating in the condensation reaction is directly exposed, which improves the efficiency of the condensation reaction. Because the DMT protecting group of the carrier has been removed in advance, the probability of the N-1 and N-2 deletions of the gene sequence after synthesis caused by the incomplete removal of the protecting group of the common carrier is greatly reduced, and the purity of the gene after synthesis is improved. DNA is synthesized by using the DNA synthesis matrix of the present application, and its accuracy rate is improved by about 10%.

[0021] On the other hand, the present application provides a DNA synthesis matrix that avoids cross-contamination and a preparation method thereof. The sample addition sites are subjected to high-intensity DMT removal before synthesis. The use of the DNA synthesis matrix that avoids cross-contamination of the present application can also improve the accuracy of DNA synthesis and reduce the probability of N-1 and N-2 deletions in the gene sequence after synthesis; and no DMT removal treatment is performed between the sample addition sites. Only the pretreated areas can perform subsequent reactions, and the areas that have not been pretreated serve as protection and spacing, thereby preventing cross-contamination during DNA synthesis, thereby preventing a reduction in the accuracy of DNA synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0023] Figure 1 It is a schematic diagram of the vector structure without DMT protection;

[0024] Figure 2 This is a micrograph of the substrate prepared in Example 1 without DMT protection. DETAILED DESCRIPTION

[0025] In order to facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the technical field of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" includes any and all combinations of one or more related listed items. "Alternatively" herein means by way of example.

[0027] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0028] On one hand, the present application provides a DNA synthesis matrix, comprising a carrier without DMT and a fixing material for fixing the carrier, wherein the carrier without DMT comprises an active group without DMT.

[0029] In a specific example, the thickness of the matrix is ​​0.01 mm to 10 mm, the average pore size of the matrix is ​​1 μm to 500 μm, and the ratio of the thickness to the average pore size is 10:1 to 100:1. For example, 0.05 mm, 0.1 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm. For example, the ratio of thickness to average pore size is 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1.

[0030] Optionally, the active group is at least one of a phosporamidite group, a universal linkerphosphoramidite group and a modified phosporamidites group.

[0031] Further optionally, the carrier has an average of 0.1 nmol / cm 2 ~10nmol / cm 2 Active groups. For example: 0.05nmol / cm 2 , 0.1nmol / cm 2 , 0.5nmol / cm 2 , 1nmol / cm 2 , 1.5nmol / cm 2 , 2nmol / cm 2 , 2.5nmol / cm 2 , 3nmol / cm 2 , 3.5nmol / cm 2 , 4nmol / cm 2 , 4.5nmol / cm 2 , 5nmol / cm 2 , 5.5nmol / cm 2 , 6nmol / cm 2 、6.5nmol / cm 2 , 7nmol / cm 2 , 7.5nmol / cm 2 , 8nmol / cm 2 、8.5nmol / cm 2 , 9nmol / cm 2 , 9.5nmol / cm 2 , 10nmol / cm 2 .

[0032] The hydrophobic contact angle of the substrate is 10 to 45 degrees, for example, the contact angle is 10 degrees, 15 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees, or 45 degrees.

[0033] In one embodiment, the steps include:

[0034] The carrier without DMT and the fixing material are heated to melt the carrier to form an intermediate, and the intermediate is subjected to calendaring and / or stretching treatment to obtain the DNA synthesis matrix without DMT protection.

[0035] On the other hand, the present application provides a DNA synthesis matrix that avoids cross-contamination, comprising at least two synthesis sites, wherein there is a gap between the synthesis sites; among the vectors in the synthesis sites, more than 95% are vectors without DMT; among the vectors between the synthesis sites, more than 95% are vectors with DMT.

[0036] In one embodiment, the steps include:

[0037] The DMT-carrying carrier and the fixing material are heated to melt the carrier to form an intermediate, and the intermediate is subjected to calendaring and / or stretching treatment to obtain the DMT-protected DNA synthesis matrix, and the synthesis site is subjected to de-DMT treatment, while the synthesis sites are not subjected to de-DMT treatment.

[0038] In a specific example, the synthesis site is also ultrasonically cleaned. In the process of preparing the DNA synthesis matrix, some plasticizers or small particles will cover the surface of the synthesis site, causing shielding on the surface of the synthesis site. Even if a strong de-DMT treatment is performed, DMT cannot be removed, resulting in a significant increase in the probability of N-1 and N-2 deletions in the gene sequence after synthesis. These surface impurities can be removed by ultrasonic cleaning. The present invention only performs ultrasonic cleaning on the synthesis site, and does not perform ultrasonic cleaning between the synthesis sites. The synthesis sites are still covered with plasticizers or small particles, which can further avoid cross contamination and improve the accuracy of DNA synthesis.

[0039] Ultrasonic cleaning refers to dripping a cleaning solution such as acetonitrile solution on the synthesis site of the DNA synthesis matrix, and ultrasonicating at 100Hz to 1000Hz and 30℃ to 60℃ for 0.5h to 3h. Then rinse with a cleaning solution such as acetonitrile solution for 5min to 30min, and dry in a vacuum drying oven at 60℃ to 90℃ for 1h to 4h. Because the cleaning solution is only dripped on the synthesis site, only the synthesis site area produces a cleaning effect during the ultrasonic process, and because there is no liquid between the synthesis sites, it will not affect the surface impurities, so that the directional ultrasonic cleaning of the synthesis site can be achieved.

[0040] According to the above DNA synthesis substrate, the above DNA synthesis substrate avoiding cross contamination is used in DNA synthesis.

[0041] The following is a detailed description in conjunction with specific examples. The following examples do not include other components except for inevitable impurities unless otherwise specified. The reagents and instruments used in the examples are conventionally selected in the art unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are implemented according to conventional conditions, such as the conditions described in the literature, books, or methods recommended by the manufacturer.

[0042] The following takes a DNA synthesis matrix without DMT as an example. It is understandable that it may also be a CPG synthesis filter element without DMT or other similar products.

[0043] Example 1

[0044] This embodiment provides a DNA synthesis matrix without DMT, which has a thickness of 0.1 mm, an average pore size of 2 μm, a thickness-to-pore size ratio of 50:1, and an average of 1 nmol / cm 2 The active group is a phosphoramidite monomer (phosporamidite) without a 4,4-dimethoxytriphenylmethyl (DMT) protecting group, as shown in the schematic diagram Figure 1 As shown, the hydrophobicity of the substrate is a contact angle of 30 degrees.

[0045] The preparation method of the above-mentioned DNA synthesis matrix without DMT is to mix and melt 1g of the carrier without DMT and 20g of the fixing material to form an intermediate, and then calender the intermediate to obtain the DNA synthesis matrix without DMT. The parameters of the calendering treatment are pressure: 300Psi, temperature: 140°C, and tension: 850N.

[0046] Figure 2 This is a micrograph of the matrix prepared in Example 1. The round particles are the fixed material and the irregular particles are the carrier.

[0047] The method of using the above-mentioned substrate for synthesizing DNA is to sequentially add reagents to the surface of the DNA substrate, the reagent dropping speed is 0.2 μl / s, the reagent volume is 0.5 μl, and the area of ​​the synthesis site is 5 mm 2 . 96 synthesis sites are set on each DNA substrate.

[0048] The specific operation steps of DNA synthesis are: first cycle: activation, reaction for 60 seconds, nucleotide monomers and activators are mixed to form active intermediates; capping reagent is added, reaction for 30 seconds; iodine oxidation reagent is added, reaction for 30 seconds.

[0049] Enter the next cycle and the process is the same as above until the target DNA is synthesized.

[0050] A DNA sequence was synthesized using the above method, and the sequences from 5' to 3' end were: ATCGAGACCGAGGAGAGG.

[0051] Example 2

[0052] This embodiment provides a DNA synthesis matrix without DMT, which has a thickness of 0.1 mm, an average pore size of 2 μm, a thickness-to-pore size ratio of 50:1, and an average of 1 nmol / cm 2 The active group is a phosphoramidite monomer (phosporamidite) without a 4,4-dimethoxytrityl (4,4-dimethoxytriphenylmethyl, DMT) protecting group, and the hydrophobicity of the matrix is ​​a contact angle of 30 degrees.

[0053] The preparation method of the DNA synthesis matrix without DMT is as follows: 1 g of a carrier without DMT and 20 g of a fixing material are mixed and melted to form an intermediate, and then the intermediate is subjected to calendering treatment to obtain the DNA synthesis matrix without DMT. The parameters of the calendering treatment are pressure: 300 Psi, temperature: 140° C., and tension: 850 Newtons.

[0054] The method for using the DNA synthesis substrate without DMT is to sequentially add reagents to the surface of the DNA synthesis substrate without DMT, the reagent dropping speed is 0.2 μl / s, the reagent volume is 0.5 μl, and the area of ​​the synthesis site is 5 mm 2 . Each DNA synthesis substrate without DMT is provided with 96 synthesis sites.

[0055] The specific operation steps of DNA synthesis are: first cycle: activation, reaction for 60 seconds, nucleotide monomers and activators are mixed to form active intermediates; capping reagent is added, reaction for 30 seconds; iodine oxidation reagent is added, reaction for 30 seconds.

[0056] Enter the next cycle and the process is the same as above until the target DNA is synthesized.

[0057] A DNA sequence was synthesized using the above method, and the sequences from 5' to 3' end were: ATTTAGGTGACACTATAG.

[0058] Example 3

[0059] This embodiment provides a DNA synthesis matrix without DMT, which has a thickness of 0.1 mm, an average pore size of 2 μm, a thickness-to-pore size ratio of 50:1, and an average of 1 nmol / cm 2 The active group is a phosphoramidite monomer (phosporamidite) without a 4,4-dimethoxytrityl (4,4-dimethoxytriphenylmethyl, DMT) protecting group, and the hydrophobicity of the matrix is ​​a contact angle of 30 degrees.

[0060] The method for preparing the synthetic matrix without DMT is as follows: 1 g of a carrier without DMT and 20 g of a fixing material are mixed and melted to form an intermediate, and then the intermediate is subjected to calendering treatment to obtain a DNA synthetic matrix without DMT. The parameters of the calendering treatment are pressure: 300 Psi, temperature: 140° C., and tension: 850 Newtons.

[0061] The method for using the DNA synthesis substrate without DMT is to sequentially add reagents to the surface of the DNA synthesis substrate without DMT, the reagent dropping speed is 0.3 μl / s, the reagent volume is 0.6 μl, and the area of ​​the synthesis site is 5 mm 2 . Each DNA synthesis substrate without DMT is provided with 96 synthesis sites.

[0062] The specific operation steps of DNA synthesis are: first cycle: activation, reaction for 60 seconds, nucleotide monomers and activators are mixed to form active intermediates; capping reagent is added, reaction for 30 seconds; iodine oxidation reagent is added, reaction for 30 seconds.

[0063] Enter the next cycle and the process is the same as above until the target DNA is synthesized.

[0064] A DNA sequence was synthesized using the above method, and the sequences from 5' to 3' end were: CAGGAAACAGCTATGAC.

[0065] Example 4

[0066] This embodiment provides a DNA synthesis matrix that avoids cross contamination, which has a thickness of 0.1 mm, an average gap size of 2 μm, a ratio of thickness to gap size of 50:1, and an average of 1 nmol / cm 2 Active groups: The active groups on the CPG at the synthesis site are phosphoramidite monomers with DMT protective groups, and the hydrophobicity of the matrix is ​​a contact angle of 30 degrees.

[0067] The preparation method of the DNA synthesis matrix avoiding cross contamination is as follows: 1 g of a carrier with DMT and 20 g of a fixing material are mixed and melted to form an intermediate, and then the intermediate is subjected to calendering treatment to obtain a DNA synthesis matrix with DMT. The parameters of the calendering treatment are pressure: 300 Psi, temperature: 140° C., and tension: 850 Newtons.

[0068] Add the de-DMT reagent dropwise onto the synthesis sites, taking care to avoid adding or diffusing between the synthesis sites.

[0069] The DMT removal reagent is a trichloroacetic acid (TCA) solution. The specific process is: add an excess of trichloroacetic acid (TCA) solution to the reaction site three times until no obvious color reaction occurs at the site. Then add anhydrous acetonitrile 2 to 3 times to rinse the removed DMT group. Finally, place it in a vacuum drying oven at 90°C for 2 hours to volatilize the acetonitrile to obtain a synthetic matrix with exposed hydroxyl groups.

[0070] The specific operation steps of DNA synthesis are: first cycle: activation, reaction for 60 seconds, nucleotide monomers and activators are mixed to form active intermediates; capping reagent is added, reaction for 30 seconds; iodine oxidation reagent is added, reaction for 30 seconds.

[0071] Enter the next cycle and the process is the same as above until the target DNA is synthesized.

[0072] A DNA sequence was synthesized using the above method, and the sequences from 5' to 3' end were: AATCATCGCAAGACCGGC.

[0073] Example 5

[0074] This embodiment provides a DNA synthesis matrix that avoids cross contamination, which has a thickness of 0.1 mm, an average gap size of 2 μm, a ratio of thickness to gap size of 50:1, and an average of 1 nmol / cm 2 Active groups: The active groups on the CPG at the synthesis site are phosphoramidite monomers with DMT protective groups, and the hydrophobicity of the matrix is ​​a contact angle of 30 degrees.

[0075] The preparation method of the synthetic matrix avoiding cross contamination is as follows: 1 g of a carrier with DMT and 20 g of a fixing material are mixed and melted to form an intermediate, and then the intermediate is subjected to calendering treatment to obtain a DNA synthetic matrix with DMT. The parameters of the calendering treatment are pressure: 300 Psi, temperature: 140° C., and tension: 850 Newtons.

[0076] Add the de-DMT reagent dropwise onto the synthesis sites, taking care to avoid adding or diffusing between the synthesis sites.

[0077] The DMT removal reagent is a trichloroacetic acid (TCA) solution. The specific process is: add an excess of trichloroacetic acid (TCA) solution to the reaction site three times until no obvious color reaction occurs at the site. Then add anhydrous acetonitrile 2 to 3 times to rinse the removed DMT group. Finally, place it in a vacuum drying oven at 90°C for 2 hours to volatilize the acetonitrile to obtain a synthetic matrix with exposed hydroxyl groups.

[0078] Perform ultrasonic cleaning on the synthesis site. Specifically, add acetonitrile solution to the synthesis site of the DNA synthesis matrix, be careful not to diffuse outside the synthesis site, and ultrasonicate at 200Hz and 60℃ for 1 hour. Then rinse with acetonitrile solution for 10 minutes and dry in a vacuum drying oven at 90℃ for 2 hours.

[0079] The specific operation steps of DNA synthesis are: first cycle: activation, reaction for 60 seconds, nucleotide monomers and activators are mixed to form active intermediates; capping reagent is added, reaction for 30 seconds; iodine oxidation reagent is added, reaction for 30 seconds.

[0080] Enter the next cycle and the process is the same as above until the target DNA is synthesized.

[0081] A DNA sequence was synthesized using the above method, and the sequences from 5' to 3' end were: AATCATCGCAAGACCGGC.

[0082] Comparative Example 1

[0083] This embodiment provides a DNA synthesis matrix with DMT, which has a thickness of 0.1 mm, an average pore size of 2 μm, a thickness-to-pore size ratio of 50:1, and an average of 1 nmol / cm 2 The active group is a phosphoramidite monomer (phosporamidite) with a 4,4-dimethoxytrityl (4,4-dimethoxytriphenylmethyl, DMT) protective group, and the hydrophobicity of the matrix is ​​a contact angle of 30 degrees.

[0084] The preparation method of the DNA synthesis matrix with DMT is as follows: 1g of the carrier with DMT and 20g of the fixing material are mixed and melted to form an intermediate, and then the intermediate is calendered to obtain the DNA synthesis matrix. The parameters of the calendering treatment are pressure: 300Psi, temperature: 140°C, and tension: 850N.

[0085] The method for using the DNA synthesis substrate with DMT is to sequentially add reagents to the surface of the DNA synthesis substrate with DMT, the reagent dropping speed is 0.2 μl / s, the reagent volume is 0.5 μl, and the area of ​​the synthesis site is 5 mm 2. 96 synthesis sites are set on each DNA synthesis matrix with DMT.

[0086] The specific operation steps of DNA synthesis are as follows: First cycle: add deprotection reagent, react for 60 seconds; add DMT modified base + catalyst, react for 120 seconds; add capping reagent, react for 30 seconds; add iodine oxidation reagent, react for 30 seconds.

[0087] Enter the next cycle and the process is the same as above until the target DNA is synthesized.

[0088] A DNA sequence was synthesized using the above method, and the sequences from 5' to 3' end were: AATCATCGCAAGACCGGC.

[0089] Comparative Example 2

[0090] This comparative example provides a DNA synthesis matrix with a thickness of 0.1 mm, an average pore size of 2 μm, a ratio of thickness to pore size of 50:1, and an average of 1 nmol / cm 2 Active groups: The active groups on the CPG at the synthesis site are phosphoramidite monomers with DMT protective groups, and the hydrophobicity of the matrix is ​​a contact angle of 30 degrees.

[0091] The preparation method of the above DNA synthesis matrix is ​​to mix and melt 1g of the carrier with DMT and 20g of the fixing material to form an intermediate, and then calender the intermediate to obtain the DNA synthesis matrix with DMT. The parameters of the calendering treatment are pressure: 300Psi, temperature: 140°C, and tension: 850N.

[0092] The DNA synthesis matrix is ​​soaked in a de-DMT reagent, and then anhydrous acetonitrile is added 2-3 times to rinse the removed DMT group. Finally, it is placed in a vacuum drying oven at 90°C for 2 hours to evaporate the acetonitrile to obtain a synthetic matrix with exposed hydroxyl groups.

[0093] The entire DNA synthesis matrix was ultrasonically cleaned. Specifically, the DNA synthesis matrix was soaked in acetonitrile solution, transferred to an ultrasonic generator, and ultrasonicated at 200 Hz and 60° C. for 1 hour. Then, it was rinsed with acetonitrile solution for 10 minutes and dried in a vacuum drying oven at 90° C. for 2 hours.

[0094] The method for using the DNA synthesis substrate without DMT is to sequentially add reagents to the surface of the DNA substrate, the reagent drop rate is 0.2 μl / s, the reagent volume is 0.5 μl, and the area of ​​the synthesis site is 5 mm 2 . 96 synthesis sites are set on each DNA substrate.

[0095] The specific operation steps of DNA synthesis are: first cycle: activation, reaction for 60 seconds, nucleotide monomers and activators are mixed to form active intermediates; capping reagent is added, reaction for 30 seconds; iodine oxidation reagent is added, reaction for 30 seconds.

[0096] Enter the next cycle and the process is the same as above until the target DNA is synthesized.

[0097] A DNA sequence was synthesized using the above method, and the sequences from 5' to 3' end were: AATCATCGCAAGACCGGC.

[0098] The specific results of Examples 1 to 5 and Comparative Examples 1 to 2 are shown in Table 1:

[0099] Table 1

[0100]

[0101]

[0102] It can be seen from the data in Table 1 that the DNA synthesis accuracy of Examples 1 to 3 is higher than that of Comparative Example 1. This is because Examples 1 to 3 are carriers without DMT protection provided by the present application, and Comparative Example 1 is a carrier with DMT protection. During the preparation process of the DNA synthesis matrix of the present invention, the surface of the carrier is shielded by impurities such as fixed materials and plasticizers. Therefore, it is difficult to remove DMT completely during use, thereby reducing the yield and accuracy of DNA synthesis. The DNA synthesis matrix without DMT protection provided by the present application has an accuracy of 70.2% to 72.3% in DNA synthesis, and the probability of N-1 and N-2 deletions is 0.02% to 0.04%. Compared with the carrier with DMT protection, the accuracy of DNA synthesis is increased by about 10%, and the probability of N-1 and N-2 deletions is reduced by about 0.2%.

[0103] In the cross-contamination-avoiding DNA synthesis carriers (without DMT + with DMT) of Examples 4 to 5, high-intensity DMT removal is performed on the sample loading sites before synthesis, and no DMT removal treatment is performed between the sample loading sites. Only the pretreated areas can undergo subsequent reactions, and the areas that have not been pretreated serve as protection and spacing. These areas that are not subjected to DMT removal will not produce a synthesis reaction even if liquid penetrates through them, thereby preventing cross-contamination during the DMT removal process. The DNA synthesis vector (without DMT + with DMT) for avoiding cross-contamination provided in the present application has an accuracy rate of 77.5% to 79.8% in DNA synthesis, and the probability of N-1 and N-2 deletion is 0.03% to 0.04%. Compared with Comparative Example 2, the DNA synthesis accuracy of Examples 4-5 is increased by about 20%; the probability of N-1 and N-2 deletion in Examples 4-5 is 0.05% higher than that in Examples 1-3. This is because even high-intensity de-DMT reagents, even ultrasonic treatment, cannot completely desorb DMT, but the DNA synthesis accuracy of Examples 4-5 is further improved because its structure has the function of preventing cross-contamination.

[0104] The comparative examples described above only express several embodiments of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the scope of protection of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings may be used to explain the contents of the claims.

Claims

1. A DNA synthesis substrate, characterized in that The invention comprises a carrier without DMT and a fixing material for fixing the carrier, wherein the carrier without DMT comprises an active group without DMT.

2. The DNA synthesis substrate according to claim 1, characterized in that The thickness of the matrix is ​​0.01 mm to 10 mm, the average void size of the matrix is ​​1 μm to 500 μm, and the ratio of the thickness to the average void size is (10:1) to (100:1).

3. The DNA synthesis substrate according to claim 1, characterized in that The active group is at least one of a phosporamidite group, a universal linker phosphoramidite group and a modified phosporamidites group.

4. The DNA synthesis substrate according to claim 1, characterized in that The carrier is immobilized with an average of 0.1 nmol / cm 2 ~10nmol / cm 2 Active groups.

5. The DNA synthesis substrate according to claim 1, characterized in that The hydrophobic contact angle of the substrate is 10 degrees to 45 degrees.

6. The method for preparing a DNA synthesis substrate according to any one of claims 1 to 5, characterized in that: The following steps are involved: heating the carrier without DMT and the fixing material to melt the carrier to form an intermediate; The intermediate is subjected to calendering and / or stretching treatment to obtain the DNA synthesis matrix without DMT protection.

7. A DNA synthesis matrix for avoiding cross contamination, characterized in that: It comprises at least two synthesis sites, wherein there is a gap between the synthesis sites; among the vectors at the synthesis sites, more than 95% are the vectors without DMT as claimed in claim 1; among the vectors between the synthesis sites, more than 95% are the vectors with DMT.

8. A method for preparing a DNA synthesis matrix to avoid cross contamination, characterized in that: Used to prepare the DNA synthesis substrate for avoiding cross contamination according to claim 7; The preparation method comprises the following steps: heating the carrier with DMT and the fixing material so that the carrier melts to form an intermediate; The intermediate is subjected to calendering and / or stretching treatment to obtain the DNA synthesis matrix with DMT protection; The synthesis sites are subjected to de-DMT treatment, and the synthesis sites are not subjected to de-DMT treatment.

9. The method for preparing a DNA synthesis substrate for avoiding cross contamination according to claim 8, characterized in that: The method also comprises performing ultrasonic cleaning on the synthesis site.

10. An application of a DNA synthesis matrix, characterized in that: The invention comprises the use of the DNA synthesis substrate according to any one of claims 1 to 5 and the DNA synthesis substrate for avoiding cross contamination according to claim 7 in DNA synthesis.

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