CN116004746A - A kit for enzymatic DNA synthesis and its application - Google Patents

Abstract

The application discloses a kit for enzymatic DNA synthesis and application thereof, and belongs to the field of DNA synthesis. The invention discloses a kit for enzymatic DNA synthesis, which comprises a chip, an initial chain and a terminal deoxynucleotidyl transferase, wherein the chip contains a probe, the probe is a single-chain DNA molecule, the 3 'terminal of the probe is modified by amino, the probe comprises a flexible arm region and a binding region combined with the initial chain, the flexible arm region is formed by connecting 6-12T, the binding region consists of 20-30 nucleotides, and the nucleotide sequence of the binding region is reversely complementary with the 5' terminal of the initial chain. The invention provides a chip for enzymatic DNA synthesis, which has high dissociation efficiency and can be repeatedly used, and has important significance for the field of enzymatic DNA synthesis.

Description

A kit for enzymatic DNA synthesis and its application

technical field

The application belongs to the field of DNA synthesis, and in particular relates to a kit for enzymatic DNA synthesis and its application.

Background technique

At present, DNA synthesis mainly adopts the solid-phase phosphoramidite chemical synthesis method developed in the 1980s, through a 4-step cycle reaction, to realize the synthesis of oligonucleotide chains from 3' to 5'. However, DNA chemical synthesis technology has its own bottleneck problems, such as cumbersome reaction steps, long time-consuming for a single cycle (6-8 minutes), high consumption and high cost of chemical reagents, large use of toxic and flammable organic reagents, pollution larger. Therefore, DNA biosynthesis based on protease catalysis has gradually become a new generation of DNA synthesis technology that is expected to replace chemical synthesis technology.

Enzymatic DNA synthesis technology refers to the use of template-independent terminal deoxynucleotidyl transferase (TdT) to add a single nucleotide to the end of the DNA chain. Its synthesis mechanism is similar to the DNA replication mechanism in organisms, and can realize oligonucleotide chain Synthesized from 5' to 3' direction. In the presence of natural nucleotides, TdT can extend single-stranded DNA fragments by thousands of nucleotides. Biological DNA synthesis technology has incomparable advantages over chemical synthesis. For example, the substrate specificity and catalytic efficiency of biological enzymes are generally significantly higher than those of chemical synthesis. The mild aqueous reaction conditions of biological methods can reduce the production of by-products.

Microfluidic chip is the main platform for the realization of microfluidic technology. The main feature of the device is that its effective structures (channels, reaction chambers and other functional components) for containing fluids are at least in the micron scale in one latitude, thereby greatly improving reaction efficiency and reducing costs. Due to the significant difference in principle between DNA chemical synthesis technology and biosynthesis technology, current microfluidic chips are mainly suitable for chemical synthesis technology, and there is no microfluidic chip for enzymatic DNA synthesis technology.

In addition, currently known devices using microfluidic chips for DNA synthesis are ubiquitous, and the dissociation efficiency of probes after immobilization is not high (enzymatic dissociation is used), the steps are cumbersome (two-step enzyme digestion is required), and the overall cost is high (The reagents used are more expensive), cannot be reused, and have disadvantages such as weak connection with the subsequent synthesis process.

Contents of the invention

The technical problems to be solved in the present invention are 1. the core problem of DNA synthesis chips with no mature enzyme method at present; Reagents are more expensive), can not be reused, and have disadvantages such as weak connection with the subsequent synthesis process.

In order to solve the above technical problems, in a first aspect, the present invention provides a kit for enzymatic DNA synthesis, the kit includes a chip and an initial strand,

The chip contains a probe, the probe is a single-stranded DNA molecule, the 3' end of the probe is modified by an amino group, and the probe includes a flexible arm region and a binding region combined with the initial chain, so The flexible arm region is connected by 6-12 Ts, the binding region is composed of 20-30 nucleotides, and the nucleotide sequence of the binding region is reverse complementary to the 5' end of the starting chain .

Further, in the above kit, the length of the probe is 26-42nt.

Further, in the above kit, the initial strand includes a pairing region that binds to the probe, the nucleotide sequence of the pairing region is reverse complementary to the binding region of the probe, and the pairing region The length is 20-30 nucleotides.

Further, in the above kit, the initiation strand further includes a priming region at the 3' end for initiating DNA synthesis, and the length of the priming region is greater than 3 nt.

Further, in the above kit, the length of the priming region of the initial chain is 4-6nt.

Further, in the above kit, the nucleotide sequence of the probe is SEQ ID No.1, and the nucleotide sequence of the pairing region of the initiation strand is the 1st-25th positions of SEQ ID No.2.

In one embodiment of the present invention, the nucleotide sequence of the probe from 3' to 5' direction is SEQ ID No.1, and the 1st-11th positions of SEQ ID No.1 are flexible arms, and SEQ ID No. 1 Positions 12-36 are the binding region to the initial chain.

_NH2-3' -TTTTTTTTTTCCGATCTCTGAGGATGCGCTGAACT-5' (SEQ ID No. 1).

In one embodiment of the present invention, the nucleotide sequence of the starting strand from 5' to 3' direction is SEQ ID No.2, and the 1st-25th positions of SEQ ID No.2 are complementary to the probe The combined pairing region, the 26th-31st position of SEQ ID No. 2 is the initiation region of the initiation chain.

5'-GGCTAGAGACTCCTACGCGACTTGAATGACG-3' (SEQ ID No. 2).

Further, in the above kit, the kit further includes terminal deoxynucleotidyl transferase (TdT).

In the present invention, the amino acid sequence of the terminal deoxynucleotidyl transferase (TdT) is SEQ ID No.3.

Further, the kit can also protect the reagents required for the hybridization reaction between the probe and the initial strand.

In order to solve the above-mentioned technical problems, the present invention protects the above-mentioned chip in a second aspect.

Further, the chip is a microfluidic chip, and the chip also includes an aldehyde-based glass slide on which probes are immobilized.

In order to solve the above-mentioned technical problems, in a third aspect, the present invention provides a method for preparing the above-mentioned chip, comprising cross-linking the above-mentioned probes with an aldehyde-modified substrate to obtain the chip.

In order to solve the above-mentioned technical problems, in a fourth aspect, the present invention provides a method for enzymatic DNA synthesis, comprising hybridizing the probe with the initial strand in the above-mentioned chip, and then using terminal deoxynucleotide transfer Enzymes and dNTPs with protective bases perform multiple rounds of cycles in the chip to achieve enzymatic synthesis of the target DNA sequence. In the last round of cycles, the chip is heated to 95-98 ° C, so that the probe The needle dissociates from the starting strand, resulting in synthetic DNA.

The cycle number of the multiple cycles is equal to the number of nucleotides of the DNA to be synthesized minus the length of the priming region of the initial strand.

The cycle number of the multiple cycles can be 1-5, but it has the potential to synthesize 50 cycles.

After the cycle is over, in the last round, the chip is heated to 95-98 degrees Celsius, and a sufficient amount of ultrapure water is passed into the reaction chamber to wash out the synthesized DNA, which is then sequenced and detected using the Illumina Miseq platform.

Beneficial effects compared with prior art:

1) In the existing technology, the dissociation efficiency of DNA immobilized on the chip is low, and the steps are cumbersome (two-step enzyme digestion is required): There are reports in the literature that ssDNA is cut from the solid phase surface by using UDG and endonuclease. This method is prone to incomplete enzyme reaction, low dissociation efficiency, and requires two-step enzyme reaction, and the operation steps are cumbersome. However, the method adopts the principle of high-temperature denaturation and separation of complementary pairing between the probe and the starting strand, and overcomes the shortcomings of the existing methods.

2) The overall cost is higher (the reagents used are more expensive): the reaction system reported in 1 contains a variety of enzymes, which increases additional costs, and some other technologies, such as the combination of biotin-streptavidin and Dissociation, although the dissociation efficiency is higher and more convenient, but the cost of streptavidin-modified slides is relatively high, and this method has no advantages compared with this method.

3) Cannot be reused: the DNA chips involved in the prior art cannot be reused, and only one batch of DNA sequences can be synthesized at a time, which causes the increase of cost and the pollution of materials. However, after the initial strand of the method is dissociated, the probe can still continue to combine with the new initial strand to carry out a new enzymatic DNA synthesis reaction, which greatly reduces the cost and saves materials. 4) The connectivity of the follow-up synthesis process is not strong: because DNA synthesis is a continuous process, steps such as mass spectrometry or sequencing detection, connection, and error correction are required after synthesis. After the DNA strands on the existing chip are dissociated, it is difficult to It will continue to be used in the next reaction, and it needs to be taken out for purification before proceeding to the next step. In this method, ultrapure water is used to dissolve DNA in the last step, and the washed out solution can directly enter the next reaction chamber for sequencing quality control, or continue to perform reactions such as connection and error correction, which greatly improves the consistency of DNA synthesis. and integration. In order to comprehensively reflect the reproducibility and coherence advantages of this method, as well as the potential for enzymatic DNA synthesis, we used the same chip to perform TdT enzyme plus T reactions three times, and the results showed that the three plus T efficiencies were all above 70%. .

Description of drawings

Figure 1 is a schematic flow chart of the enzymatic DNA synthesis device.

Figure 2 shows the accuracy of enzymatic synthesis of single-stranded DNA.

Fig. 3 is the verification result of the binding immobilization and dissociation of the starting strand and the probe.

Fig. 4 is the fluorescent intensity measured during the binding and dissociation process of the fluorescent probe and the fluorescent initiator chain.

Fig. 5 is the verification result of repeated combination of the starting strand and the probe on the chip.

Figure 6 shows the fluorescence intensity of the initial strand binding and dissociation for three times.

Figure 7 shows the effect of DNA synthesis of the chip combined three times.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The SSC buffers in the following examples are all purchased 20×SSC solutions (pH 7.4) from Coolaber, the product number of which is SL3030, and are diluted with ultrapure water to the corresponding concentration when used.

The following examples adopt Graphpad v8.0 statistical software to process the data, and the experimental results are represented by mean ± standard deviation, adopting T test test, * represents that there is a significant difference (P＜0.05), and ** represents that there is extreme significance Difference (P<0.01), *** indicates extremely significant difference (P<0.001).

The terminal deoxynucleotidyl transferase used in the following examples is a TDT variant enzyme, which is a protein whose amino acid sequence is SEQID No.3, and the coding gene (SEQ ID No.4) of this TDT variant enzyme is constructed into pET -28a (Novagen, Kan+) was cut between NdeI and XhoI to obtain a recombinant plasmid named pET-28a-TdT. And express and purify TdT enzyme according to the following method.

1. Gene expression

In order to detect the activity of TDT enzyme in vitro, the enzyme was expressed and purified exogenously in Escherichia coli. The host bacterium is E.coli BL21(DE3) (Novazyme C504), but the host bacterium is not limited thereto, and all hosts that can be used to express proteins are included.

(1) Transfer the E. coli expression recombinant plasmid pET-28a-TDT into E. coli BL21(DE3) to obtain recombinant bacteria. Positive clone selection (Kan+, 100 mg/mL) was carried out on a kanamycin-resistant plate, and cultured overnight at 37°C;

(2) Pick a single clone into 5mL LB liquid medium (Kan+, 100mg/mL), and culture at 37°C, 220r/min until the OD600 is 0.6-0.8. Transfer the bacterial liquid in 5mL LB medium to 800mL 2YT medium (Kan+, 100mg/mL), culture at 37°C and 220rpm until the OD600 is 0.6-0.8, cool down to 16°C, add IPTG to a final concentration of 0.5mM, Induce expression for 16h;

(3) The above-mentioned cultured bacteria liquid is collected in the bacteria collection bottle, and centrifuged at 5500r/min for 15min;

(4) Discard the supernatant, suspend the obtained bacterial pellet with 35mL protein buffer (50mM Tris-HCl, 2mM EDTA, 0.1% Triton X-100, pH7.4), pour it into a 50mL centrifuge tube, and store at -80°C Store in the refrigerator.

2. Protein purification

(1) Bacteria destruction: use a high-pressure and low-temperature crushing apparatus to destroy the bacterial cell precipitate obtained in the above-mentioned 3 twice under the condition of a pressure of 1200 bar and 4°C. Centrifuge at 4°C and 10000r/min for 45min, take the precipitate and supernatant after centrifugation, and prepare samples;

(2) Purification: the supernatant was suction-filtered through a 0.45 μm microporous membrane, and then purified by nickel affinity chromatography. The specific steps were as follows:

a: Column equilibration: before supernatant, wash 2 column volumes with ddH2O, and then equilibrate 1 column volume of Ni affinity chromatography column with protein buffer;

b: Sample loading: slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 0.5mL/min, and repeat again;

c: Elution of impurity proteins: Rinse 1 column volume with protein buffer, then use 50mL protein buffer containing 50mM imidazole to elute strongly bound impurity proteins, take the first few drops of flow-through samples, and prepare samples;

d: Elution of the target protein: use 20mL of 100mM, 200mM, 300mM imidazole protein buffer to elute the target protein respectively, take the first few drops of the flow-through sample, prepare the sample, and detect it with 12% SDS-PAGE. The results are shown in Figure 2 Show.

(3) Concentration and liquid replacement: the collected target protein was concentrated by centrifugation (4°C, 3400r/min) with a 50mL Amicon ultrafiltration tube (30kDa, Millipore Company) to 1mL. Add 10mL of protein buffer, concentrate to 1mL, and repeat this process once to obtain purified protein TDT.

(4) The protein concentration after concentration was detected with a Nondrop 2000 micro-spectrophotometer, which was 4 mg/mL. That is, the purified and concentrated TDT protein is obtained.

>TdT enzyme, SEQ ID No.3

MDRFKAPAVISQRKRQKGLHSPKLSCSYEIKFSNFVIFIMQRKMGLTRRMFLMELGRRKGFRVESELSDSVTHIVAENNSYLEVLDWLKGQAVGDSSRFELLDISWFTACMEAGRPVDSEVKYRLMEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMaeNYEFKENEIF CLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEIEIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKEAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLY CDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA*

>TdT enzyme coding gene, SEQ ID No.4

ATGGACCGCTTCAAAGCCCCTGCTGTGATTAGCCAGCGTAAACGTCAGAAAGGTCTGCACTCTCCGAAGCTGTCCTGCAGCTATGAGATTAAATTCTCCAACTTCGTTATTTTTTATCATGCAGCGTAAAATGGGCCTGACCCGCCGTATGTTTCTGATGGAACTGGGTCGCCGTAAAGGTTTTTCGTGTTGAATCTGAACTGAGCGATTCCGTTACCCACA TCGTTGCTGAAAACAACAGCTACCTGGAAGTTCTGGACTGGCTGAAAGGCCAGGCAGTTGGTGACAGCTCTCGTTTCGAGCTGCTGGACATCTCCTGGTTCACCGCGTGCATGGAGGCTGGTCGTCCGGTTGATTCCGAGGTTAAATATCGCCTGATGGAGCAGTCCAGTCTCTGCCGCTGAACATGCCTGCGCTGGAAATGCCGGCGTTT ATTGCAACTAAAGTGTCCCAGTATTCCTGCCAGCGTAAAACGACTCTGAATAACTACAACAAAAATTCACTGACGCGTTCGAAGTAATGGCCGAGAACTACGAATTCAAGGAAAACGAAATTTTCTGCCTGGAATTCCTGCGTGCGGCATCCCTGCTGAAATCCCTGCCGTTCTCCGTCACCCGTATGAAGGATATCCAGGGCCTGCCGTGCGTGGGTGACCAA GTGCGTGACATCATCGAAGAAATCATCGAAGAAGGCGAGTCTTCCCGTGTTAACGAAGTTCTGAACGATGAACGTTACAAAGCATTCAAACAGTTCACCTCTGTATTCGGTGTTGGCGTGAAAACCTCTGAAAAATGGTACCGCATGGGCCTGCGTACCGTGGAAGAGGTTAAAGCTGACAAAACCCTGAAACTGAGCAAAATGCAGAAGGCGGGTCTGCTGTGT TACGAAGACCTGGTTTCCTGCGTATCTAAAGCGGAAGCAGACGCCGTTAGCCTGATTGTAAAGAACACGTTTGCACCTTCCTGCCGGATGCCCTGGTGACCATCACTGGTGGCTTCCGTCGTGGCAAAAACATCGGTCACGACATCGACTTCCTGATCACGAACCCGGGTCCGCGTGAAGACGATGAACTGCTGCATAAGGTGATCGACCTGTGGAAG AAACAGGGTCTGCTGCTGTACTGCGATATTATTGAGTCCACCTTCGTTAAGGAGCAGCTGCCGTCCCGCAAAGTGGATGCGATGGATCATTTTCAGAAATGTTTTGCAATTCTGAAACTGTACCAGCCGCGCGTCGACAACTCCACCTGCAATACCCTCCGAGCAACTGGAAATGGCCGAAGTGAAAGATTGGAAAGCTATTCGTGTGGATCTGGTTATC ACCCCGTTCGAACAGTACCCATACGCTCTGCTGGGTTGGACCGGTAGCCGTCAGTTCGGTCGCGATCTGCGCCGTTACGCCGCTCACGAGCGTAAAATGATCCTGGACAACCACGGTCTGTACGACCGTCGTAAACGTATTTTCTGAAAGCTGGTTCCGAAGAAGAAATCTTCGCTCATCTGGGTCTGGACTACGTTGAACCGTGGGAACGTA ACGCGTGA.

Embodiment 1, the method for non-template-dependent enzymatic DNA synthesis

In this embodiment, the synthetic nucleotide sequence is the single-stranded DNA described in 5'-TCTCT-3' as an example.

The workflow of the template-independent enzymatic DNA synthesis method is shown in Figure 1.

1.1. Probe design and initial strand design

The probe is a full-length 30-60nt ssDNA chain with amino modification at the 3' end of the DNA, which can dehydrate with the aldehyde site on the glass slide to form a Schiff base covalently bonded. From the 3' to the 5' direction, there is a 6-12nt polyT sequence first, which acts as a flexible arm to reduce the steric hindrance during the enzyme reaction, and then a binding region (20-30nt) that binds to the initial chain. The initial strand is a 20-40nt full-length ssDNA strand, the 20-30nt sequence at the 5' end is a pairing region that can be reversely complementary to the probe binding region, and the 3' end is the trigger region for initiating DNA synthesis , the number of nucleotides in the priming region is greater than 3nt, such as 4-6nt.

According to the above design requirements, design the probe and initial strand with the nucleotide sequence as follows:

Probe NH ₂ -3'-TTTTTTTTTTTT -CCGATCTCTGAGGATGCGCTGAACT -5' (the nucleotide sequence is SEQID No.1);

Initiating strand 5'- GGCTAGAGACTCCTACGCGACTTGA ATGACG-3' (nucleotide sequence is SEQ ID No. 2).

The above-mentioned probes and starting strands were artificially synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

The 3' end of the above probe sequence is modified by amino group, the 1st-11th position of the nucleotide sequence is the flexible arm, and the 12th-36th position is the binding region of the probe. The 1st-25th nucleotides of the above-mentioned starting chain are the pairing region that can be reverse-complementary paired with the probe binding region, and the 26th-31st nucleotides are the priming region for initiating DNA synthesis.

1.2. Cross-linking the probe with the aldehyde-modified substrate

Immobilizing probes on the surface of aldehyde-modified glass slides is a relatively mature method, and the probes are tightly bound to the glass slides, which cannot be directly dissociated by high temperature and other means. The specific operation steps are as follows:

a) Probes were dissolved in 3×SSC, 1.5 mM betaine. Probes (2 μM) were dissolved and mixed;

Among them, the composition of 1.5mM betaine is betaine as the solute (CAS number is 107-43-7), and the solvent is 3×SSC;

b) Use a pipette gun to drop the prepared probe solution (2 μL) onto an aldehyde-modified glass slide (purchased from Shanghai Bio-Tech Co., Ltd., referred to as aldehyde-based slide);

c) After spotting, place at room temperature for 30 minutes, transfer the slide to a metal bath at 100°C (Beaver 2016C) and dry for 30 minutes;

d) In the ultraviolet crosslinking instrument, 600J crosslinks two to three times;

e) Cleaning the slides, washing them twice with 0.2% SDS at room temperature for 2 minutes each time; washing them twice with deionized water at room temperature for 2 minutes each time;

Among them, 0.2% SDS is compared with 0.2% SDS aqueous solution, and the content of SDS is 2g/L;

f) Soak the slide in the aldehyde blocking solution for 15 minutes;

Among them, the configuration method of the aldehyde blocking solution is: take 0.12g of sodium borohydride (belonging to the catalog of precursors to poison and explosives, its CAS number is 16940-66-2, purchased from sigma) dissolved in 30mL of PBS and then add 10mL of absolute ethanol Prepared by mixing;

Among them, the PBS solution was purchased from Coolaber, and its product number was SL6113;

g) Rinse the slide again: rinse twice in 0.2% SDS, 2 minutes each time; rinse once in deionized water, 2 minutes each time, and finally spin dry with a slide centrifuge.

1.3. Hybridization of the probe to the initial strand

a) Configuration of hybridization solution: Formamide (30%, purchased from coolaber, its product number is CF5391), 1.8mL; 20×SSC 1.8mL; SDS (0.5%) 0.03g; 50×Denhardt (5×, purchased from invitrogen, its Product number is 750018) 0.6mL, add water to make up to a total volume of 6mL;

Solution A: 1×SSC; 0.1% SDS;

Liquid B: 0.05×SSC;

Solution C: 95% ethanol;

b) Dilute the starting strand to 10 μM with hybridization solution, preheat to 45-50°C, and mix thoroughly.

c) Pass the prepared starting chain solution into the reaction chamber, and react at 45-50° C. for 2-4 hours.

d) Wash with liquid A for 3 minutes; liquid B for 3 minutes; liquid C for 90 seconds.

e) After drying, store at 4°C until use.

1.4. Initial strand DNA synthesis reaction and dissociation

Cycle step 1: use TdT enzyme and dNTPs with protected bases (purchased from Firebird BiomolecularSciences) for enzyme reaction, the enzyme reaction system is: 1μM initial chain, 0.1mM dNTPs, 0.25mM CoCl2, 100mMNaCl, 50mM phosphate buffer (pH 6.8), the enzyme reaction condition is 30 degrees Celsius, 5-10min.

Cycle step 2: use a deprotection solution (0.7M sodium nitrite solution) to elute the protected bases, and perform multiple rounds of cycles to achieve enzymatic synthesis of the target DNA sequence.

The number of cycles of multiple rounds of circulation is equal to the number of nucleotides to be synthesized DNA minus the length of the priming region of the initial chain, the number of cycles of multiple rounds of circulation can be 1-5, that is, steps 1 and 2 cycle 1- 5 rounds.

Among them, the dNTP with protected bases added in the first, third and fifth rounds is Thymine (Firebird Biomolecular Sciences, TONH2-171;

The dNTP with protected bases added in the second and fourth rounds is Cytosine (Firebird Biomolecular Sciences, CONH2-172;

After the cycle ends, the chip is heated to 95-98°C in the last round, and a sufficient amount of ultrapure water is passed into the reaction chamber to wash out the synthesized DNA.

1.5. Sequencing detection.

The Universal DNA Library Prep Kit for Illumina V3 (Novazyme, ND607) was used for library construction, and the Illumina Miseq platform was used for sequencing detection.

A total of 3 repeated experiments were performed, and the accuracy was calculated after sequencing the synthesized single-stranded DNA.

The accuracy of adding 5'-TCTCT-3' sequence after 5 rounds of sequencing reactions is shown in Figure 2. In the first round of reaction in Fig. 2, the ratio of successfully added was 86.64%, and the ratio of not added was 13.11%; the ratio of successfully added in the second round was 85.74%, and the ratio of not added was 14.03%; The proportion of 85.39% was 85.39%, and the proportion of no addition was 14.21%; the proportion of successful addition in the fourth round was 83.41%, and the proportion of no addition was 16.01%; the proportion of successful addition in the fifth round was 81.99%, and the proportion of no addition was 15.64%; the results show that: the proportion of successful addition in the five rounds of reaction is higher than 80%.

Embodiment 2, effect measurement of enzymatic DNA synthesis device

2.1. Probe immobilization and initial chain heating dissociation effect verification experiment

In the existing technology, the dissociation efficiency of DNA immobilized on the chip is low, and the steps are cumbersome (two-step enzyme digestion is required): there are reports in the literature that ssDNA is cut from the solid phase surface by using UDG and endonuclease. This method is prone to incomplete enzyme reaction, low dissociation efficiency, and requires two-step enzyme reaction, and the operation steps are cumbersome.

However, the method adopts the principle of high-temperature denaturation and separation of complementary pairing between the probe and the starting strand, and overcomes the shortcomings of the existing methods.

Detection of immobilization effect using fluorescent probes: the specific operation steps are the same as the above-mentioned probe immobilization method.

Among them, the fluorescent probe is based on the nucleotide shown in SEQ ID No.1, and a fluorescent modification group Cy3 (Suzhou Jinweizhi) is added at its 5' end;

The results are shown in A and B in Figure 3: A in Figure 3 is a blank control. First, immobilize the 3'-terminal amino-modified probe on the surface of the aldehyde-modified glass slide, and use the fluorescent probe to detect the immobilization effect. The results are shown in Figure 3 B. The results show that the fluorescent probe is evenly combined on the aldehyde-based glass slide .

Use ordinary probes and fluorescent starting strands to detect the binding effect after hybridization, and the specific operation steps are the same as the above-mentioned probe-primer hybridization method.

The modification group and modification position of the fluorescent initial chain are: Cy3, which is modified at the 3' end.

The results are shown as C in Figure 3, and the results show that the initial strand can be well combined with the probe.

In order to prove the efficiency of thermal dissociation of the initial strands, they were washed with double distilled water at 95°C and observed again with a fluorescence microscope.

The results are shown in D in Figure 3, almost no fluorescence can be detected in D in Figure 3, indicating that the initial chain and the probe have been dissociated, and the results show that the dissociation effect of the aldehyde-based glass slide is very good. Three groups were carried out for each experiment, and the statistical results of the fluorescence intensity data are shown in Figure 4. Compared with the blank control group and the fluorescent probe group, the fluorescence intensity increased significantly, indicating that the probe could be successfully fixed on the glass slide. However, when the non-fluorescent probe is combined with the fluorescent initial strand, the fluorescence intensity of the blank control is also more significant, indicating that the initial strand can be successfully fixed on the glass slide by hybridizing with the probe. Afterwards, through high temperature dissociation, comparing the fluorescent initial chain group with the high temperature dissociation group, the fluorescence is significantly reduced, indicating that the initial chain can be dissociated from the glass slide smoothly.

2.2. Reusability effect verification experiment

None of the DNA chips involved in the prior art can be reused, and only one batch of DNA sequences can be synthesized at a time, which causes an increase in cost and contamination of materials. However, after the initial strand of the method is dissociated, the probe can still continue to combine with the new initial strand to carry out a new enzymatic DNA synthesis reaction, which greatly reduces the cost and saves materials.

In order to verify whether the chip can be reused, after the first dissociation, the fluorescence intensity did not decrease significantly when combined with the fluorescent initiation chain again. After dissociation and binding again, the fluorescence intensity does not decrease significantly, which reflects the reusable characteristics of the present invention.

The results are shown in Figure 5. A in Figure 5 is the first binding of the fluorescent initiation chain to the probe, B in Figure 5 is the first dissociation of the fluorescent initiation chain, and C in Figure 5 is the second binding of the fluorescent initiation chain. The first time combined with the probe, D in Figure 5 is the second dissociation of the fluorescent initial strand, E in Figure 5 is the third binding of the fluorescent initial strand to the probe, and F in Figure 5 is the third time of the fluorescent initial strand Dissociate. The results showed that the fluorescence could be detected for three times of binding, but no fluorescence could be detected after dissociation, which proved that the combination and dissociation operation effected three times were good. Three groups were carried out for each experiment, and the statistics of fluorescence conversion are shown in Figure 6. The results in Figure 6 show that there is no significant difference between the fluorescence intensities of the initial chain after three bindings, and there is no significant difference between the fluorescence intensities after the three initial chain dissociations, indicating that there is no significant difference between the binding and dissociation steps of the three repeated initial chains. Significantly affects probe and initiator strand functionality.

2.3. Repeatedly used T-adding efficiency verification experiment

Because DNA synthesis is a continuous process, steps such as mass spectrometry or sequencing detection, connection, and error correction are required after synthesis. After the DNA strands on the existing chip are dissociated, it is difficult to continue to be used in the next step. Carry out the next step after purification. In this method, ultrapure water is used to dissolve DNA in the last step, and the flushed solution can directly enter the next reaction chamber for sequencing quality control, or continue to perform reactions such as connection and error correction, which greatly improves the consistency of DNA synthesis. and integration.

In order to comprehensively reflect the reproducibility and coherence advantages of this method, as well as the potential for enzymatic DNA synthesis, we used the same chip to perform TdT enzyme plus T reaction three times. The specific operation steps were the same as the above enzyme reaction steps, the difference The dNTPs with protective bases added only in three rounds were Thymine (Firebird Biomolecular Sciences, TONH2-171; T enzyme reaction was repeated after each dissociation.

The result is shown in Figure 7. In Fig. 7, the ratio of successful addition in the first T experiment was 85.93%, the ratio of no addition was 13.87%, and the ratio of other errors was 0.2%; the ratio of successful addition in the second T experiment was 74.16%, The proportion of no addition was 25.77%, and the proportion of other errors was 0.07%; in the third experiment of adding T, the proportion of successful addition was 72.66%, the proportion of no addition was 27.19%, and the proportion of other errors was 0.15%; it can be seen that , The efficiency of adding T for three times is above 70%.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

Claims (10)

1. A kit for enzymatic DNA synthesis, characterized in that: the kit comprises a chip and a starting chain,

the chip comprises a probe, wherein the probe is a single-stranded DNA molecule, the 3 '-end of the probe is modified by an amino group, the probe comprises a flexible arm region and a binding region combined with the initial chain, the flexible arm region is formed by connecting 6-12T, the binding region consists of 20-30 nucleotides, and the nucleotide sequence of the binding region is reversely complementary with the 5' -end of the initial chain.

2. The kit of claim 1, wherein: the length of the probe is 26-42nt.

3. Kit according to claim 1 or 2, characterized in that: the initial strand comprises a pairing region bound to the probe, the nucleotide sequence of the pairing region is reversely complementary to the binding region of the probe, and the length of the pairing region is 20-30 nucleotides.

4. A kit according to any one of claims 1-3, wherein: the initiation strand further comprises a initiation region at the 3' end for initiating DNA synthesis, the initiation region being greater than 3nt in length.

5. The kit of any one of claims 1-5, wherein: the initiation region of the initiation strand has a length of 4-6nt.

6. The kit of any one of claims 1-5, wherein: the nucleotide sequence of the probe is SEQ ID No.1, and the nucleotide sequence of the pairing region of the initial strand is SEQ ID No.2 at positions 1-25.

7. The kit of any one of claims 1-6, wherein: the kit further comprises a terminal deoxynucleotidyl transferase.

8. The chip of any one of claims 1-7.

9. A method for preparing a chip comprising crosslinking the probe of any one of claims 1 to 6 with an aldehyde-modified substrate to obtain the chip.

10. A method of enzymatic DNA synthesis comprising hybridizing the probe to the starting strand in the chip of any one of claims 1-7, and then performing a plurality of cycles of enzymatic synthesis of the DNA sequence of interest in the chip using a terminal deoxynucleotidyl transferase and dntps with protected bases, and heating the chip to 95-98 ℃ in the last cycle to dissociate the probe from the starting strand to obtain a synthesized DNA.

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