CN110922434A

CN110922434A - Deoxynucleotide primer synthesis method

Info

Publication number: CN110922434A
Application number: CN201911236612.XA
Authority: CN
Inventors: 刘振新; 华权高; 李立; 杨涛涛; 朱松林; 舒芹
Original assignee: WUHAN GENECREATE BIO-ENGINEERING Co Ltd
Current assignee: WUHAN GENECREATE BIO-ENGINEERING Co Ltd
Priority date: 2019-12-05
Filing date: 2019-12-05
Publication date: 2020-03-27
Anticipated expiration: 2039-12-05
Also published as: CN110922434B

Abstract

The invention provides a method for synthesizing a deoxynucleotide primer, which comprises the following steps of: step S1, removing protecting group; step S2, activation and coupling; step S3, oxidizing the intermediate; step S4, with a cap; step S5, washing the residual solution; step S6, repeating the steps S1 to S5; step S7, separating the deoxynucleotide primer pre-product from the carrier to obtain the deoxynucleotide primer. Compared with the prior art, the invention has the beneficial effects that: (1) by changing the sequence of the oxidation reaction and the cap reaction, the mutation rate of the primers is reduced, and the quality of the synthesized primers is improved; (2) the existing deoxynucleotide synthesis reagent and instrument are adopted, so that the method is easy to popularize; (3) the method can be used for synthesizing a trace product, and the waste of the product is avoided; (4) the dosage of the synthetic reagent is less, and the synthetic period is shortened.

Description

Deoxynucleotide primer synthesis method

Technical Field

The invention belongs to the field of biomolecular science, and particularly relates to a method for synthesizing a deoxynucleotide primer.

Background

At present, the primer synthesis basically adopts a solid-phase phosphoramidite triester method. There are many DNA synthesizers, and whatever machine is used for synthesis, the synthesis is carried out by a solid phase phosphoramidite method: the phosphoramidite monomer in solution forms a 3 '-5' phosphodiester linkage by condensation reaction, which is attached to a solid support. And sequentially extended until synthesis is complete at the last 5' base of the sequence. The whole synthesis process is automatically completed by an instrument, and each cycle is sequentially completed according to five steps of deprotection, activation, coupling, capping and oxidation.

In the existing reaction procedures, the activation and coupling reaction can generate unstable nucleoside phosphite intermediate, and the reaction is often the reason of primer mutation due to unstable chemical property; in order to reduce the probability of mutation caused by coupling reaction, a new coupling activator is reported in patent CN 110115963. However, the reaction using tetrazole as a coupling activator is well-established in the existing synthesis apparatus, the new coupling activator is difficult to adapt to the existing synthesis apparatus, and the new reagent is also difficult to purchase in the market, so that the further promotion is difficult.

Moreover, most of the solid phase carriers for synthesizing the primers used at present are powdery controllable microporous glass beads (CPG), the process for preparing the carriers is complicated, the filling amount required during CPG synthesis is large, the reaction with reagents is insufficient, the synthesis efficiency is low, the amount of the primers for synthesizing is small, the amount of the primers for failure is large, the effect is poor, and the cost is high. When CPG powder is used as a carrier for synthesis, the prepared carrier is generally a fixed powder amount (generally 4mg), the synthesized primer is about 5OD, while the primers for experiment are generally only 1-2OD, even 0.1OD, and the primers are difficult to sell again after being stored as products, so that the synthesis mode causes great waste.

The time required for synthesizing one base in the synthesizer is about 8 minutes, and if 192 primers of 30nt are synthesized, the synthesis time is about 4 hours, so that the synthesis efficiency is low.

Disclosure of Invention

In order to reduce the failure rate of primer synthesis in the prior art, the invention provides a method for synthesizing a deoxynucleotide primer.

The specific technical scheme is as follows:

a method for synthesizing a deoxynucleotide primer is characterized by comprising the following steps of:

step S1, removing the protecting group of the nucleotide monomer which is connected with the protecting group and is connected with the carrier in advance to obtain the free 5 '-hydroxyl, wherein the protecting group is a dimethoxytrityl group for protecting the 5' -hydroxyl;

step S2, activating the 3 'end of a new base monomer on a carrier by using phosphoramidite and an activating agent to obtain a nucleoside phosphite activation intermediate, and carrying out condensation reaction on the nucleoside phosphite activation intermediate and the free 5' -hydroxyl in the step S1 to obtain a nucleoside phosphite intermediate, wherein the nucleoside phosphite intermediate contains a phosphite bond, and the activating agent is tetrazole or a tetrazole derivative;

step S3, the phosphorous acyl of the nucleoside phosphite intermediate in the step S2 is oxidized into phosphoric triester;

step S4, carrying out a capped reaction to eliminate unreacted free 5' -hydroxyl;

step S5, cleaning the residual reagent in the steps S1 to S4 by using a cleaning solution;

step S6, repeating the steps S1 to S5;

step S7, separating the deoxynucleotide primer pre-product from the carrier to obtain the deoxynucleotide primer.

Compared with the prior art, the invention has the beneficial effects that: (1) by changing the sequence of the oxidation reaction and the cap reaction, the mutation rate of the primers is reduced, and the quality of the synthesized primers is improved; (2) the existing deoxynucleotide synthesis reagent and instrument are adopted, so that the method is easy to popularize; (3) the method can be used for synthesizing a trace product, and the waste of the product is avoided; (4) the dosage of the synthetic reagent is less, and the synthetic period is shortened.

Further, the steps S1 to S6 are completed in a deoxynucleotide synthesizer.

Further, the method for synthesizing the deoxynucleotide primer further comprises a step S7 of purifying the deoxynucleotide primer.

Furthermore, the carrier is a solid-phase cylindrical carrier formed by sintering controllable microporous glass beads and a binder.

Further, in the step S1, the dimethoxytrityl group is removed with trichloroacetic acid, and the trichloroacetic acid is washed with the washing solution after the dimethoxytrityl group is removed.

Further, in the step S3, iodine is added as an oxidizing agent to oxidize the phosphorouside of the nucleoside phosphite intermediate into a phosphotriester.

Further, in the step S4, acetic anhydride and azomethiazole are added to acetylate the 5 '-hydroxyl group to eliminate the unreacted free 5' -hydroxyl group.

Further, the washing solution is acetonitrile.

Further, in the step S7, the deoxynucleotide primer pre-product is separated from the carrier by treating with ammonia water at 90 to 95 ℃.

The beneficial effects of adopting the further technical scheme are as follows: in the solid-phase cylindrical carrier, the reaction is carried out more fully, the impurity content after the reaction is reduced, the product quality is improved, and the reaction rate is also improved.

Drawings

FIG. 1 illustrates the use of a solid phase column support in accordance with an embodiment of the present invention;

FIG. 2 shows a comparative example using a CPG support;

FIG. 3 is a running chart of DNA primers synthesized by the method of example and the method of comparative example;

FIG. 4 is a running chart of DNA primers synthesized by the method of example and the method of comparative example;

FIG. 5 is a mass spectrum of the synthesis method of example one;

FIG. 6 is a mass spectrum of a synthetic method using comparative example I;

FIG. 7 shows a conventional solid phase phosphoramidite triester method for primer synthesis;

wherein, 1-A200 nmol solid phase column carrier, 1-B100 nmol solid phase column carrier, 1-C50nmol solid phase column carrier, 1-D5 nmol solid phase column carrier, 3-A embodiment method synthesis DNA primer glue running picture, 3-B comparison example method synthesis DNA primer glue running picture, 4-A embodiment method synthesis DNA primer glue running picture, 4-B comparison example method synthesis DNA primer glue running picture.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

The current DNA synthesizer sets up a cycle according to the phosphoramidite triester method by the following steps (as shown in FIG. 7):

the first step is that nucleotide with protected active group connected to the solid phase carrier CPG in advance reacts with trichloroacetic acid, the protecting group Dimethoxytrityl (DMT) of the 5 '-hydroxyl is removed, free 5' -hydroxyl is obtained, and then acetonitrile is used for washing once, and residual trichloroacetic acid on the carrier is washed away; (Step1)

And secondly, mixing the raw materials for synthesizing the DNA, the phosphoramidite protected nucleotide monomer and an activator tetrazole to obtain a nucleoside phosphite activated intermediate (Step2), wherein the 3' end of the nucleoside phosphite activated intermediate is activated, the 5' -hydroxyl is still protected by DMT, and the nucleoside phosphite activated intermediate and the free 5' -hydroxyl in the solution are subjected to condensation reaction (Step 3).

Third, a capping reaction, in which very few 5' -hydroxyl groups may not be reacted (less than 2%) and which is terminated with acetic anhydride and 1-methylimidazole and then continued, can be isolated during purification (Step 4).

In the fourth Step, oxidation, the phosphorylidene form is converted to a more stable phosphotriester by the action of the oxidant iodine (Step 5).

Through the above four steps, one deoxynucleotide is linked to the nucleotide on the solid phase carrier.

And then the five steps are circularly carried out until the synthesis of the primer is finished.

To better terminate the continued reaction of unnecessary fragments, a second cap and acetonitrile wash step was added to the cycle.

The DNA primer synthesis program was designed as follows:

a method for synthesizing a deoxynucleotide primer comprises the following steps of:

step S1, removing the protecting group from the base monomer which is connected with the protecting group and is connected with the carrier in advance to obtain the free 5 '-hydroxyl, wherein the protecting group is DMT for protecting the 5' -hydroxyl;

removing DMT by using TCA, and washing the trichloroacetic acid by using a washing solution after DMT is removed;

and step S5, washing the residual reaction solution with a washing solution.

Step S6, repeating the steps S1 to S5;

the steps S1 to S6 are completed in a deoxynucleotide synthesizer.

Further, the method for synthesizing the deoxynucleotide primer further comprises a step S7 of purifying the deoxynucleotide primer. The purity of the synthesized primer can reach more than 85 percent after the detection improvement, most experiments can be satisfied without purification, and a purification step can be added if special requirements exist.

In the step S3, iodine (OX) is added as an oxidizing agent to oxidize the phosphorouside of the nucleoside phosphite intermediate into a phosphotriester.

Further, in the step S4, acetic anhydride (Cap a) and azomethiazole (CapB) are added to acetylate the 5 '-hydroxyl group to eliminate the unreacted free 5' -hydroxyl group.

In the step S1 and the step S5, the washing solution is acetonitrile.

In the step S7, the deoxynucleotide primer pre-product is separated from the carrier by ammonia high-temperature treatment.

The designed primers were synthesized in one cycle as follows:

step S1 is programmed as:

1 Start, DeBlock, wait, little drawer, big drawer

2 Wash waiting, small drawer and big drawer

Step S2 is programmed as

3 Couple waiting, small drawing s and large drawing s

Step S3 is programmed as:

4 Oxidize Plate A, Ox-1, wait for s, little draw s, big draw s

Step S4 is programmed as:

5 Cap Plate A: Cap-B Cap-A, wait, little pump, big pump

Step S5 is programmed as:

6 Wash, wait, small drawer, big drawer

Wherein, the large and small pumping represents the pressure of the pumping reagent in the instrument.

The reagent dosage and the reaction conditions of each step are adjusted according to the size of the carrier.

In the following examples and comparative examples, TCA is dichloromethane and trichloroacetic acid prepared in a certain ratio, ACT is a thiotetrazolium acetonitrile solution, CapA is a mixed reagent of acetic anhydride and tetrahydrofuran, CapB is a mixed reagent of pyridine, azomethimazole, and tetrahydrofuran, and iodine solution is a mixed reagent of iodine, pyridine, and water;

a, G, C or T and ACT are simultaneously driven into the corresponding carrier.

The synthetic content and the reagent dosage are controlled by different carrier sizes.

Example one

Oligo192dna synthesizer was used to synthesize DNA primers

The solid phase carrier is adopted as a solid phase cylindrical carrier, and the CPG content is 5 nmol.

The designed primers were synthesized in one cycle as follows:

step S1 is programmed as:

start, DeBlock 60ul, wait 12s, little 1s, big 4s

Wash 70ul wait for 2s, small draw for 4s, and large draw for 4s

Step S2 is programmed as

The Couple waits for 50s, the small drawer lasts for 0.6s, and the large drawer lasts for 3s

Step S3 is programmed as:

oxidize Plate A, Ox-120 ul, wait 17s, little draw 1s, big draw 2s

Step S4 is programmed as:

cap Plate A, Cap-B15 ul Cap-A15 ul, wait for 15s, draw for 1s little, draw for 2s big

Step S5 is programmed as:

wash 30ul, wait for 2s, draw 4s little, draw 4s big

The amounts of reagents are shown in table 1:

TABLE 1 example-reagent dosage corresponding table

Synthetic reagent	Content (ul)
		ACT	12
dA	8
		dC	8
dG	8
		dT	8
OX	20
		CapA	15
CapB	15
		TCA	60
Acetonitrile	100
		Total up to	254

Step S6, repeating the steps S1 to S5;

and S7, separating the deoxynucleotide primer pre-product from the carrier by ammonia water treatment at 90-95 ℃.

The time required for synthesizing 192 30nt pieces of product was 2 to 3 hours more.

Example two

Oligo192dna synthesizer was used to synthesize DNA primers

The solid phase carrier is adopted as a solid phase column carrier, and the CPG content is 50 nmol.

The designed primers were synthesized in one cycle as follows:

step S1 is programmed as:

star, DeBlock 150ul, wait for 20s, little for 1.5s, big for 5s

Wash 160ul wait for 2s, small draw for 4s, and large draw for 4s

Step S2 is programmed as

The Couple waits for 50s, the small drawer lasts for 0.9s, and the large drawer lasts for 4s

Step S3 is programmed as:

oxidize Plate A, Ox-140 ul, wait for 20s, draw 2s little, draw 3s big

Step S4 is programmed as:

cap Plate A, Cap-B35 ul Cap-A35 ul, wait for 18s, little pump for 1.2s, big pump for 3s

Step S5 is programmed as:

wash 120ul, wait for 2s, draw 4s little, draw 4s big

The amounts of reagents are shown in table 2:

TABLE 2 corresponding table of the amount of reagent used in the example II

The time required for synthesizing 192 strands of 300nt product is 2.5 to 3.5 hours.

Step S6, repeating the steps S1 to S5;

EXAMPLE III

Oligo192dna synthesizer was used to synthesize DNA primers

The solid phase carrier is adopted as a solid phase column carrier, and the CPG content is 100 nmol.

The designed primers were synthesized in one cycle as follows:

step S1 is programmed as:

star, DeBlock 180ul, wait for 30s, little for 3s, big for 6s

Wash 200ul waiting for 2s, small drawing for 4s and large drawing for 4s

Step S2 is programmed as

The Couple waits for 60s, the small drawer lasts for 1.2s, and the large drawer lasts for 4s

Step S3 is programmed as:

oxidize Plate A, Ox-155 ul, wait 24, draw 3s little, draw 4s big

Step S4 is programmed as:

cap Plate A, Cap-B45 ul Cap-A45 ul, wait for 20s, draw down for 1.5s, draw up for 4s

Step S5 is programmed as:

wash 150ul, wait for 2s, draw for 4s, draw for 5s

The amounts of reagents are shown in table 3:

TABLE 3 corresponding table of three reagent dosages in the examples

Synthetic reagent	Content (ul)
		ACT	30
dA	22
		dC	22
dG	22
		dT	22
OX	55
		CapA	45
CapB	45
		TCA	180
Acetonitrile	350
		Total up to	793

The synthesis of 192 pieces of 600nt product by the above method takes 3.5 to 4.5 hours more.

Step S6, repeating the steps S1 to S5;

Example four

Oligo192dna synthesizer was used to synthesize DNA primers

The solid phase carrier is adopted as a solid phase column carrier, and the CPG content is 200 nmol.

The designed primers were synthesized in one cycle as follows:

step S1 is programmed as:

star, DeBlock 200ul, wait 40s, little 4s, big 8s

Wash 220ul waiting for 5s, small drawing for 4s and large drawing for 6s

Step S2 is programmed as

The Couple waits for 70s, the small drawer lasts for 1.6s, and the large drawer lasts for 5s

Step S3 is programmed as:

oxydize Plate A, Ox-165 ul, wait for 30s, draw 2s little, draw 5s big

Step S4 is programmed as:

cap Plate A, Cap-B55 ul Cap-A55 ul, wait for 24s, draw for 2s, draw for 5s

Step S5 is programmed as:

wash 180ul, wait for 4s, draw for 6s

Step S6, repeating the steps S1 to S5;

The amounts of reagents are shown in table 4:

TABLE 4 corresponding table of three reagent dosages in the examples

The time required for synthesizing 192 strands of 1200nt product is 4.5 to 5.5 hours more.

Comparative example 1

DNA primer Synthesis was carried out according to the following procedure

Preparation with 100nmol CPG

Deprotection group

Step S1 Start, DeBlock 180ul, wait for 20S, little 3S, big 6S

Step S2 Wash 200ul wait for 2S, small draw 4S, large draw 4S

Coupling of

Step S3 Couple waits for 55S, 3S for small drawer and 4S for large drawer

With cap

Step S4 Cap Plate A, Cap-B45 ul Cap-A45 ul, wait for 15S, little draw for 1S, big draw for 4S

Oxidation by oxygen

Step S5 Oxidize Plate A, Ox-155 ul, waiting for 17S, small drawing for 1S, and large drawing for 4S

With cap

Step S6 Cap Plate A, Cap-B45 ul Cap-A45 ul, wait for 15S, little draw for 1S, big draw for 4S

Step S7 Wash 120ul, wait for 2S, little 4S, big 4S

Step S8, repeating the steps S1 to S7;

and S9, separating the deoxynucleotide primer pre-product from the carrier by ammonia water treatment at 90-95 ℃.

Comparative example-reagent dosage table

TABLE 5 corresponding table of the amounts of comparative examples and reagents

Synthetic reagent	Content (ul)
		ACT	30
dA	22
		dC	22
dG	22
		dT	22
OX	55
		CapA	90
CapB	90
		TCA	180
Acetonitrile	320
		Total up to	853

The time required for synthesizing 192 30nt pieces of product was 4 hours more.

Comparative example No. two

DNA primer Synthesis was carried out according to the following procedure

Preparation with 150nmol CPG

Deprotection group

Step S1 Start, DeBlock 220ul, wait for 30S, little 3S, big 6S

Step S2 Wash 250ul waiting for 4S, small drawing 4S and large drawing 4S

Coupling of

Step S3 Couple waits for 65S, small drawing for 3S and large drawing for 4S

With cap

Step S4 Cap Plate A, Cap-B55 ul Cap-A55 ul, wait for 15S, little draw for 1S, big draw for 4S

Oxidation by oxygen

Step S5 Oxidize Plate A, Ox-165 ul, waiting for 17S, small drawing for 1S, and large drawing for 4S with cap

Step S6 Cap Plate A, Cap-B55 ul Cap-A55 ul, wait for 15S, little draw for 1S, big draw for 4S

Step S7 Wash 150ul, wait for 2S for small drawing 4S and big drawing 4S

Step S8, repeating the steps S1 to S7;

Comparative example reagent dosage table

TABLE 6 corresponding table of the amounts of the second reagents in the comparative examples

The time required for synthesizing 192 strands of 45nt product was 5 hours more.

Comparative example No. three

DNA primer Synthesis was carried out according to the following procedure

Preparation with 200nmol CPG

Deprotection group

Step S1 Start, DeBlock 260ul, wait for 30S, little 3S, big 6S

Step S2 Wash 280ul waiting for 5S, small drawing 4S and large drawing 4S

Coupling of

Step S3 Couple waits for 75S, small drawing for 3S and large drawing for 4S

With cap

Step S4 Cap Plate A, Cap-B65 ul Cap-A65 ul, wait for 15S, little draw for 1S, big draw for 4S

Oxidation by oxygen

Step S5 Oxidize Plate A, Ox-175 ul, waiting for 17S, small drawing for 1S, large drawing for 4S, waiting for 17S, small drawing for 1S, and large drawing for 4S

With cap

Step S6 Cap Plate A, Cap-B65 ul Cap-A65 ul, wait for 15S, little draw for 1S, big draw for 4S

Step S7 Wash 200ul, wait for 2S for small drawing 4S and big drawing 4S

Step S8, repeating the steps S1 to S7;

Comparative example three reagent dosage table

TABLE 7 corresponding table of the amounts of the three reagents of the comparative example

Synthetic reagent	Content (ul)
		ACT	45
dA	35
		dC	35
dG	35
		dT	35
OX	75
		CapA	130
CapB	130
		TCA	260
Acetonitrile	480
		Total up to	1260

The time required for synthesizing 192 60nt pieces of product was 6 hours more.

The primers synthesized in each example and comparative example were the same.

The property detection of each embodiment and each pair of proportional synthetic primers is carried out

The detection and statistical method of the variation rate comprises the following steps: 192 sequences are synthesized, the same sequences are obtained in the examples and the comparative examples, carrier amplification is carried out after synthesis, sequencing is carried out, 3 samples are selected for each gene for sequencing, the number of mutation deletions of the samples is detected, the mutation rate is obtained by dividing the number by the total base number of the samples, for example, 7 gene segments (primers designed by 7 gene sequences, 11328 bases) are sent for each gene, 3 samples are sent for each gene, 21 mutation deletion bases are detected, and the mutation rate is 21/(11328 3) ═ 0.00062, so that the mutation rate is 0.62 ‰. The lower this ratio, the higher the quality of the primer.

The results are shown in Table 8.

TABLE 8 detection results of purity and variation rate of synthesized product

Synthesis ofProduct(s)	Purity (%)	Rate of variation (‰)
			Example one	85	0.63
Example two	86	0.55
			EXAMPLE III	84	0.58
Example four	85	0.65
			Comparative example 1	81	1.4
Comparative example No. two	80	1.2
			Comparative example No. three	81	1.3

From the above data and figures, it can be shown that the present invention is suitable for synthesis of very trace amount of product, for example, 1-2OD product can be synthesized by 5nmol carrier, which avoids waste of experiment, and the purity is higher than that of the traditional method, and most experiments can be satisfied without purification.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for synthesizing a deoxynucleotide primer is characterized by comprising the following steps of:

step S6, repeating the steps S1 to S5;

2. The method of claim 1, wherein the steps S1 to S6 are performed in a deoxynucleotide synthesizer.

3. The method of claim 1, further comprising a step of purifying the deoxynucleotide primer (S7).

4. The method for synthesizing a deoxynucleotide primer according to claim 1, wherein the carrier is a solid-phase cylindrical carrier formed by sintering controllable microporous glass beads and a binder.

5. The method of claim 1, wherein the dimethoxytrityl group is removed with trichloroacetic acid in step S1, and the trichloroacetic acid is washed with the washing solution after the dimethoxytrityl group is removed.

6. The method of claim 1, wherein in step S3, iodine is added as an oxidant to oxidize the phosphorouside of the nucleoside phosphite intermediate into a phosphotriester.

7. The method of claim 1, wherein in step S4, acetic anhydride and azomethimazole are added to acetylate the 5 '-hydroxyl group to eliminate unreacted free 5' -hydroxyl group.

8. The method of claim 5, wherein the washing solution is acetonitrile.

9. The method of claim 1, wherein the deoxyribonucleotide primer synthesis product is isolated from the support by treating with ammonia water at 90 to 95 ℃ in step S7.