阅读说明：本技术 生物素标记的dna及其制备方法、试剂盒 (Biotin labeled DNA, preparation method and kit thereof ) 是由 高晨燕 王光亮 祝亮 何凡 王晓炜 于 2019-10-12 设计创作，主要内容包括：本发明涉及一种生物素标记的DNA及其制备方法、试剂盒。该生物素标记的DNA的制备方法包括以下步骤：将活化剂、含有咪唑基的化合物及DNA混合后进行反应,得到含有咪唑基的DNA,其中,活化剂包括碳二亚胺；及将连接有氨基的生物素与含有咪唑基的DNA反应,得到生物素标记的DNA。上述生物素标记的DNA的制备方法具有能够大规模生产,产量较高且成本低等优点。(The invention relates to biotin-labeled DNA, a preparation method thereof and a kit. The preparation method of the biotin-labeled DNA comprises the following steps: mixing an activating agent, a compound containing an imidazolyl group and DNA, and then reacting to obtain DNA containing the imidazolyl group, wherein the activating agent comprises carbodiimide; and reacting the biotin connected with the amino group with DNA containing imidazolyl to obtain biotin-labeled DNA. The preparation method of the biotin-labeled DNA has the advantages of large-scale production, high yield, low cost and the like.)

1. A method for preparing biotin-labeled DNA, comprising the steps of:

mixing an activating agent, a compound containing an imidazolyl group and DNA, and then reacting to obtain DNA containing the imidazolyl group, wherein the activating agent comprises carbodiimide; and

and reacting biotin connected with amino with the DNA containing the imidazolyl to obtain biotin-labeled DNA.

2. The method of producing a biotin-labeled DNA, according to claim 1, wherein the compound having an imidazole group is a monocyclic imidazole compound; or the imidazole group-containing compound is at least one selected from imidazole, methylimidazole and N, N-carbonyldiimidazole.

3. The method for producing a biotin-labeled DNA according to claim 1 or 2, wherein in the biotin having an amino group attached thereto, the amino group is attached to the biotin via a linker arm.

4. The method of claim 3, wherein the linker is at least one member selected from the group consisting of polyethylene glycol and α -aminoglutaric acid;

preferably, the raw material of the connecting arm is HO (CH)₂CH₂O)_nH and n are any integer of 3-10.

5. The method for producing a biotin-labeled DNA as claimed in claim 1 or 2, wherein the carbodiimide is at least one selected from the group consisting of dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N, N' -diisopropylcarbodiimide.

6. The method of claim 1 or 2, further comprising a step of adding a thiol compound to terminate the reaction of the activator with the imidazole group-containing compound and the DNA after the step of mixing the activator, the imidazole group-containing compound, and the DNA and then reacting the mixture.

7. A biotin-labeled DNA, wherein the biotin-labeled DNA is linked to biotin via a phosphate group of the DNA.

8. The biotin-labeled DNA according to claim 7, wherein the phosphate group is linked to the biotin via a linker arm; the raw material of the connecting arm is at least one of polyethylene glycol and alpha-aminoglutaric acid.

9. The biotin-labeled DNA according to claim 8, wherein the linker arm is- (CH)₂CH₂O)_nN is an integer of 3 to 10.

10. A kit comprising the biotin-labeled DNA obtained by the method for producing the biotin-labeled DNA according to any one of claims 1 to 6 or the biotin-labeled DNA according to any one of claims 7 to 9.

Technical Field

The invention relates to the technical field of biology, in particular to biotin-labeled DNA, a preparation method thereof and a kit.

Background

Recently, detection has been made more sensitive due to the high affinity, robust binding between biotin-labeled antigen or antibody and avidin bound to the enzyme, and the multi-step amplification effect. Therefore, biotin-labeled antigens or antibodies are widely used.

Currently, for antigens or antibodies of proteins, NHS- (PEG) n-biotin is mainly used for labeling the antigens or antibodies. The method is simple and convenient, the group of N-hydroxysuccinimide (NHS) can react with the amino group of an antigen or an antibody under mild conditions, then an antigen/antibody- (PEG) N-biotin compound is generated, the biotin (biotin) in the compound can be combined by Streptavidin (SA) reaction, and the compound can be widely applied to chemiluminescence immunoassay.

However, currently, non-proteinaceous antigens are labeled by biological methods. For example, double-strand DNA (dsDNA), is primarily labeled biotin by PCR amplification: adding a certain proportion of dUTP labeled with biotin into dNTPs of PCR, and adding the dUTP labeled with biotin into newly synthesized dsDNA double strands by PCR to obtain the dsDNA labeled with biotin. However, the method of biotin labeling by PCR amplification requires PCR, and the total volume of the amplification system in general PCR is 10. mu.L or 50. mu.L. Therefore, the amount per completion is small, and it is difficult to meet industrial demands. And the method for labeling biotin by PCR amplification requires biotin-labeled dUTP, dNTP and enzyme, which are relatively expensive, so that the production cost is too high and industrial popularization is difficult.

Disclosure of Invention

In view of the above, there is a need for a method for preparing biotin-labeled dsDNA, which is aimed at the problems of low yield and high cost of biotin-labeled dsDNA.

A method for preparing biotin-labeled DNA, comprising the steps of:

mixing an activating agent, a compound containing an imidazolyl group and DNA, and then reacting to obtain DNA containing the imidazolyl group, wherein the activating agent comprises carbodiimide; and

and reacting biotin connected with amino with the DNA containing the imidazolyl to obtain biotin-labeled DNA.

The preparation method of the biotin-labeled DNA connects biotin to DNA through a chemical method, and has the advantages of large-scale production, higher yield, low cost and the like compared with the traditional biological method for obtaining the biotin-labeled DNA through PCR amplification.

In one embodiment, the imidazole-containing compound is a monocyclic imidazole compound.

In one embodiment, the imidazole-containing compound is selected from at least one of imidazole, methylimidazole, and N, N-carbonyldiimidazole.

In one embodiment, in the biotin having an amino group attached thereto, the amino group is attached to the biotin through a linker arm.

In one embodiment, the material of the connecting arm is selected from at least one of polyethylene glycol and alpha-aminoglutaric acid.

In one embodiment, the material of the connecting arm is HO (CH)₂CH₂O)_nH and n are any integer of 3-10.

In one embodiment, the carbodiimide is selected from at least one of dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and N, N' -diisopropylcarbodiimide.

In one embodiment, after the step of mixing the activator, the imidazole group-containing compound, and the DNA and then reacting, the method further comprises the step of adding a thiol compound to terminate the reaction of the activator with the imidazole group-containing compound and the DNA.

A biotin-labeled DNA linked to biotin through a phosphate group of the DNA.

In one embodiment, the phosphate group and the biotin are linked by a linker arm; the raw material of the connecting arm is at least one of polyethylene glycol and alpha-aminoglutaric acid.

In one embodiment, the linker arm is- (CH)₂CH₂O)_nN is an integer of 3 to 10.

A kit comprising DNA labeled with the above biotin.

Drawings

FIG. 1 is a synthetic route of a method for preparing biotin-labeled DNA according to an embodiment;

FIG. 2 is a synthetic route for generating an imidazole-containing DNA in the method for preparing biotin-labeled DNA of FIG. 1;

FIG. 3 is a histogram of the dsDNA antibody concentration of example 8 versus the luminescence of the corresponding test solution;

FIG. 4 is a plot of dsDNA antibody concentration versus luminescence for the corresponding test solutions of example 8.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

One embodiment of the present invention provides a biotin-labeled DNA linked to biotin via a phosphate group of the DNA. Further, the phosphate group is linked to biotin via a linker arm. The distance between biotin and DNA molecules can be increased through the connecting arm, and the influence of biotin on DNA is reduced. Specifically, the raw material of the connecting arm is at least one selected from polyethylene glycol and alpha-aminoglutaric acid. Of course, in some of these embodiments, the starting material of the linker arm may also be other compounds.

In one embodiment, the linker arm is- (CH)₂CH₂O)_nN is an integer of 3 to 10. Further, n is 3, 5 or 10. Further, n is 3.

Experiments prove that the luminous intensity of the DNA marked by the biotin is higher than that of the traditional biology, so that the sensitivity of the DNA marked by the biotin when applied to chemiluminescence immunoassay detection is higher.

Referring to fig. 1, one embodiment of the present invention provides a method for preparing biotin-labeled DNA, including steps S110 to S120.

Step S110, the activator, the imidazole group-containing compound, and the DNA are mixed and reacted to obtain the imidazole group-containing DNA. The activating agent comprises a carbodiimide.

Specifically, the activating agent includes a carbodiimide. The activator serves to activate the phosphate group of the DNA to enable the DNA to react with the imidazole-containing compound. Further, an activator is used to activate the hydroxyl group of the phosphate group of DNA.

In one embodiment, the carbodiimide is selected from at least one of dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and N, N' -diisopropylcarbodiimide. Further, the activating agent includes 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. EDC is short for 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. In the illustrated embodiment, the activator is EDC.

Specifically, an imidazole-containing compound is used to activate the phosphate group of DNA so that biotin can be linked to DNA. In the present embodiment, the imidazole group-containing compound is a monocyclic imidazole compound. Of course, in other embodiments, the imidazole-group-containing compound may also be a non-monocyclic imidazole compound, such as a bicyclic imidazole compound or a heterocyclic ring-containing compound, as long as the contained imidazole group is active without being interfered by other structures. In one embodiment, the imidazole-containing compound is selected from at least one of imidazole, methylimidazole, and N, N-carbonyldiimidazole.

In this embodiment, the DNA refers to double-stranded DNA (dsDNA), because dsDNA is often used as an antigen in autoimmune detection, and is of great significance for detection of immune diseases. Of course, the specific nucleotide sequence structure of the DNA is not limited as long as it is dsDNA. It is understood that in other embodiments, the DNA may be single-stranded DNA, so long as the DNA has a phosphate group. Note that the phosphoric acid group herein refers to a group having a structure shown below:

in the illustrated embodiment, the dsDNA is

The DNA having an imidazole group is

Referring to FIG. 2, the mixing of the activator, the imidazole-containing compound and the DNA and the reaction include: the activating agent reacts with the DNA to activate the hydroxyl of the phosphate group of the DNA to obtain the DNA with the activated hydroxyl; then, the hydroxyl group-activated DNA is reacted with an imidazole group-containing compound to obtain an imidazole group-containing DNA. Further, the reaction of the activator with the DNA is an addition reaction. The reaction between the hydroxyl group-activated DNA and the imidazole group-containing compound is a substitution reaction.

In the illustrated embodiment, the DNA after hydroxyl group activation is

Specifically, the conditions under which the activator reacts with the imidazole group-containing compound and the DNA are: reacting for 0.5-2 h at 16-30 ℃. Furthermore, the reaction temperature is 22-28 ℃, and the reaction time is 0.75-1.5 h.

In one embodiment, after the step of mixing the activator, the imidazole group-containing compound, and the DNA and then reacting, the method further comprises the step of adding a thiol compound to terminate the reaction of the activator with the imidazole group-containing compound and the DNA. The sulfhydryl compound can quench the activator carbodiimide, and further reaction between the imidazole-containing compound and DNA is avoided. Further, the sulfhydryl compound is at least one selected from mercaptoethanol, mercaptothreitol, cysteine and cysteine hydrochloride. Further, the mercapto compound is mercaptoethanol.

In one embodiment, the step of mixing an activator, an imidazole group-containing compound, and DNA and reacting to obtain imidazole group-containing DNA comprises: mixing an activating agent, a compound containing an imidazolyl group and DNA, and then reacting to obtain a first reaction liquid of the DNA containing the imidazolyl group; and purifying the first reaction solution of DNA containing imidazolyl to obtain DNA containing imidazolyl. The purification treatment is intended to separate DNA containing an imidazole group from other substances in the first reaction solution. For example, an imidazole group-containing DNA is separated from an unreacted imidazole group-containing compound and/or an activating agent. Of course, there are various purification methods, such as ultrafiltration purification, desalting column purification, dialysis purification, and the like. When the first reaction solution containing an imidazole group-containing DNA is subjected to purification treatment, one of the above-mentioned purification methods may be selected, or a combination of a plurality of purification methods may be used. In the present embodiment, the first reaction solution for purifying DNA containing an imidazole group is purified by desalting column purification or ultrafiltration purification.

Step S120, reacting biotin having an amino group attached thereto with DNA having an imidazole group to obtain biotin-labeled DNA.

Specifically, the amino group in biotin having an amino group attached thereto is reacted with an imidazole group in DNA having an imidazole group to obtain biotin-labeled DNA.

In one embodiment, the biotin to which the amino group is attached is further attached with a linker arm, one end of which is attached to the amino group and the other end of which is attached to the biotin. Connecting armThe method has the effects of increasing the distance between DNA and biotin, reducing the steric hindrance of the DNA to the biotin and facilitating the subsequent reaction of avidin and the biotin. Further, the raw material of the connecting arm is selected from at least one of polyethylene glycol and alpha-aminoglutaric acid. Furthermore, the material of the connecting arm is polyethylene glycol. In the present embodiment, the material of the linker arm is HO (CH)₂CH₂O)_nH and n are any integer of 3-10. Further, n is 3, 5 or 10. Further, n is 3.

In one embodiment, the linker arm is- (CH)₂CH₂O)_nN is an integer of 3 to 10. The biotin to which the amino group and linker arm are attached is: amino group- (CH)₂CH₂O)_nBiotin (i.e., Amine- (CH)₂CH₂O)_n-biotin)。

In the illustrated embodiment, the biotin to which the amino group and linker arm are attached is

The biotin-labeled DNA is

In one embodiment, the step of reacting biotin having an amino group attached thereto with DNA having an imidazole group to obtain biotin-labeled DNA comprises: reacting biotin connected with amino with DNA containing imidazolyl to obtain a second reaction solution containing biotin-labeled DNA; then, the second reaction solution containing the biotin-labeled DNA is subjected to purification treatment. The purification treatment is intended to separate the biotin-labeled DNA from other substances in the second reaction solution. For example, biotin-labeled DNA is separated from non-reacted biotin having an amino group attached thereto. Of course, there are various purification methods, such as ultrafiltration purification, desalting column purification, dialysis purification, and the like. When the second reaction solution of biotin-labeled DNA is subjected to purification treatment, one of the above-mentioned purification methods may be selected, or a combination of a plurality of purification methods may be used. In this embodiment, the second reaction solution containing the biotin-labeled DNA is purified by desalting column purification or ultrafiltration purification.

The preparation method of the biotin-labeled DNA at least comprises the following advantages:

(1) large-scale production can be realized, and the yield is high: the traditional biological method is to obtain biotin-labeled DNA by PCR amplification, and the total volume of the amplification system of the general PCR is 10 or 50 mu L, and the amount of each amplification is small. Compared with the traditional biological method for preparing the DNA marked by the biotin, the preparation method of the DNA marked by the biotin marks the biotin on the DNA by a chemical method, can realize large-scale production and has higher yield.

(2) The cost is low: when the traditional biological method obtains the biotin-labeled DNA through PCR amplification, the biotin-labeled dUTP, dNTP and enzyme are needed, the prices of the biotin-labeled dUTP, dNTP and enzyme are higher, and the PCR amplification needs to be carried out in a PCR instrument, so that the production cost is increased. Compared with the traditional biological method, the preparation method of the biotin-labeled DNA has the advantages of easily obtained raw materials, lower raw material cost, easily satisfied whole preparation conditions and lower production cost.

(3) The prepared biotin-labeled DNA has higher luminous intensity than that of the traditional biology in chemiluminescence immunoassay detection, so that the sensitivity of the biotin-labeled DNA in chemiluminescence immunoassay detection is higher.

In addition, an embodiment of the present invention provides a biotin-labeled DNA prepared by the method for preparing a biotin-labeled DNA according to any one of the above embodiments.

An embodiment of the present invention further provides an application of the biotin-labeled DNA of any of the above embodiments in the preparation of a kit.

An embodiment of the present invention also provides a kit comprising the above biotin-labeled DNA.

Specifically, the kit comprises a marker, and the marker comprises the biotin-labeled DNA.

In one embodiment, the kit comprises a carrier, a coating antigen and a marker; the kit realizes the detection of the antibody to be detected by detecting the amount of the formed coating antigen-antibody to be detected-marker compound.

Specifically, the coating antigen includes the biotin-labeled DNA described above. The label is an antibody (anti-antibody or secondary antibody) of a labeled antibody to be detected, the label can be specifically bound with the antibody to be detected but not bound with the biotin-labeled DNA, and a chemiluminescent or bioluminescent substance is labeled on the label so as to be capable of emitting light under corresponding conditions for detection. The carrier is used for separating the coating antigen-antibody to be detected-marker compound formed by the reaction of the coating antigen-antibody to be detected-marker from other substances. In this embodiment, the carrier is an avidin-coated magnetic bead. Of course, in other embodiments, the carrier may also be a cellulose film coated with avidin.

The kit comprises the DNA marked by the biotin, the combination between the avidin and the biotin has extremely high affinity, the reaction has high specificity, and the multi-stage amplification effect between the avidin and the biotin can be utilized, so that the detection sensitivity of the kit is higher.