KR100851764B1 - Oligonucleotide marker which contains phase transfer catalyst and the material differentiating or discerning method which employs the same - Google Patents

Abstract

The present invention relates to an oligonucleotide derivative dissolved in an organic solvent and a method for identifying or identifying an object using the same, and more particularly, to an oligonucleotide derivative combined with a phase transfer catalyst (PTC) and a label thereof. A method of identifying or identifying objects used as sieves.

Oligonucleotide derivatives

Description

Oligonucleotide marker which contains phase transfer catalyst and the material differentiating or discerning method which employs the same}

1 is a schematic diagram showing a route for dissolving an oligonucleotide in an organic solvent and applying it to a paint and then recovering it again.

2 is a diagram showing a combination of an oligonucleotide derivative having an amide and ester bond with a base portion of an oligonucleotide or an alcohol at 5 'and 3' positions, and a phase inversion catalyst (R is saturated according to the components and uses of the paint). Hydrocarbons, aromatic hydrocarbons, unsaturated hydrocarbons, saturated or unsaturated hydrocarbons containing heteroatoms).

Figure 3 is a photograph of the electrophoresis on agarose gel after the oligonucleotide was diluted by concentration to perform a polymerase chain reaction (PCR).

Figure 4a is a photo of electrophoresis on agarose gel after the polymerase chain reaction (PCR) was carried out by coating and recovering the oligonucleotide derivative protected by acryloyl chloride and the phase inversion catalyst mixed with paint.

Figure 4b is a photograph analyzing the nucleotide sequence of the oligonucleotide of Figure 4a.

FIG. 5A is a photograph of electrophoresis on agarose gel after polymerase chain reaction (PCR) was performed by coating and recovering an oligonucleotide derivative protected by acetyl chloride and a phase conversion catalyst by mixing with paint.

Figure 5b is a photograph analyzing the nucleotide sequence of the oligonucleotide of Figure 5a.

FIG. 6a shows that the oligonucleotide derivative (B) coupled with the phase inversion catalyst was coated and recovered by coating and recovering the oligonucleotide (A) coupled with the phase inversion catalyst, followed by polymerase chain reaction (PCR). Photographed on agarose gel electrophoresis.

Figure 6b shows the result of analyzing the base sequence of the conjugate A and conjugate B of Figure 6a.

Figure 7a is a photograph of three kinds of oligonucleotide derivative-PTC conjugate consisting of different nucleotide sequences at the same time mixed with paint to coat and recover the object to perform a PCR electrophoresis on agarose gel.

Figure 7b shows the result of analyzing the base sequence of the three oligonucleotide derivative-PTC conjugate of Figure 7a.

The present invention relates to oligonucleotide derivatives dissolved in organic solvents and methods for identifying or identifying objects using the same, and more particularly, oligonucleotide derivatives combined with a phase transfer catalyst (PTC) and a label thereof. It is about the identification or identification of the goods used.

Oligonucleotides can be amplified in large quantities into oligonucleotides of the same nucleotide sequence through polymerase chain reaction (PCR) even if the amount is extremely small, and the original oligonucleotides present in extremely small amounts by analyzing the nucleotide sequences of the amplified oligonucleotides. There is a unique advantage that can identify the base sequence of. Thus, by adding a very small amount of such oligonucleotides to various materials or products, such as oils, paints, food, gunpowder, and expensive works of art, it is possible to accurately determine the original source of the materials or goods, the transport route or the authenticity of the works of art.

Since the oligonucleotides have the property of being negatively charged by deprotonation in neutral conditions, the oligonucleotides themselves composed of a plurality of phosphodiester bonds exhibit strong hydrophilic properties.

Thus, the oligonucleotides themselves are well soluble in aqueous solutions but hardly soluble in common organic solvents. This property is not a problem when preparing an aqueous solution of oligonucleotides, but when the oligonucleotides are dissolved in an organic solvent, poor solubility problems occur.

However, methods for using such oligonucleotides as labeling bodies have already been disclosed. For example, International Patent Publication Nos. WO87 / 06383, WO90 / 14441, WO91 / 17265, WO94 / 04918 and the like. WO87 / 06383 suggested that nucleotides can be used as markers, but no method of identifying objects through DNA amplification or sequencing has been described, and the method of dissolving hydrophilic DNA in an organic solvent is not described. Not listed. WO90 / 14441 discloses a technique for introducing oligonucleotides into an organic layer using a detergent to dissolve hydrophilic oligonucleotides in oil.

However, the patent only confirms the presence of DNA by confirming whether or not it is amplified using a specific primer, and does not mention the identification function of the DNA base sequence. WO91 / 17265 discloses the identification of a nucleotide sequence by amplification of a gene by a specific primer set forth in WO90 / 14441, mentioning that oligonucleotides can covalently bind to a solid support or material.

However, the information described in the above patent is that when nucleotides are directly bound to paint or oil, the covalent bonds must be broken at the extraction and recovery stage of the oligonucleotide, resulting in a modification of the base and thus the correct sequence at the gene amplification stage. The problem of not being amplified occurred and could not be commercialized. WO94 / 04918 discloses a technique for amplifying genes, analyzing sequences, and using two or more luminescent or color compounds other than oligonucleotides as markers in a more improved manner. However, this method also does not consider the reactivity of the hydroxyl or base amine groups of the oligonucleotide, and has the original sequence when PCR or sequencing is performed due to the reaction of hydroxyl or amine groups. Problems arise in that oligonucleotides cannot be obtained.                         

In addition, the methods disclosed in the above patents only mention that oligonucleotides can be used in paints or oils, more specifically, vehicle paints, and the like, and methods for tracking and verifying the original vehicle using them have not been specifically described. .

Accordingly, it is an object of the present invention to provide an oligonucleotide derivative combined with a phase inversion catalyst having improved solubility in lipophilic substances so that it can be used as an identification label of an object.

It is another object of the present invention to provide a method for preparing an oligonucleotide derivative combined with the phase inversion catalyst.

Still another object of the present invention is to provide a method for labeling an object to be identified by using an oligonucleotide derivative combined with the phase inversion catalyst.

Still another object of the present invention is to provide a method of identifying or identifying an object by extracting and isolating the oligonucleotide from an object labeled with an oligonucleotide derivative to which the phase inversion catalyst is bound and analyzing the nucleotide sequence through an amplification reaction. It is.

The object of the present invention as described above is achieved by providing a method for identifying or identifying an oligonucleotide derivative coupled with a phase inversion catalyst and an object using the same as a label.                     

Oligonucleotide derivatives combined with the phase inversion catalyst of the present invention

1) forming an ionic bond between a phase inversion catalyst and an oligonucleotide in an organic solvent to prepare an oligonucleotide to which a phase inversion catalyst is bound, 2) an acyl halide such as an oligonucleotide conjugate to a phase inversion catalyst Prepared by a method comprising the step of binding a reactive blocking protecting group to remove reactivity.
In addition, the oligonucleotide is preferably composed of 20 to 1000 base pairs.

In the present invention, a compound having a quaternary ammonium salt or a cationic surfactant is used as the phase inversion catalyst.

Since the phase inversion catalyst has a compound having a quaternary ammonium salt or a cationic surfactant structure, the phase inversion catalyst forms an ionic bond with an oligonucleotide of an anion to cancel the anion charged state of the oligonucleotide. Oligonucleotides whose charge is canceled through the coupling with the catalyst may be converted from water soluble to fat soluble to be dissolved in an organic solvent.

In this way, when an oligonucleotide having a property of dissolving in an organic solvent is added to an organic solvent and mixed with an lipophilic product such as an oil paint, it is dispersed in a uniform state to produce an lipophilic object containing an extremely low concentration of oligonucleotide.

However, the oligonucleotide combined with the phase inversion catalyst obtained in step 1) is reacted with a reactive blocking protecting group such as an acyl halide, in which the 5 'or 3' hydroxyl group or base amine group of the sugar of the oligonucleotide is reacted in an organic solvent, The hydroxyl group moieties are esterified and the amine groups of the base are amidated.

The acyl halide may be appropriately selected depending on the type of the organic solvent or the use of the oligonucleotide. For example, if the oligonucleotide is simply to be dissolved in the paint, halogenated acyl (e.g. acetyl chloride) with an unreactive substituent is used to block the reactivity and to prevent loss of the coating for a long time. When a chemical bond of is desired, it is preferable to use a halogenated acyl (eg acryloyl chloride) having a reactivity capable of inducing a chemical bond.

In addition, the reactive blocking protecting group is a carbonyl compound having an amide bond with nitrogen and an ester bond with oxygen, a silanyl compound having an N-Si and O-Si bond, a sulfonyl compound having an NS and OS bond, and a NC; It is selected from the group consisting of saturated hydrocarbons, aromatic hydrocarbons, unsaturated hydrocarbons, saturated hydrocarbons containing heteroatoms, or unsaturated hydrocarbons containing heteroatoms, which can break NC and OC bonds during ammonia treatment with OC bonds.

For this reason, when the oligonucleotide combined with the phase inversion catalyst obtained in step 1) is mixed with the lipophilic substance as it is, a chemical reaction occurs with the lipophilic substance according to the type of the oily product, and the oligonucleotide is later recovered in the oligonucleotide later recovery step. This is because a problem occurs that is not recovered as it is.

Therefore, in the present invention, the reaction is blocked by introducing a reactive blocking protecting group such as acyl halide before adding the oligonucleotide to which the phase inversion catalyst is bound to the oily material, thereby securing chemical stability in the lipophilic material. The loss of oligonucleotides through chemical bonds with oily materials can be reduced.                     

It is still another object of the present invention to provide a method for adding an identification label of a lipophilic substance by adding an oligonucleotide derivative to which the phase inversion catalyst is bound to a lipophilic substance.

The lipophilic materials include oily products such as automotive coating paints, lacquers, road lane marking paints, petroleum, paint thinners, gunpowder, natural oils, construction paints, organic solvents, adhesives, oil paints, meat and seafood.

Still another object of the present invention is to 1) extract the identification label from the object labeled by the object identification label protecting the oligonucleotide derivative to which the phase inversion catalyst and the oligonucleotide are bound by a reactive blocker; 2) treating the extracted identification label with ammonia to remove the reactive blocking protecting group bound to the oligonucleotide derivative; 3) recognizing the nucleotide sequence of the identification label portion of the oligonucleotide derivative; 4) an object identification method comprising the step of recognizing an object labeled with a identification label having a base sequence recognized in 3).

The method further comprises amplifying the identification label portion oligonucleotide through a polymerase chain reaction prior to recognizing the nucleotide sequence.

In addition, the method of the present invention may further comprise the step of cloning the amplified oligonucleotide into a vector (vector) after the amplification step by the polymerase chain reaction.

The oligonucleotides bound to the phase change catalysts dissolved in the organic solvent according to the present invention, and the oligonucleotide derivatives and the phase change catalysts (PTC) conjugates protecting them with reactive blockers can be applied to various fields. The oligonucleotide derivative and a phase inversion catalyst (PTC) conjugate may be used as a marker in various oils, paints, foods, security systems, and vehicles.

When an oligonucleotide having a specific sequence is used as a vehicle marker for the exterior coating of a vehicle, when the vehicle is mixed with a vehicle paint, the oligonucleotide is separated from a small piece of paint away from the vehicle when the vehicle escapes in a traffic accident. After amplification by PCR, the nucleotide sequence of the oligonucleotide can be analyzed to track the vehicle corresponding to the nucleotide sequence.

In the above method, the oligonucleotide sequence can function as an identification label (password) by four base combinations of adenine (A), cytosine (C), guanine (G), and thymine (T). For example, if the size of the oligonucleotide is 40 base pairs, the 15 base pair portions at both ends are each made of a base sequence known as a template sequence to which a primer is attached during amplification by PCR. The part can be selected from the above four bases and combined to obtain the number of 4 ¹⁰ cases. Accordingly, the oligonucleotide of 10 base pairs in length can serve as 4 ¹⁰ kinds of identification markers, and thus the identification marker portion of each object can be labeled using oligonucleotides having different base sequences.

Therefore, after amplifying the oligonucleotide collected from the object, analyzing the nucleotide sequence, it is possible to identify the original object corresponding to the identification label partial nucleotide sequence.

In addition, when a large number of objects to be searched, such as a vehicle, the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide may be used in combination. If the identification label portion of each oligonucleotide is 10 base pairs, 4 ¹⁰ kinds of combinations can be made. If the identification label portions of three types of oligonucleotides are combined, the number of 4 ¹⁰ × 4 ¹⁰ × 4 ¹⁰ cases can be obtained. However many vehicles can be identified using this combination. Here, the 15 base pairs of the sock ends of the three oligonucleotides may be designed so that different primers may be used, and the terminal sequence should be designed so that the terminal sequence is not the same sequence as that of the intermediate identification label region.

When using an oligonucleotide having an identification label portion obtained through the base sequence combination as a label, the method for labeling the oligonucleotide and the method for recognizing the identification label through analysis of the oligonucleotide label are as follows.

In the present invention, the oligonucleotide base sequence is designed in consideration of amplification through polymerase chain reaction (PCR). More specifically, the part to which an identification label is assigned synthesizes 10 base pair oligonucleotides by combination of 10 base pairs (4 ¹⁰ types of identification labels can be given), and both bases of the 15 base pair whose base sequence are already known Oligonucleotides are linked to result in the synthesis of 40 base pairs of oligonucleotides. The 15 base pair portions of the sock end, of which the nucleotide sequence is already known, serve as a template for the forward primer and the backward primer during polymerase chain reaction (PCR). Depending on the object to be labeled, by lengthening the base sequence of the identification labeling portion 10mer by 10 base pairs or more, the number of cases of the identification label can be increased, so that the identification label can be given to more objects.

The oligonucleotide to be used was designed as follows.

1) Identification Labeling Region Sequence Design

The basic design algorithm used Smith et al. First, a unique identification label sequence is generated randomly for each oligomer. Local alignment of identification label sequences excludes similarities between the identification label sequences and those that cross hybridize with each other (Smith, TF, and MS Waterman, 1981. Identification of common molecular) subsequences. J. Mol. Biol. 147, 195-197). This process was implemented by dynamic programming, and the variables used to measure similarity were defined as mismatch penalty 3, match score 10, and gap panel 3. Only local alignment values obtained through the above procedure were lower than 75. Through this procedure, the identification oligomeric sequence was generated and the results were databased.

2) edge sequence design                     

As described above, after randomly generating oligomeric sequences of 15 base pairs (15mer), only base sequences having a melting point (Tm, Melting Temperature) value between 50 and 55 ° C are selected. At this time, the melting point value is obtained by a neighbor-neighbor method (Breslauer, KJ, Frank, R., Blocker, H., and Marky, LA, 1986, Predicting DNA duplex stability from the base sequence.Proc.Natl Acad.Sci. USA, 83, 3746-3750). This method is known to be more accurate than the method using the Guanine-Cytosine content value. The oligomers thus obtained can cause fatal results in the case of cross hybridization with each other during amplification through polymerase chain reaction (PCR). Therefore, in order to solve the problem, a local alignment was performed between the forward primer and the backward primer to obtain an oligomer having a value lower than 50. In addition, only the sum of the value of the left oligomer (forward primer, FOR), the identification sequence value, and the value of the right oligomer (rear primer, REV) is less than 100. It was not. Variables performed at this time were determined as mismatch panel 5, match score 10, and gap panel 5.

As described above, the synthesized oligonucleotide is synthesized and purified using an automatic oligonucleotide synthesizer, and then an aqueous oligonucleotide solution and an organic solvent (using toluene or ethyl ether) are added together with a phase inversion catalyst (PTC), followed by sufficient mixing. After separation, only the organic solvent layer is separated and the process is repeated until the oligonucleotides are not visible in the aqueous solution layer. The oligonucleotides combined with the phase inversion catalyst obtained through this process form a uniform phase with respect to the organic solvent, and can be dispersed in the organic solvent at extremely low concentration. This makes it possible to convert existing water-soluble oligonucleotides into fat-soluble in a simple way.

Oligonucleotides combined with the phase inversion catalyst are lipophilic materials, such as automotive coating paints, lacquers, road lane marking paints, petroleum, paint thinners, gunpowder, natural oils, construction paints, organic solvents, adhesives, oil paints Oily products such as fish, meat and seafood are available for selection.

However, the amine group of the base of the oligonucleotide combined with the phase inversion catalyst or the hydroxyl group of oxygen and sugar have reactivity with the lipophilic substance according to the type of lipophilic substance, and when the oligonucleotide is directly mixed with the lipophilic substance, There is a problem that the oligonucleotide is not properly recovered in the oligonucleotide recovery step by causing a chemical reaction with the substance.

Therefore, prior to adding the oligonucleotides associated with the phase inversion catalyst to the lipophilic substance, a reactive blocking protecting group such as acyl halide is introduced to protect the portion capable of reacting with the lipophilic substance, thereby blocking the reactivity. It is preferable. In the present invention, acetyl chloride having an inactive substituent is used as the halogenated acyl introduced into the oligonucleotide combined with the phase inversion catalyst, or acryloyl chloride having a substituent capable of strong chemical bonding with the constituent resin of the paint is used. . As a result, all of the original oligonucleotides were recovered as shown in the following Examples.

The method for recovering the oligonucleotide from the oligonucleotide derivatives combined with the phase inversion catalyst to the lipophilic substance and then again is as follows. For example, the process of extracting an oligonucleotide from a piece of paint to which an oligonucleotide derivative to which a phase inversion catalyst is bound is extracted by dissolving a small amount of oligonucleotide derivative by treating the paint piece with an organic solvent, and then extracting the extracted oligonucleotide derivative. Is treated with ammonia to remove the reactive blocking protecting group and reduced to the phosphodiester structure originally possessed by the oligonucleotide.

The method of tracking and confirming the original object from the extracted oligonucleotide is as follows. The oligonucleotide obtained by treatment with ammonia to remove the protecting group is amplified by polymerase chain reaction (PCR). At this time, 15 base pairs (15mer) of the sock end of the base sequence of 40 base pairs (40mer) is known, so that the polymerase chain reaction using primers prepared according to the base sequence as the forward primer and the backward primer, respectively After amplification through PCR, the amplified product is subjected to sequencing to identify the base sequence of 10 base pairs (10mers) located at the center, and to track and confirm the object labeled with the identification label corresponding to the sequence. . Through this process, it is possible to find out the identity of an object.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the following Examples, but the general scope of the present invention is not limited to the following Examples.

Example 1

Determination of Oligonucleotide Concentration

To determine the concentration of the oligonucleotide to be added to the paint, 40-base pair (40mer) oligonucleotides were synthesized, and serial dilution was performed 10 times from 10 pmole / μl to 1ztmole / μl to polymerase chain reaction (PCR). Amplified by. After amplification, the product was electrophoresed on agarose gel, and the results are shown in FIG. 3. As shown in FIG. 3, even when the concentration of the oligonucleotide was 1 ztmole, it was confirmed that the amplification was well through the polymerase chain reaction (PCR).

Therefore, in this example, the oligonucleotide concentration to be mixed with the paint was determined to be 100 atmole in consideration of the amount of oligonucleotide to be added to the paint and the amount to be lost when separating the oligonucleotide from the paint piece, and was used in the next experiment. In Figure 3, each lane represents the oligonucleotide concentration, lane 1 is 10pmole, lane 2 is 1pmole, lane 3 is 100ftmole, lane 4 is 10ftmole, lane 5 is 1ftmole, lane 6 is 100atmole, lane 7 is 10atmole, lane 8 Is 1 atmole, lane 9 is 1 atmole, lane 10 is 100 ztmole, lane 11 is 10 ztmole, and lane 12 is 1 ztmole.

Example 2

Solubility Test of Organic Oligonucleotides by Phase Inversion Catalyst (PTC).

The oligonucleotide having the desired base sequence is synthesized and purified using an automatic oligonucleotide synthesis apparatus, and then sufficiently mixed with an aqueous oligonucleotide solution and an organic solvent such as toluene or ethyl ether together with a phase inversion catalyst (PTC), followed by layer separation. Then, only the organic solution layer was separated and the process was repeated until the oligonucleotides in the aqueous layer were not visible in the ultraviolet (UV). As a phase shift catalyst (PTC), it was tested by ultraviolet (UV) light absorption using tetra butyl ammonium hydroxide and hexadecyl trimethyl ammonium bromide. It was confirmed that the phase shift catalyst (PTC) was added to the oligonucleotide dissolved only in 5 ml) and extracted from the organic solvent layer (5 ml of toluene or ethyl ether).

After the extraction of the organic solvent layer was repeated five times or more (5 ml), the aqueous layer was examined by ultraviolet (UV), and it was found that most of the oligonucleotides were dissolved in the organic solvent layer and hardly remained in the aqueous layer. In addition, oligonucleotides coupled to the phase inversion catalyst (PTC) dissolved in the organic solvent layer were measured by Maldi-Tof mass spectrum, and as a result, it was confirmed that the oligonucleotide was the first oligonucleotide.

Example 3

Protective reaction experiment to introduce protecting group into oligonucleotide

A reaction experiment was carried out to protect the oligonucleotides before adding 40 base pairs (40mer) of oligonucleotides combined with a phase inversion catalyst (PTC) dissolved in the organic layer to the paint. The organic solvent layer containing the oligonucleotide combined with the phase inversion catalyst obtained in Example 2 was reacted with excess acryloyl chloride. The salt generated through the reaction was extracted and removed by an aqueous solution, and then the organic solvent layer was examined by using a Maldi-Tof mass spectrum. Substituted with acryloyl group. The acryloyl group contains a ketone and a double bond, and thus can be covalently bonded by addition reaction with the resin component of the paint.

Example 4

Experiment of coating and recovering a mixture of oligonucleotide derivatives combined with a phase inversion catalyst and paint

40 base pairs (40mer) oligonucleotide derivatives combined with the phase conversion catalyst after the process of Example 3 was mixed with a coating lacquer for automobiles, and then coated and recovered. After mixing the coating lacquer for automobile and the oligonucleotide derivative combined with the phase inversion catalyst, and coating it on the glass surface, the coated lacquer is dried in an oven at about 80 ° C. or more for at least 12 hours, cooled to room temperature, and then detergent And washed several times with water. Then, in order to recover the oligonucleotide from the lacca again, the remaining lacca fragment is dissolved with acetonitrile and dimethylformamide (DMF), and then the protecting group bound to the amine group of the base of the lacca and oligonucleotide and the hydroxyl group of the sugar To be removed, ammonia treatment was performed at 80 ° C. for at least 12 hours. Thereafter, the lacca component was extracted using ethyl ether, and only an aqueous layer was obtained, and then passed through a short reverse-phase column to recover the original oligonucleotide form. The recovered oligonucleotide was amplified by polymerase chain reaction (PCR), and then subjected to sequencing reaction with the amplified product.                     

The reaction of analyzing the polymerase chain reaction (PCR) and the nucleotide sequence of the oligonucleotide was performed as follows. The oligonucleotide sequence of the 40 base pair (40mer) was 5'- agc att ttg tgg ggc gtg ata gcc tcc ttg gcc gca aag a -3 '( SEQ ID NO: 1 ). '-agc att ttg tgg ggc-3' (15mer, SEQ ID NO: 2 ) was used, and the sequence of the backward primer was 5'-ccg cga ggt ggt ggt ctt tgc ggc caa gg (29mer) -3 '. In order to increase the efficiency of the polymerase chain reaction (PCR), the rear primer sequence was set to 29 base pairs (29mer, SEQ ID NO: 3 ) instead of 15 base pairs (15mers). Direct sequencing on 10% polyacrylamide gel after purification with DNA PrepMate II (Bionia Co., Ltd.) for sequencing oligonucleotides amplified by polymerase chain reaction (PCR). Sequencing reactions were performed. The results are shown in FIGS. 4A and 4B. Figure 4a shows the result of electrophoresis on agarose gel after amplification by a polymerase chain reaction (PCR) by coating and then mixed with a coating coating car coating lacquer according to the present invention. As shown in Figure 4a, it can be seen that the oligonucleotide is normally recovered by the present invention. Figure 4b shows the results of sequencing the amplification product of Figure 4a. As shown in Figure 4b, it can be seen that the first nucleotide sequence and the resulting nucleotide sequence. In particular, it can be confirmed that the base sequences of ten (gtg ata gcc t, SEQ ID NO: 4 ) representing the identification marker region except for the forward primer and the backward primer are identical, and by differently identifying the 10 base sequences It was confirmed that it can be used as a label.

However, when the amplification product through PCR was applied directly to the sequencing reaction, impurities appeared and the photograph was not clear, and the base immediately after the sequencing primer was not analyzed. The amplification product was cloned into a vector without performing sequencing immediately and then subjected to sequencing.

First Sequence: 5'-agc att ttg tgg ggc gtg ata gcc tcc ttg gcc gca aag a -3 '

Result Sequence: 5'- g ata gcc tcc ttg gcc gca aag a cc acc acc -3 '( SEQ ID NO: 5 )

In FIG. 4A, M denotes a size marker, and lanes 1 to 7 show results of experiments with 100 atmole / μL oligonucleotides. In FIG. 4B, the left four lanes show the nucleotide sequences of the oligonucleotides recovered according to the present invention, and the right four lanes show the nucleotide sequences as they are, without being mixed with a lacquer for automobile coating.

Example 5

Experiment of coating and recovering oligonucleotide derivative-PTC conjugate with acetyl substituent with paint

The procedure of Example 3 was carried out in the same manner except that acetyl chloride was used instead of acryloyl chloride. Thereafter, 40 base pair (40mer) oligonucleotide derivatives combined with a phase inversion catalyst were carried out in the same manner as in Example 4 to perform polymerase chain reaction (PCR) and sequencing. Thereafter, the oligonucleotides amplified by the polymerase chain reaction (PCR) were purified with DNA PrepMate II (Bionia Co., Ltd.) for sequencing, and then cloned into T-vectors. Sequences were analyzed using a T7 primer complementary to the T7 promoter in a T-vector on a 10% polyacrylamide gel. The results are shown in Figures 5a and 5b. Figure 5a shows the result of amplification by PCR after coating and coating the mixture with car coating lacquer. As shown in Figure 5a, it can be seen that the oligonucleotide is normally recovered according to the present invention. 5b shows that the product of FIG. 5a is identical to the first nucleotide sequence as a result of analyzing the nucleotide sequence.

First Sequence: 5'-agc att ttg tgg ggc gtg ata gcc tcc ttg gcc gca aag a-3 '

Result Sequence: 5'-ggt gg t ctt tgc ggc caa gga ggc tat cac gcc cca caa aat gct -3 '(cloned in reverse, SEQ ID NO: 6 )

Analytical Sequence: 5'- agc att ttg tgg ggc gtg ata gcc tcc ttg gcc gca aag a cc acc-3 '( SEQ ID NO: 7 )

In Figure 5a, M is a size marker, lanes 1 to 7 are oligonucleotide derivatives combined with a phase inversion catalyst mixed with a coating lacquer for automobiles, and then recovered and amplified by polymerase chain reaction (PCR), followed by agarose gel. It is photographed electrophoresis on. 5B shows the result of analyzing the nucleotide sequence of the product of FIG. 5A according to the present embodiment, and it can be seen that the nucleotide sequence is identical to the first sequence according to the present invention.

Example 6

Experiment to coat and recover oligonucleotide (A) combined with phase inversion catalyst and oligonucleotide derivative (B) combined with phase inversion catalyst by mixing with paint

After mixing and drying the oligonucleotide (A) combined with the phase inversion catalyst after the reaction of Example 2 and the oligonucleotide derivative (B) combined with the phase inversion catalyst after the process of Example 3 with different kinds of paints, After the same recovery process as in Example 4, polymerase chain reaction (PCR) and sequencing reactions were performed. The results are shown in Figures 6a and 6b. In FIG. 6A, lane 1 shows a result obtained by mixing binder A with a urethane paint, and lane 2 shows a result obtained by mixing binder A with a coating paint for automobiles, and lane 3 shows a result of combining binder B with a coating paint for automobiles. As shown in the figure, the result of recovery after mixing was shown. As shown in FIG. Thereafter, the base sequence was analyzed by analyzing the base sequence, and the results were as follows. Expected Sequence: 5'-agc att ttg tgg ggc tgc ctg gcg ccc ttg gcc gca aag acc acc tcg cgg-3 '( SEQ ID NO: 8 )

Resulting sequence of lane 1 (A): 5'-agc att ttg tgg ggc tgc ctg gcg ccc ttg gcc gca aag acc acc tcg c-3 '( SEQ ID NO: 9 )

Result nucleotide sequence of lane 3 (B): 5'-agc att ttg tgg ggc tgc ctg gcg gcc cac aaa atc gt-3 '( SEQ ID NO: 10 )

Conjugate A recovered after mixing with automotive coating paint was observed after electrophoresis on agarose gel during the purification of amplification products through polymerase chain reaction (PCR). -Cloning using vector did not work properly. In this embodiment, the forward primer sequence used during PCR was agc att ttg tgg ggc, and the next 10 sequences (tgc ctg gcg c) were sequenced as markers, and the base sequence was exactly identical. It was confirmed. The sequence of the backward primer was used 5-cc ttg gcc gca aag acc acc tcg cgg-3 '(29mer, SEQ ID NO: 11 ). The result of binder A was different according to the type of paint, but when it was mixed with urethane paint, the recovery was good and the sequence was well analyzed later. When it was mixed with automotive coating paint, the sequence was not properly cloned. I couldn't. This shows that the base of the oligonucleotide reacts directly when mixed with the automotive coating paint because the protection reaction of the amine or the oxygen moiety is not performed, and the recovery is poor. In FIG. 6B, the left four lanes show the oligonucleotide sequence of lane 1 of FIG. 6A, the four lanes in the middle, the oligonucleotide sequence of lane 2, and the right four lanes show the oligonucleotide sequence of lane 3.

Example 7

Combination of three different oligonucleotides

In this example, a combination of 40 base pairs (40mer) of oligonucleotides consisting of different base sequences was used. Here, each oligonucleotide has a different sequence so that different primers can be used, and the central identification region is also designed not to overlap with the edge sequence. The three oligomeric sequences thus designed were as follows.

Oligo SEQ ID NO: 1: ctg atg ggc cgc aac ctt cag tac att ttg ggc gca cca t ( SEQ ID NO: 12 )

Oligo sequence 2: tca ttc gac ccc cgg agc tgg cgt agt cga acc ggg ttc t (standing column No. 13)
Oligonucleotide SEQ ID 3: cgc gcg gtg ttg aat tca tgg cca gtg gaa cgc ttt ccg c (standing column No. 14)

delete

Each of the three oligonucleotides were subjected to the procedures of Examples 2 and 3, and then the three oligonucleotide derivatives combined with the phase inversion catalyst were mixed with the coating lacquer for automobiles and coated according to the procedure of Example 4. After drying, the oligonucleotides were recovered from the coated lacquer again. The recovered oligonucleotides were amplified by polymerase chain reaction (PCR). The results are shown in Figure 7a. Here, primers were used in three different primer pairs to match the three oligonucleotides. Primer sequences used were as follows.

Primer 1 (forward: ctg atg ggc cgc aac SEQ ID NO: 15 , backward: atg gtg cgc cca aaa SEQ ID NO: 16 )

Primer 2 (forward: tca ttc ccc gac cgg SEQ ID NO: 17 , backward: acc cgg tga aac gcc SEQ ID NO: 18 )

Primer 3 (forward: cgc gcg gtg ttg aat SEQ ID NO: 19 , backward: gcg gaa agc gtt cca SEQ ID NO: 20 )

Subsequently, sequencing reactions were performed with the PCR amplification products. The results are shown in Figure 7b. In FIG. 7A, M is a size marker, lanes 1 and 2 are PCR amplified by primer 1, lanes 3 and 4 are amplified by primer 2, and lanes 5 and 6 are amplified by primer 3. In FIG. 7B, the four lanes on the left represent oligonucleotide 1, the four lanes in the middle represent oligonucleotide 2, and the four lanes on the right represent oligonucleotide 3. Analysis of the nucleotide sequence confirmed that it is identical to the original oligonucleotide sequence. Therefore, the original sequence was confirmed by combining three different oligonucleotides. Therefore, in practical applications, oligonucleotide derivatives bound to two or more phase inversion catalysts of different sequences could be used in combination. Therefore, more combinations are generated and can be used efficiently when there are many objects to identify.

The present invention is to provide an oligonucleotide that maintains strong stability and binding in the oil-based product to minimize modification and loss due to external conditions.

According to the present invention, when an oligonucleotide derivative combined with a phase inversion catalyst dissolved in an organic solvent is added to various lipophilic substances, the object can be tracked and confirmed by later extracting and analyzing the oligonucleotide from these objects. That is, when the oligonucleotide derivatives combined with the phase inversion catalyst are added to the paint and then coated on the car, the identification label sequence can be later identified from a small amount of paint debris, so that the original vehicle can be traced. It can be used for a variety of similar uses.

In today's socially increasing traffic accidents, it is not only possible to motivate to reduce social crime, but also to obtain conclusive evidence by being able to track and verify the original vehicle from pieces of paint away from the vehicle when the accident vehicle escapes. It can reduce cases and at the same time secure a safety net from social crime.

Attach sequence list electronic file  

Claims (24)

Oligonucleotide for object identification label in which a phase transfer catalyst (PTC) and an oligonucleotide are combined with a compound having a quaternary ammonium salt or a cationic surfactant. According to claim 1, wherein the oligonucleotide is an object identification label oligonucleotide, characterized in that consisting of 20 to 1,000 base pairs. delete The oligonucleotide according to claim 1, wherein the phase inversion catalyst is tetrabutylammonium hydroperoxide or hexadecyl trimethyl ammonium bromide. The method according to claim 1, wherein the object identification label oligonucleotide is a halogenated acyl or a carbonyl compound having an amide bond with nitrogen and an ester bond with oxygen, a silanyl compound having an N-Si and O-Si bond, NS and OS Reactive blocking protecting group, which is any one selected from the group consisting of a sulfonyl compound to be bonded, a saturated or unsaturated hydrocarbon which is OC-bonded with NC, an aromatic hydrocarbon, and a saturated or unsaturated hydrocarbon containing a hetero atom, is further bonded. Oligonucleotide for object identification label to be. delete delete The oligonucleotide for object identification label according to claim 5, wherein the halogenated acyl is selected from the group consisting of acetyl chloride and acryloyl chloride. 1) forming a bond between the oligonucleotide and the phase inversion catalyst which is a compound having a quaternary ammonium salt or a cationic surfactant in an organic solvent; 2) Carbonyl compounds having halogenated acyl or amide bonds with nitrogen and ester bonds with oxygen, silanyl compounds having N-Si and O-Si bonds to the oligonucleotides to which the phase inversion catalyst is bound, and OS bonds with NS Combining a reactive blocking protecting group selected from the group consisting of a sulfonyl compound, a saturated or unsaturated hydrocarbon having an OC bond with NC, an aromatic hydrocarbon, and a saturated or unsaturated hydrocarbon including a hetero atom to remove reactivity; Method for producing an object identification label oligonucleotides is coupled to a phase inversion catalyst comprising a. The method of claim 9, wherein the phase inversion catalyst is tetrabutylammonium hydroperoxide or hexadecyl trimethyl ammonium bromide. delete 10. The method of claim 9, wherein the halogenated acyl is selected from the group containing acetyl chloride or acryloyl chloride. Oligonucleotide combined with a compound having a quaternary ammonium salt or a phase inversion catalyst, which is a cationic surfactant, has an amide bond with a halogenated acyl or nitrogen and an ester bond with oxygen, and an N-Si and O-Si bond. Object identification label protected by a reactive blocker selected from the group consisting of silanyl compounds, sulfonyl compounds which combine with NS and OS, saturated or unsaturated hydrocarbons which combine with NC and OC, aromatic hydrocarbons, and saturated or unsaturated hydrocarbons containing heteroatoms And adding the sieve to the object to be identified. The method of claim 13, wherein the object is made of paint for car coating, lacquer, paint for road lane marking, petroleum, paint thinner, gunpowder, oil derived from nature, construction paint, organic solvent, adhesive, oil paint, meat and seafood. Object identification labeling method characterized in that the lipophilic substance selected from the group. 1) A compound having a quaternary ammonium salt or a carbonyl compound having an amide bond with an acyl halide or nitrogen and an ester bond with oxygen, and an oligonucleotide to which an oligonucleotide is bound to a phase inversion catalyst, which is a cationic surfactant, and N-Si and O -A reactive blocker selected from the group consisting of silanyl compounds having Si bonds, sulfonyl compounds having OS bonds with NS, saturated or unsaturated hydrocarbons having NC and OC bonds, aromatic hydrocarbons, and saturated or unsaturated hydrocarbons containing heteroatoms; Extracting the identification label from the object labeled by the protected object identification label; 2) treating the extracted identification label with ammonia to remove reactive blocking protecting groups bound to the oligonucleotides; 3) recognizing the nucleotide sequence of the identification label portion of the oligonucleotide; 4) object identification method comprising the step of recognizing the object labeled with the identification label having the base sequence recognized in 3). 16. The method of claim 15, further comprising amplifying the identification label portion oligonucleotide through a polymerase chain reaction prior to recognizing the nucleotide sequence. 17. The method of claim 16, further comprising cloning the amplified oligonucleotide into a vector after the amplification step by the polymerase amplification reaction. The method according to claim 15, wherein the oligonucleotide consists of 20 to 1,000 base pairs. delete The object identification method according to claim 15, wherein the phase inversion catalyst is tetrabutylammonium hydroperoxide or hexadecyl trimethyl ammonium bromide. The method according to claim 15, wherein the object identification label oligonucleotide is a halogenated acyl or a carbonyl compound having an amide bond with nitrogen and an ester bond with oxygen, a silanyl compound having an N-Si and O-Si bond, NS and OS Recognition of the object further characterized in that the reactive blocking protecting group selected from the group consisting of a sulfonyl compound to be bonded, a saturated or unsaturated hydrocarbon with an OC bond with NC, an aromatic hydrocarbon, a saturated or unsaturated hydrocarbon containing heteroatoms. Way. delete delete 22. The method of claim 21, wherein said acyl halide is selected from the group consisting of acetyl chloride and acryloyl chloride.

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