KR100740869B1 - Method and apparatus for amplification of nucleic acid sequences using immobilized dna polymerase - Google Patents

Abstract

The present invention relates to a method and apparatus for amplifying nucleotide sequences using immobilized DNA polymerases, which is easy to separate and recover from DNA polymerases after nucleotide amplification, and to not only die polymerases having high temperature stability but also high-temperature diene polymerases. It is an object of the present invention to provide an apparatus and method which can use an enzyme, which is simpler in construction and processing, and which can be easily implemented in a complex apparatus such as a lab-on-a-chip. The present invention a) maintains the first reaction zone in the temperature range for the double strand of the DNA to be separated into single-stranded DNA molecules, respectively, and the complementary primer is bonded to a specific site of the single-stranded DNA molecule to form a partial double strand Maintaining a second reaction zone at a temperature range for the second reaction zone, and maintaining the third reaction zone at a temperature range for the primer extension product to be synthesized by the DNA polymerization in the partial double strand; b) immobilizing the diene polymerase Positioning a die between the first reaction zone, the second reaction zone, and the third reaction zone so that a specific DNA sequence can be amplified. It is characterized by repeating the step of moving at least one or more times.

Sequence amplification, immobilized DNA polymerase, polymerase chain reaction,

Description

METHODS AND APPARATUS FOR AMPLIFICATION OF NUCLEIC ACID SEQUENCES USING IMMOBILIZED DNA POLYMERASE}             

1 is a block diagram showing the concept of a nucleotide sequence amplification method using an immobilized DNA (Deoxyribonucleic Acid: DNA) polymerase according to the present invention,

Figure 2a is a block diagram of a nucleotide sequence amplification method using an immobilized DNA polymerase, characterized in that to move the DNA by thermal convection,

2B and 2C are a perspective view and a cross-sectional view of a nucleotide sequence amplification apparatus using immobilized DNA polymerase, characterized in that the DNA is moved by thermal convection;

Figure 3a and Figure 3b is a block diagram of a nucleotide sequence amplification method using the immobilized DNA polymerase, characterized in that for moving the DNA by the electric field generating means,

4 is a block diagram of a nucleotide sequence amplification method using immobilized DNA polymerase, characterized in that the die is moved by a pressure difference generating means;

5 is a block diagram of a nucleotide sequence amplification method using an immobilized die polymerase, characterized in that the die is moved by stirring means;

6 is a diagram showing an example of temperature distribution in the case of forming three reaction zones in a reaction sample of the experimental example of the present invention;

Figure 7 is agarose gel electrophoresis picture showing the results of nucleotide sequence amplification using immobilized diene polymerase according to the present invention.

<Explanation of symbols for the main parts of the drawings>

1: first reaction zone

2: second reaction zone

3: third reaction zone

4: immobilized diene polymerase

5: reaction vessel

6: square wave generator

7, 7 ': electrode

8, 8 ': piston

9: magnetic rod

10: electromagnet type stirring drive device

101: first conductive block

102: second conductive block

103: reaction vessel

104: heating device

105: temperature control fluid inlet

106: temperature control fluid outlet

107: insulation

111, 117: through hole

112: opening

The present invention relates to a method and an apparatus for amplifying nucleotide sequences using immobilized DNA polymerase. More specifically, the present invention forms a plurality of reaction zones in which a polymerase chain reaction (PCR) can occur and places the diene polymerase immobilized in a specific region among the plurality of reaction zones, The present invention relates to a method and apparatus for amplifying a specific nucleotide sequence by moving a DNA between reaction regions.

The sequencing technique is widely used for research and development and diagnostic purposes in the life sciences, genetic engineering, and medical fields, and in particular, sequencing by polymerase chain reaction (hereinafter, referred to as 'PCR sequencing technique'). ) Is widely used. Details of the PCR sequencing technique are described in US Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188.

Various devices and methods have been developed and used to automate PCR sequencing to amplify different types of genetic samples more efficiently and quickly. The basic principles of operation are as follows.

In commercially available PCR sequencing, template DNA to be amplified, oligonucleotide primer pairs having a sequence complementary to a specific sequence of each single strand of the template DNA, and a high temperature stable DNA polymerase ( Samples containing thermostable DNA polymerase, and deoxyribonucleotide triphosphates (dNTPs) are prepared and amplified a specific site sequence of the template diene by repeating a temperature cycle that sequentially changes the temperature of the sample. Specifically, three or two stages of temperature cycling cycles are used, and the process of achieving sequencing by temperature change is as follows. The first step is the deaturation step, in which the double stranded DNA is separated into single stranded DNA by heating the sample to a high temperature. The second step is an annealing step, in which the single stranded DNA and the primer are double-stranded and partially double-stranded DNA-primer complexes by cooling the sample passed through the degassing step to an appropriate temperature. (DNA-primer complex) is formed. The third step is a polymerization step, in which the original template is obtained by extending the primer of the diene-primer complex by the polymerase by polymerizing the primer of the diene-primer complex by maintaining the sample subjected to the annealing step at an appropriate temperature. Cloning a new single stranded DNA having a sequence complementary to the DNA. By repeating these three steps about 20-40 times in sequence, the base sequence between the two primers is replicated every cycle to achieve amplification of several million times or more.

The temperature in the desulfurization step mainly uses a value in the range of 90 ° C to 94 ° C, and the temperature in the annealing step is appropriately adjusted according to the melting point ( T _m ), that is, the T _m value of the primer used. Typically, values in the range of 40 ° C. to 60 ° C. are used. Temperature in the poly-head localization step according to the optimal 72 ℃ effective temperature of sseomeoseu aqua tea kusu high temperature stability chosen DNA polymerase (Taq DNA Polymerases) extracted from (Thermus aquaticus), often used, using a three-step temperature cycle Cycle The most common is the active temperature range of the tack die polymerase, so a two-stage temperature cycle is also used in which the temperature of the annealing step and the polymerization step is equalized to circulate the temperature.

The conventional nucleotide sequence amplification method changes the temperature of the entire sample containing the DNA polymerase according to a three-stage or two-stage temperature cycle cycle, the following problems exist.

First, in the conventional nucleotide sequence amplification method, since the DNA polymerase is included in the sample, the process of removing the enzyme added for purification of the sample after completion of the reaction is not simple, and it is difficult to reuse the already used enzyme.

Second, the conventional nucleotide sequence amplification method can use only a diene polymerase having a high temperature stability, such as tack diene polymerase. It is because the conventional nucleotide sequence amplification apparatus includes the process of heating the whole sample to high temperature.

Third, it is difficult to implement a conventional temperature cycle type amplification method into a complex device such as a lab-on-a-chip. Complex devices, such as lab-on-a-chip, are miniaturized devices that allow multiple stage reactions and processes to be performed simultaneously or sequentially on a single chip. Conventional sequence amplification methods require a process to change the temperature of the entire sample. Therefore, due to the complexity of the configuration and process, it is a disadvantageous method for miniaturization, and it is also difficult to implement in a composite device in which a sudden temperature change is undesirable.

One of several solutions to these problems is to use immobilized DNA polymerase. Immobilized enzyme refers to an enzyme that is physically or chemically bound to a support and maintains catalytic activity. The general advantage obtained by using the immobilized enzyme is, firstly, since the enzyme can be easily separated and recovered from the reaction solution by removing the support on which the enzyme is immobilized after the reaction, thereby simplifying the purification process of the sample. In addition, the cost can be reduced by reusing the recovered immobilized enzyme. Third, the efficiency of the reaction process can be improved by simplifying the multi-step processes including the enzymatic reaction. In addition, as the enzyme is immobilized, the utilization efficiency may be improved by secondary effects such as physical stability of the enzyme or changes in the conditions of the enzyme reaction. Therefore, by using the immobilized DNA polymerase, it can be expected that it will be possible to solve the above problems with the conventional sequence amplification method.

However, there is no known method to solve the above problems through the immobilization of enzymes, and the main causes can be broadly divided into the problem of the activity of the immobilized enzyme and the process problem using the same. First, various methods for immobilizing enzymes have been reported. However, a substantial product such as a polymerase chain reaction is produced, and the preparation of immobilized DNA polymerase is maintained while maintaining a high enough activity to detect the result. No method has been reported. That is, in order to implement a method and apparatus for amplifying a nucleotide sequence using immobilized DNA polymerase, development of a method capable of immobilizing the DNA polymerase with high activity must be preceded. Second, even if the use of immobilized die polymerase while maintaining high activity, it is difficult to expect a great effect by going through the existing temperature cycle cycle. First, the conventional temperature cycling cycle method requires a step of heating the entire sample to a high temperature, and thus has a limitation that it is impossible to use a diene polymerase which is not high temperature stability. In addition, in the conventional temperature cycling method, the diene polymerase is polymerized only in the temperature cycling step suitable for the polymerization step, and the diene polymerization reaction is performed only at a part of the total reaction time. The temporal efficiency of is limited by the rate of temperature change of the temperature cycling cycle. Therefore, there is a need for the development of a new method of sequencing and apparatus for sequencing the advantage of using an immobilized die polymerase rather than the conventional temperature cycling cycle method of sequentially changing the temperature of the entire reaction sample.

It is an object of the present invention to provide a method and apparatus for amplifying a new sequence using immobilized DNA polymerase.

More specifically, an object of the present invention is to provide a method and apparatus for amplifying a sequence which is easy to separate and recover the DNA polymerase after base sequence amplification by using an immobilized DNA polymerase. Through this, it is easy to purify a sample, and also to provide a method and apparatus for amplifying a sequence capable of reusing DNA polymerase.

Another object of the present invention is to provide a method and a device for sequencing a sequence using an immobilized die polymerase capable of using not only a die polymerase having high temperature stability but also a die polymerase not having a high temperature stability. The use of various DNA polymerases can be used to expand the utilization of sequencing methods and devices. Especially, the use of DNA polymerases, rather than high temperature stability, can be used to improve the accuracy of sequence replication by DNA polymerization. It is intended to provide a method and apparatus for amplifying a sequence.

In addition, the present invention provides a method and apparatus that does not require a temperature change process used in a conventional sequence amplification method and apparatus, thereby using a sequence amplification method and apparatus using an immobilized DNA polymerase having a simpler structure and process. For the purpose of Accordingly, an object of the present invention is to provide an apparatus and method that can be easily miniaturized and implemented in a complex device such as a lab-on-a-chip as compared to the conventional nucleotide sequence amplification method and apparatus.

Those skilled in the art to which the present invention pertains can easily recognize other objects and advantages of the present invention from the drawings, the detailed description of the invention, and the claims.

In order to achieve the above object, the present invention provides a method and apparatus for amplifying a new sequence using immobilized DNA polymerase as follows.

More specifically, the present invention provides a method for amplifying at least one specific diene nucleotide sequence by a polymerase chain reaction.

a) maintaining the first reaction zone in the temperature range for the double strand of the DNA to separate into single stranded DNA molecules, respectively;

Complementary primers are coupled to specific regions of the single-stranded DNA molecule to maintain a second reaction zone in the temperature range for forming partial double strands,

Maintaining a third reaction zone in a temperature range for the primer extension product in the partial double strand to be synthesized by DNA polymerization;

b) placing the immobilized diene polymerase in the third reaction zone; And

c) moving the die between the first reaction region, the second reaction region, and the third reaction region so that a specific die nucleotide sequence can be amplified.

Provided is a method for amplifying a sequence using an immobilized die polymerase, characterized in that repeating the step of moving the die at least one or more times.

The present invention also provides a device for amplifying at least one specific DNA sequence by a polymerase chain reaction,

a) reaction vessel;

b) maintaining the first reaction zone of the reaction vessel in a temperature range for separating the double strands of the DNA into single stranded DNA molecules, respectively,

The second reaction zone of the reaction vessel is maintained at a temperature range for forming partial double strands by binding complementary primers to specific sites of the single stranded DNA molecule,

Constant temperature maintenance means for maintaining a third reaction region of the reaction vessel in a temperature range for the primer extension product in the partial double strand to be synthesized by a diene polymerization reaction;

c) moving means for moving a die between said first reaction zone, said second reaction zone, and said third reaction zone so that a specific die base sequence can be amplified;

An immobilized die polymerase is positioned in the third reaction region, and the moving unit provides a nucleotide sequence amplification apparatus using the immobilized die polymerase, characterized in that the die is repeatedly moved at least once.

The present invention also provides a device for amplifying at least one specific DNA sequence by a polymerase chain reaction,

a) reaction vessel;

b) maintaining the first reaction zone of the reaction vessel in a temperature range for separating the double strands of the DNA into single stranded DNA molecules, respectively,

The second reaction zone of the reaction vessel is maintained at a temperature range for forming partial double strands by binding complementary primers to specific sites of the single stranded DNA molecule,

And a constant temperature maintaining means for maintaining a third reaction region of the reaction vessel in a temperature range for synthesizing the primer extension product in the partial double strand by die polymerization.

An immobilized die polymerase is positioned in the third reaction region, and a relatively high temperature region of the first region, the second region, and the third region is located at a lower height than a region having a relatively low temperature, and Sequence amplification using an immobilized DNA polymerase, wherein the DNA is repeatedly moved at least one time between the first region, the second region, and the third region by thermal convection so that the DNA sequence can be amplified. Provide the device.

In the present invention, the immobilized die polymerase refers to a die polymerase which is bound while maintaining activity on a solid phase. There may be a number of methods for preparing immobilized DNA polymerase, but immobilized DNA polymerase whose activity is high enough to detect the result of amplification of the base DNA of the template DNA as a result of the polymerase chain reaction. It should be possible to manufacture it. In the present invention, an immobilized diene polymerase having high activity was prepared by masking the active site of the diene polymerase with a diene substrate and immobilizing the covalent bond on the gold surface. The specific procedure is described in the experimental example of the present specification. As a result of the present invention, the activity of the immobilized enzyme is about 60-80% compared to the same amount of the solution phase enzyme, which can be used for the polymerase chain reaction. Enough activity. However, the immobilized diene polymerase used in the present invention is not limited to that immobilized by the above method, and may be used even if the diene polymerase immobilized by another method.

In general, a sample used in the method for amplifying a sequence by a DNA polymerase includes four types of triphosphorylated deoxynucleotides such as a template die, dATP, dCTP, dGTP, and dTTP as a substrate, a primer for starting a polymerization reaction, and a DNA. The DNA polymerase which catalyzes the polymerization reaction is contained in a buffer that is adjusted to maintain an appropriate salt concentration and hydrogen ion concentration.

The composition of the sample used in the present invention is different from the sample used in the conventional sequencing method. That is, unlike the conventional sequencing method in which the DNA polymerase is dissolved in an aqueous solution, such as a sample, the DNA on the aqueous solution contains the DNA polymerase because the present invention uses the DNA polymerase immobilized on a solid support. Not. Therefore, unlike the conventional method, after the reaction is completed, the separation of the enzyme and the purification of the sample are simple, and the advantage of enabling the reuse of the enzyme can be obtained.

The template die of the sample used in the present invention may be present in any form of single strand, double strand, and partial double strand, and may be present in a mixture of several kinds of dies having different lengths or shapes. When amplifying sequences in a sample containing messenger Ribonucleic Acid (mRNA), reverse transcriptase is used to convert Ribonucleic Acid (RNA) into DNA. Complementary Deoxyribonucleic Acid (cDNA) can also be used as a template.

In the present invention, the primer uses at least one pair of oligonucleotides each containing a 5'-terminal portion of the DNA double strand corresponding to the specific nucleic acid sequence to be amplified. These primer pairs are each configured to complementarily bind to the 3'-terminus of a specific base sequence of the template diene to start the diene polymerization reaction, and when the polymerase reaction proceeds in series, Add in excess molar ratio as compared to When amplifying a specific sequence of the template die and some modified sequences, bases partially substituted, deleted, or added within the primer sequence are used within a range that allows the primer to maintain a structure capable of binding to the template die. It is apparent to those skilled in the art that the present invention can be used as a primer even though it contains a base sequence that is not partially complementary to the template die.

The biggest feature of the present invention is to form a plurality of reaction zones, each maintained at a specific temperature, in the sample, and to allow the die to be circulated between these reaction zones.

The reaction of the DNA polymerase is 1) a double step of separating the double strand of the DNA contained in the sample into single-stranded DNA molecules; 2) an annealing step in which complementary primers are bound to specific sites of a single stranded DNA molecule to form partial double strands; 3) consists of a polymerisation step in which the primer extension product is synthesized in the partial double strands, and the sequence is amplified by the sequential and repeated occurrence. In the conventional sequence amplification method, the above steps are performed by a temperature cycle method of sequentially changing the temperature of the entire sample.

However, in the present invention, the steps are performed by forming a plurality of reaction zones having a temperature range in which the steps can be performed, and circulating a die between the plurality of reaction zones. In the present invention, the plurality of reaction zones, 1) maintaining the first reaction zone in the temperature range for each double strand of the DNA to be separated into single-stranded DNA molecules, 2) complementary to a specific site of the single-stranded DNA molecules The second primer retains the second reaction region at a temperature range for forming a partial double strand by combining the primers. 3) The primer extension product at the partial double strand has a temperature range for replication with a base sequence complementary to the template die. 3 Maintain the reaction zone. The first reaction zone, the second reaction zone, and the third reaction zone may overlap all or a part of each other, and each temperature range may also partially or all overlap.

The die is annealed in a single strand in the first reaction zone, annealed with the primer in the second reaction zone, and the polymerization reaction occurs by placing the die polymerase immobilized in the third reaction zone, thereby extending the primer. The product is duplicated. The base sequence of the primer extension product generated at this time may have a base sequence identical to or partially substituted, deleted or added to a specific base sequence of the DNA used as a template according to the primer construction method. When the polymerase chain reaction occurs, primer extension products are also used as templates in addition to the diene used as the first template, and then primer extension products having the same nucleotide sequence are amplified.

In the present invention, the DNA sequence is amplified by moving the DNA between the first reaction region, the second reaction region, and the third reaction region so that a specific sequence can be amplified. In addition to the DNA used as a template, the DNA includes a primer extension product generated through the movement between the reaction zones. The DNAs repeatedly moved between the reaction zones, and the sample containing other buffers, trioxydeoxyribonucleotides, etc. may be fixedly located in the respective reaction zones, and move between the reaction zones with the dies. You may be. The term "moving a die so that a specific sequence can be amplified" means moving the die at least once between the three reaction zones so that a polymerase chain reaction can occur.

In the present invention, the immobilized enzyme of the immobilized enzyme is E. coli diene polymerase I, E. coli diene polymerase I having a function of replicating a sequence complementary to the template diene, Klenau fragment, T4 diene polymerase, tag The DNA polymerase and derivatives and variants thereof are selected and used.

In the conventional DNA cycle amplification method and apparatus by a temperature cycling cycle method, the entire sample is heated to a high temperature, so that only the enzyme having high temperature stability is used substantially. If not, the enzyme must be added at every temperature cycling cycle due to heat impairing the activity of the enzyme. The present invention solves a problem in which a diene polymerase is damaged by heat by placing the immobilized diene polymerase in a specific temperature range (third reaction zone). That is, in the present invention, by placing the enzyme in the reaction zone having a temperature range where the polymerization occurs so as not to be affected by the degassing process requiring a high temperature of 90 ℃ or more, the DNA polymerase is not exposed to a high temperature state Thus, the DNA polymerase having no high temperature stability was also available.

The above objects, features and advantages will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. In describing the present invention, when it is determined that the detailed description of the related known technology may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a block diagram showing the concept of a nucleotide sequence amplification method using an immobilized DNA polymerase according to the present invention. As shown in a) of FIG. 1, the first reaction region (1) in which the double strand of the DNA is maintained at a temperature range for separating into single-stranded DNA molecules, respectively, having a sequence complementary to a specific site of the single-stranded DNA molecule The primers are joined to form a second reaction zone (2) maintained at a temperature range for forming partial double strands, and a third reaction zone (3) maintained at a temperature range for synthesis of primer extension products in the partial double strand. After the immobilized diene polymerase is placed in the third reaction zone (3), the die is moved between the first reaction zone, the second reaction zone, and the third reaction zone. The present invention can also be configured such that the temperature range of each reaction zone is partially overlapped (b) of FIG. 1, and a position is changed to facilitate movement between the reaction zones (FIG. 1c). )).

As an example of the present invention, the case where the immobilized tack diene polymerase is used will be described in more detail with reference to a of FIG. 1 as follows.

The first reaction zone is maintained at the lowest height to maintain the temperature range of 90 to 94 DEG C so that the degassing takes place, and the second reaction zone is maintained at the temperature range of 40 to 60 DEG C. so that annealing of the primer occurs. At a high height, a third reaction zone is placed in the middle, for example, to maintain a temperature range of, for example, 72 ° C., which is the optimum temperature of the tack die polymerase. When configuring the reaction zone as described above, since the relatively high temperature region is located at a lower height than the relatively low temperature region, the movement of the die can be achieved by thermal convection by the temperature gradient.

That is, in the first reaction region 1, the de- activation step takes place primarily, and the annealing step is secondary by moving the de-digested die to the second reaction zone 2 by thermal convection in the presence of a primer. In the course of passing the diene-primer complex produced by annealing through the third reaction zone (3) by thermal convection, the immobilized diene polymerase (L) was placed in the third reaction zone. 4), so that the three steps of the polymerization, annealing, and polymerization of the polymerase reaction occur sequentially and repeatedly so as to efficiently amplify specific site sequences of the sample DNA. do.

Referring to b) of Figure 1 of another example of the present invention, using a DNA polymerase chosen as follows. Selecting DNA polymerase has a optimum activity at 72 ℃ However, it is known to have a broad activity to a low temperature region. Thus, the tack to the case of using a DNA polymerase, the temperature range, the polymerization reaction takes place as in b) of Figure 1 to be the same as the temperature range of the annealing takes place, it may be left for the second reaction zone and a third reaction zone at the same position have. Even in this case, the movement of the die can be achieved by thermal convection by the temperature gradient.

As another example of the present invention, the case of using the immobilized E. coli diene polymerase will be described with reference to FIG. The optimum temperature of E. coli DNA polymerase is 37 ° C, which is lower than the temperature range where annealing of the primer occurs. Therefore, when E. coli diene polymerase is used, it may be preferable to configure the third reaction region where the polymerization reaction takes place as shown in c of FIG. 1 to be higher than the second reaction region where the annealing occurs. Even in this case, the movement of the die can be achieved by thermal convection by the temperature gradient.

In the examples of the invention illustrated in Figures a), b) and c) of the die when the temperature arrangement of the reaction zones 1, 2 and 3 having a specific temperature range is not suitable for the formation of thermal convection It would be possible to additionally use the means to make this happen.

Figure 2a is a configuration used in the experimental example of the present specification, as one embodiment of the present invention. In this configuration, the die is moved by heat convection, and a region having a relatively high temperature is positioned at a lower level than a region having a relatively low temperature, but the second reaction region and the third reaction region partially overlap. to be.

As shown in FIGS. 2B and 2C, after keeping the lower first conductive block 101 at a high temperature and the upper second conductive block 102 at a low temperature, the conductive blocks 101 and 102 are insulated. Insulating by 107 causes the first reaction zone 1 to be hot in the reaction vessel 103 and the second reaction zone 2 to be low in temperature, and the third reaction zone 3 due to the temperature gradient therebetween. Is formed. At this time, since the sample in the high temperature region has a relatively low density than the sample in the low temperature region, the die is moved by buoyancy from the high temperature region located at the low height to the low temperature region located at the high height, and due to the influence of gravity By causing the movement of the die in the opposite direction, natural heat convection caused by the temperature difference causes a natural circulation of the die between the plurality of reaction zones 1, 2, and 3 in the specific temperature range. Although the heating device 104 for maintaining the first conductive block 101 at a high temperature is shown only as a block, and the circulation type constant temperature water tank for maintaining the second conductive block 102 at a low temperature is not shown, the specific configuration thereof These can be easily predicted by those of ordinary skill in the art. As the temperature maintaining means for maintaining the first reaction zone, the second reaction zone, and the third reaction zone of the present invention in a specific temperature range, in the present embodiment, the conductive blocks 101, 10 2, the heating device, and the circulating constant temperature I use a water tank. However, the means for maintaining the temperature of the present invention is not limited to the configuration consisting of the conductive blocks 101 and 102, the heating device, and the circulating constant temperature water bath. For example, without using the conductive block, a proper temperature such as liquid or gas may be used. It is also possible to form the reaction zones in such a way that the fluid comes into contact with a specific part of the reaction vessel or directly heats the sample using an infrared ray generator. The specific configuration of the constant temperature maintenance means may be variously modified according to industrial reality, and any modification may naturally fall within the scope of the present invention as long as the configuration functions to maintain the first, second, and third reaction zones in a specific temperature range. Will belong.

3A and 3B are still other embodiments of the present invention. In the present embodiments, the die is moved using an electric field.

The DNA contains many functional groups with charges in its structure, but in terms of its overall structure, the phosphate group contained one per unit nucleotide has the greatest effect, and a large amount of negative charge is generated in the buffer maintained close to neutrality. . Accordingly, as shown in FIGS. 3A and 3B, after the electrodes 7 and 7 ′ are introduced into both end regions 1 and 3 of the reaction solution, the potential difference in the reaction sample is obtained by using the square wave generator 6. By forming a and periodically changing the direction of the potential difference, the negatively charged die is sequentially moved between the reaction zones (1, 2, 3) to circulate, thereby amplifying the base sequence. Although not shown in FIGS. 3A and 3B, an electrode may be introduced into each of the reaction regions 1, 2, and 3, or a configuration in which the arrangement of the reaction regions 1, 2 and 3 may be modified.

 Since the ultimate target of die- sion, annealing, and polymerization during the diene polymerization reaction is diene, which is used as a substrate, the target material to be moved between the reaction zones is diene. However, when an electric field is applied to move dies, transfer of primers and triphosphate deoxyribonucleotides contained in the sample may occur at the same time, and in this case, the substrate is concentrated at a high concentration in each corresponding reaction region. As such, annealing and polymerisation of the DNA provides an advantageous condition that can occur more easily. The strength and retention time of the electric field formed in the sample can be adjusted according to the size of the die and the shape of the reaction vessel. If the polymerase is immobilized on the metal surface, the immobilized diene polymerase may be used as an electrode. It is possible to change the configuration depending on the reaction process, the characteristics of the sample and the shape of the reaction vessel, which is not limited to the examples of FIGS. 3A and 3B shown.

Figure 4 is another embodiment of the present invention, the step of moving the DNA is a block diagram of the nucleotide sequence amplification method using the immobilized die polymerase, characterized in that for moving the die by the pressure difference generating means.

As shown in FIG. 4, the pressure applied to one end of the reaction vessel 5 is higher or lower than the other end to generate a pressure difference, thereby maintaining the reaction zones 1, 2, and 3 maintained in a specific temperature range. You can make the DNA move in between. In FIG. 4, a case of using the reciprocating motion of the pistons 8 and 8 'is specifically illustrated, but a person having ordinary skill in the art to which the present invention pertains may use the pistons 8 and 8' as a pressure difference generating means. In addition, it is well known that it is possible to use various kinds of liquid or gas pumps that can generate pressure differences.

Figure 5 is another embodiment of the present invention, the step of moving the die is a block diagram of a nucleotide sequence amplification method using an immobilized die polymerase, characterized in that for moving the die by stirring means.

The die can be moved by rotating the magnet rod 9 placed in the reaction vessel 5 with the electromagnet type stirring drive 10 as shown in FIG. In addition, it is possible to achieve the object of the present invention by moving the die between the reaction zones by a stirring means such as using a vane stirrer or a method of periodically vibrating a thin film in contact with the solution.

Experimental Example

A. Preparation of Immobilized DNA Polymerase

The phosphate buffer of pH 8.3 containing 65 base single-stranded DNA and KS primer in a 1: 1 molar ratio was left at 94 ° C. for 10 minutes, and then cooled slowly until it reached 35 ° C. or less. . The 65 base single stranded diene and the KS primer are annealed to produce a partially doubled diene. In this solution, a proper number of moles of tack diene polymerase (AmpliTaq Gold, Perkin Elmer, USA) was added, left in a 72 ° C. dry bath for 10 minutes, and then transferred to a 50 ° C. dry bath. The partial double-stranded DNA was coupled to the site to prepare a masked diene polymerase solution.

KS Primer: 5'-CGAGGTCGACGGTATCG-3 '(SEQ ID NO: 1)

65-mer:

3'-CCAGCTGCCATAGCTATTTTCTTTTCTTTCTTAAGTTCTTTTCTTTTCCTAGGTGATCAAGATCT-5 '(SEQ ID NO: 2)

A gold wire having a diameter of 4.7 mm and a diameter of 0.1 mm was prepared to have an outer diameter of 1.5 mm and a spiral of about 4 mm so that the maximum amount of die polymerase immobilized on the surface was 0.26 pmol. In order to clean the surface, the solution was placed in a Piranha solution adjusted to 60 to 70 ° C. immediately before the reaction for 10 to 15 minutes, followed by washing with deionized water followed by absolute ethanol.

In order to introduce an immobilized reactive functional group on the gold surface, a thiol molecular monolayer film was introduced to the gold surface by using a thiolate formation reaction between the linking material having a thiol group and gold, that is, Au-S bond formation, to form a support. At this time, by using a solution in which two thiol molecules having an immobilized reactive functional group and a non-reactive end group were mixed, the mole fraction of the thiol molecules having an immobilized reactive functional group was adjusted to 5%. In order to introduce a carboxyl group as an immobilized reactive functional group, 12-mercaptododecanoic acid, a thiol molecule having a relatively long alkyl chain, was used as a linking material, and 6-mercapto-1-hexane, a thiol molecule having a non-reactive end group, was used as a linking material. Ol or 1-heptanethiol was used as matrix material. A gold wire was added to 100 μl of an ethanol solution having a total thiol molecule concentration of 2 mM, and reacted at room temperature for 2 hours. After the gold wire was taken out and washed with absolute ethanol, a carboxyl group was introduced to the surface of the gold wire.

A gold wire having a carboxyl group introduced therein was 120 µl of an ethanol solution containing 10 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 5 mM N-hydroxysuccinimide (NHS). The reaction was carried out at room temperature for 2 hours. The carboxyl group is activated by reaction with N-hydroxysuccinimide in the presence of carbodiimide to form NHS-ester.

After activating the carboxyl group of the thiol molecular monolayer membrane, the gold wire was pulled out and the active site of the DNA polymerase was placed in a masked enzyme solution and reacted. At this time, an amide bond (-CO-NH-) is formed by the reaction of the activated carboxyl group (NHS-ester) of the thiol molecular monolayer membrane with the primary amine group of the protein, and the tack diene polymerase is immobilized on the support.

When the polymerase chain reaction was carried out using the immobilized diene polymerase prepared by the above method by a conventional temperature cycling cycle, the activity of the enzyme in aqueous solution was 60-80%.

B. Preparation of Reaction Samples

As the reaction vessel, one closed glass tube was used. The length of the glass tube was 55 to 60 mm, the inner diameter was 2 mm, the outer diameter was 8 mm, and the glass thickness of the closed bottom surface was about 3 mm. The inner wall of the glass tube was coated with a polytetrafluoroethylene coating of a spray method, thermally cured, and used as a surface treatment.

pBluescript II KS (+) was used as a template die to amplify 164 bp sequences from T7 primer region 627 to T3 primer region 790. Samples used for the polymerase chain reaction were 40 ng of template die, T3 primer (5'-ATTAACCCTCACTAAAG-3 '(SEQ ID NO: 3)) and T7 primer (5'-AATACGACTCACTATAG-3' (SEQ ID NO: 4). 10 mM Tris buffer solution at pH 8.3 containing 40 pmol of each), 4 nmol triphosphate deoxyribonucleotide mixture, and 100 μl total volume containing 250 nmol magnesium chloride and 50 mM potassium chloride. It was prepared as possible. This prepared sample was placed in a reaction vessel, and the tack diene polymerase immobilized on the gold wire prepared in step A was placed in a low temperature region and used for the reaction. Using the sample into a chosen DNA polymerase of 0.26 pmol in a solution state on the prepared in the same manner as a control to measure the immobilized enzyme after the sample of DNA polymerase was carried out the polymerase chain reaction according to the temperature cycle cycle type .

C. Execution of diene polymerization

This will be described with reference to FIGS. 2B and 2C. First, the lower first conductive block 101 was heated at 96 ° C. by heating the electrothermal heating device, and the upper second conductive block 102 was fixed at 45 ° C. using a circulating constant temperature water bath. Inject the sample prepared by the method of step B into the reaction vessel and insert it into the receiving portion (111, 117, 112), and measured the temperature change in each region in the reaction vessel, 90 ° C or more so that the degassing occurs It was confirmed that the maintained high temperature region, the low temperature region maintained at about 50 ° C. so that annealing can occur, and the convection region in which the temperature gradient is formed so that heat convection can occur therebetween (Fig. 6). Accordingly, it can be expected that polymerization will occur in the upper layer of the low temperature region and the convection region.

In order to perform the polymerization reaction, the center of the spiral gold wire (1.5mm in outer diameter, 4mm in length) to which the DNA polymerase is immobilized is placed in the region where the temperature in the reaction vessel is about 55 ° C, and then the sample is It was left as it is for a certain reaction time. The reaction vessel was taken out and cooled, then the reaction product was developed using 1.0% agarose gel electrophoresis, stained with ethidium bromide dye, and the diene product was visualized by fluorescence appearing upon UV irradiation. Quantitation was performed with a densitometer. Figure 7 is an electrophoresis picture showing the results obtained by changing the reaction time up to 4 hours in 30 minutes intervals. The reaction product is a 164 bp double helix diene. As can be seen in Figure 7, it can be seen that the polymerase chain reaction began to saturate from the reaction time 120 minutes.

The present invention described above is not limited to the above-described embodiments and the accompanying drawings, and various substitutions, modifications, and changes are possible in the technical field of the present invention without departing from the technical spirit of the present invention. It is evident to those of ordinary knowledge. Therefore, the above-described embodiments and drawings are merely examples in all respects, and should not be interpreted limitedly. The scope of the present invention is shown by the claims, and is not limited by the specification text.

According to the present invention has the following effects.

First, the present invention can be repeated use of the enzyme by using the immobilized DNA polymerase, to simplify the process of removing the enzyme in a continuous process, it is possible to reduce the cost and simplify the process.

Second, the present invention can be used in the DNA amplification method and apparatus, such as Klenow fragment, T7 diene polymerase without high temperature stability in the sequence amplification method and apparatus, thereby improving the utilization of the sequence amplification method and apparatus Make it extensible. In particular, by enabling the use of die polymerase rather than high temperature stability provides a way to improve the accuracy of the polymerization to replicate the sequence.

Third, the present invention provides a method and apparatus for sequencing sequences that do not require the temperature change process used in the conventional methods and apparatuses, thereby simplifying the construction and the process, thereby providing a method for miniaturization and implementation in a complex apparatus. . In addition, thereby enabling a continuous polymerization reaction, thereby providing a method capable of improving the sequence amplification rate.

<110> AHRAM BIOSYSTEMS INC. <120> METHOD AND APPARATUS FOR AMPLIFICATION OF NUCLEIC ACID SEQUENCES          USING IMMOBILIZED DNA POLYMERASE <130> Apa23-1 <150> KR-10-2002-0055905 <151> 2002-09-13 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 1 cgaggtcgac ggtatcg 17 <210> 2 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 2 tctagaacta gtggatcctt ttcttttctt gaattctttc ttttctttta tcgataccgt 60 cgacc 65 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 3 attaaccctc actaaag 17 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <400> 4 aatacgactc actatag 17

Claims (12)

In the method of amplifying a specific diene base sequence by a polymerase chain reaction, a) injecting a reaction mixture comprising a template die, a primer, and a deoxyphosphoryl trioxide (dNTP) into the reaction vessel; b) forming first to third reaction zones in the reaction mixture, Maintaining the first reaction zone in the temperature range for the double strand of the DNA to separate into single-stranded DNA molecules, Complementary primers are coupled to specific regions of the single-stranded DNA molecule to maintain a second reaction zone in the temperature range for forming partial double strands, Maintaining a third reaction zone in a temperature range for the primer extension product in the partial double strand to be synthesized by DNA polymerization; c) placing an immobilized diene polymerase in the third reaction zone; And d) moving the die between the first reaction zone, the second reaction zone and the third reaction zone in the reaction vessel so that a specific die base sequence can be amplified; A method of amplifying a sequence using an immobilized diene polymerase, characterized in that repeating the step of moving the die one or more times. The method of claim 1, wherein the immobilized enzyme of the immobilized diene polymerase is E. coli diene polymerase I, E. coli diene polymerase I having the function of replicating a sequence complementary to the template diene polymerase I, T4 diene polymerization A method for amplifying a sequence using an immobilized diene polymerase, characterized in that it is selected from the group consisting of enzymes, tag diene polymerase and fragments having these activities. The method of claim 1, wherein the moving of the die comprises: positioning a relatively high temperature of the first, second, and third regions at a lower height than a relatively low temperature region. A base sequence amplification method using immobilized DNA polymerase, characterized in that to move the DNA. The method of claim 1, wherein the moving of the die is a nucleotide sequence amplification method using an immobilized die polymerase, characterized in that for moving the die by the electric field generating means. The method of claim 1, wherein the moving of the die is a nucleotide sequence amplification method using the immobilized die polymerase, characterized in that for moving the die by a pressure difference generating means. The method of claim 1, wherein the moving of the die is a nucleotide sequence amplification method using an immobilized die polymerase, characterized in that for moving the die by stirring means. In the device for amplifying a specific DNA sequence by a polymerase chain reaction, a) reaction vessel; b) maintaining a first reaction region formed in the reaction mixture injected into the reaction vessel at a temperature range for separating double strands of DNA into single stranded DNA molecules, respectively; The second reaction zone formed in the reaction mixture injected into the reaction vessel is maintained at a temperature range for forming partial double strands by binding complementary primers to specific sites of the single stranded DNA molecule, A constant temperature maintaining means for maintaining a third reaction region formed in the reaction mixture injected into the reaction vessel in a temperature range for the primer extension product synthesized by the DNA polymerization reaction in the partial double strand; c) moving means for moving a die between said first reaction zone, said second reaction zone, and said third reaction zone in a reaction vessel so that a specific die base sequence can be amplified. An immobilized die polymerase is located in the third reaction region, and the moving means is a sequence amplification apparatus using the immobilized die polymerase, characterized in that the die is repeatedly moved one or more times. In the device for amplifying a specific DNA sequence by a polymerase chain reaction, a) reaction vessel; b) maintaining a first reaction region formed in the reaction mixture injected into the reaction vessel at a temperature range for separating double strands of DNA into single stranded DNA molecules, respectively; The second reaction zone formed in the reaction mixture injected into the reaction vessel is maintained at a temperature range for forming partial double strands by binding complementary primers to specific sites of the single stranded DNA molecule, And a constant temperature maintaining means for maintaining a third reaction region formed in the reaction mixture injected into the reaction vessel in the partial double strand at a temperature range for synthesizing the primer extension product by the DNA polymerization reaction. An immobilized die polymerase is positioned in the third reaction region, and a relatively high temperature region of the first region, the second region, and the third region is located at a lower height than a region having a relatively low temperature, and In the reaction vessel so that the DNA sequence can be amplified The base sequence amplification apparatus using the immobilized die polymerase, characterized in that the die is repeatedly moved at least once by thermal convection between the first reaction zone, the second reaction zone and the third reaction zone. The method according to claim 7 or 8, wherein the immobilized enzyme of the immobilized DNA polymerase is a Klenau fragment of Escherichia coli DNA DNA polymerase I, E. coli DNA DNA polymerase I having a function of replicating a sequence complementary to the template DNA And a T4 diene polymerase, a tack diene polymerase and a fragment having an activity thereof. A nucleotide sequence amplification apparatus using an immobilized diene polymerase. 8. The nucleotide sequence amplification apparatus according to claim 7, wherein the moving means is an electric field generating means. The nucleotide sequence amplification apparatus using immobilized DNA polymerase according to claim 7, wherein the moving means is a pressure difference generating means. The nucleotide sequence amplification apparatus using immobilized die polymerase according to claim 7, wherein the moving means is a stirring means.

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