KR101424192B1

KR101424192B1 - Composition for Hot-start PCR Comprising Blocking Oligonucleotide

Info

Publication number: KR101424192B1
Application number: KR20110017226A
Authority: KR
Inventors: 이준희; 최소라; 김남일; 박한오
Original assignee: (주)바이오니아
Priority date: 2011-02-25
Filing date: 2011-02-25
Publication date: 2014-07-28
Also published as: KR20120097793A; WO2012115464A3; WO2012115464A2

Abstract

The present invention relates to a composition for hot start PCR, and more particularly, to a composition for PCR comprising a reaction buffer solution, MgCl ₂ , four kinds of dNTPs, and a DNA polymerase, wherein the hydroxyl group at the 3 'end is blocked, The present invention relates to a composition for hot start PCR, which comprises a blocking oligonucleotide having a base sequence. Since the composition for hot start PCR of the present invention dissociates the blocking oligonucleotide at a temperature lower than the melting temperature of the primer, primer-dimer formation at a low temperature or the attraction of the PCR amplification product due to nonspecific reaction is suppressed, The result can be obtained.

Description

[0002] Composition for Hot-start PCR Comprising Blocking Oligonucleotide [

The present invention relates to a composition for a hot start PCR comprising a blocking oligonucleotide (sometimes referred to as "blocking oligonucleotide" hereinafter), and more particularly, to a composition for blocking a 3 'end of a conventional PCR composition, And further comprising a predetermined concentration of a blocking oligonucleotide having a complementary base sequence.

The specificity in the PCR reaction is determined by the high stringency of primers that bind to the target gene sequence. However, all the components required for gene amplification during PCR are mixed at room temperature before the initial denaturation of the gene, so that priming of low string expression occurs under this condition. Priming of these low stringency primers, along with the complexity of the target DNA sequence, low reaction temperature, etc., is a major factor in increasing nonspecific amplification, since the activity of the polymerase is maintained at low temperatures, And nonspecific amplification consumes primers and other required components of limited concentration in repeated PCR, resulting in a competitive inhibitor. Nonspecific amplification has been pointed out as a major problem especially in performing low number of copy number target DNA detection, amplification of low concentration DNA sample, and multiplux PCR using various primers simultaneously.

"Hot start PCR" has been developed as a result of efforts to solve these problems in general PCR. Hot start PCR is one of the methods for obtaining more accurate PCR products. By mixing the reactants at high temperature conditions, priming of low stringency occurring at room temperature and thereby preventing oligomerization of nonspecific primers, . &Lt; / RTI >

The simplest method of performing hot start PCR is to open hot reaction tubes and add the missing components, but this method has the disadvantage of exposing the reaction mixture to contamination, forming aerosols, and causing evaporation. Another hot start PCR method is to create a necessary component, for example, a physical blocking film between the primer and the template. Among these barrier films, paraffin wax has usually been used as an electrophysical barrier film. That is, when the paraffin wax is covered on the reaction mixture and the wax is hardened, a reagent (starting reagent) is added thereto, and then mineral oil is added and the temperature of the gene amplifying device is raised to 70 to 90 ° C., And the starting reagent are mixed, and the PCR reaction proceeds. As such, since the wax dissolution and the mixing of the essential components occur precisely at high temperatures, only the priming of the high string expression can be performed well, the amplification of various gene samples can be performed at the same time, And the storage of the reaction mixture is easy. However, there is a problem that the above method is troublesome to handle and takes a long time (Non-Patent Document 1).

In addition, since the mineral oil used as the evaporation blocking film contaminates the PCR sample and appears as a non-DNA-containing band, it becomes difficult to quantitatively analyze the PCR data. Therefore, an improvement has been made to use only paraffin beads for hot start PCR. Since the paraffin beads form a solid layer at 55 ° C or lower, the primer and the template DNA are mixed only when the primer and the template DNA are not mixed at room temperature and the reaction temperature is higher than the melting point of paraffin, Can be increased. In addition, the use of paraffin beads has been preferred as a method for improving hot start PCR because paraffin is present at the bottom of a microcentrifuge tube and is easy to collect and sample at low cost (Non-Patent Document 2).

Another hot start PCR method is to use petroleum jelly such as AmpliGrease instead of paraffin wax. This method is also analogous to the method of using wax and oil, in which the reaction mixture is separated into two layers, a bottom mix and a top mix, with petroleum jelly therebetween, Is different from the method using a conventional wax in that petroleum jelly starts to melt at a lower temperature condition (melting point of about 50 ° C) than wax to form a solution and is not hardened again by cooling (Non-Patent Document 3). However, this method has a limitation that it can be applied only to a small volume sample because the mixing efficiency does not occur due to the difference in density between the upper and lower mixture when the amount of the sample is large.

In addition, various organic solvents (PEG, DMSO, Glycerol, etc.) used as a PCR promoter may be used in combination with a reaction bead prepared by coating a reaction mixture dried with a trehalose solution with wax (Non-Patent Document 4) To increase the efficiency of hot start PCR (Non-Patent Document 5).

Currently, the most widely used hot start method is to induce hot start PCR reaction by controlling the activity of DNA polymerase using an antibody against DNA polymerase. That is, at a low temperature (20 ° C to 40 ° C), the anti-DNA polymerase antibody binds to the DNA polymerase to inactivate the function of the DNA polymerase, and at a high temperature (70 ° C to 80 ° C) The DNA polymerase which has been activated by the destruction of the antibody participates in the polymerization reaction, so that the production of primer-dimer or nonspecific product can be reduced and the yield and sensitivity of PCR can be increased (Non-Patent Document 6). The primer dimer and nonspecific reaction generally occur from the mixing of the PCR reaction to the DNA denaturation step, and also occur generally in the DNA denaturation step in the PCR reaction, the primer annealing step, the primer annealing step, and the DNA polymerase activity step .

In addition, there is an example in which a new DNA polymerase (AmpliTaqGold, Perkin-Elmer, USA) is applied to human platelet alloantigen typing. Since this polymerase is activated only by heat, hot start PCR is possible, It has been reported that the amplification can be prevented (Non-Patent Document 7).

In addition, there is a method of inducing hot start PCR reaction by suppressing the reaction of Mg ² ⁺ at low temperature by using pyrophosphate and pyrophosphatase. As fatigue phosphatase and sikineunde Looking at the hot start PCR mechanism of pyrophosphate, phosphate blocks the Mg ² ⁺ ions in the reaction components for PCR reaction fatigue, the Mg ²⁺ ions are components required when the DNA polymerase exhibit activity, As a result, the activity of the DNA polymerase gradually decreases. At this time, since pyrophosphate and Mg ² ⁺ ions have high affinity, when pyrophosphate is added to the PCR mixture, Mg ² ⁺ ions required for performing the PCR reaction are captured by pyrophosphate, and the PCR reaction is stopped The reaction by the DNA polymerase is no longer carried out. As a result, even if the priming of the low string expression occurs, the resulting nonspecific amplification product is not generated. Inhibition of the reaction by added pyrophosphate can be resolved by adding a certain amount of pyrophosphatase to the PCR mixture, which slowly dissociates the bond between pyrophosphate and Mg ² ⁺ . At this time, pyrophosphatase removes the bond between pyrophosphate and Mg ² ⁺ to generate free Mg ²⁺ ions, so that the PCR reaction inhibited by pyrophosphate can proceed again. Fatigue phosphatase Since stable heat at a specific temperature (above 70 ℃), fatigue phosphatase and DNA polymerase by expressing the enzyme activity under the same temperature conditions, the glass by fatigue phosphatase Mg ² ⁺ ion broken away is a DNA polymerase To appropriately amplify the desired product by using the PCR reaction to inhibit the production of nonspecific amplification products (Patent Document 1).

In addition, a method using DNA plasmids binding to DNA polymerase has been developed (Patent Document 2). As for the mechanism of action, DNA plasmids are used as a substrate for DNA polymerase to bind with DNA polymerase. The bound DNA polymerase stops the polymerization reaction, and the polymerization reaction does not occur at the temperature before the DNA aptamer is denatured. When the reaction temperature rises, the DNA aptamer is denatured, and thus the DNA polymerase is activated to initiate the polymerization reaction. However, the above method has an advantage that the PCR reaction can be carried out immediately, but it is possible to cause a nonspecific reaction due to the long length of the extramammary.

There is also known a PCR method using a DNA polymerase inhibitor consisting of a nucleic acid sequence that partially forms a double bond, which plays a role of preventing DNA polymerase activity by binding to a DNA polymerase at a specific temperature or lower Literature 3).

As described above, many studies have been conducted on hot start PCR, and although it has recognized the necessity, it has not been applied to various experiments because of economical efficiency and various limited factors in use, and also DNA polymerase, Antibody, and pyrophosphatase are all proteins, so that they require a lot of cost and time in terms of stability and supply and demand. In addition, since the above conventional methods have a fundamental limitation in preventing extension of the primer through formation of a primer-dimer or the like, it is somewhat insufficient to prevent generation of a nonspecific amplification product due to extension of the primer there was.

Korean Patent Publication No. 10-0292883 U.S. Patent No. 6,183,967 U.S. Published Patent Application No. 2007/0212704

DAquila et al., Nucleic Acids Res., 19: 3749, 1991 Wainwright, L.A. et al., Biotechniques, 14: 34-36, 1993 Horton, R.M. et al., Biotechniques, 16: 42-43, 1994 Kaijalainen, S. et al., Nucleic Acids Res., 21: 2959-2960, 1993 Pomp, D. et al., Biotechniques, 10: 58-59, 1991 Sharkey D.J. et al., Bio / Technology, 12: 506-509, 1994 Chen, D.F. et al., Vox Sang 72: 192-196, 1997

The present invention has been made to overcome the problems of the prior art as described above, and it is an object of the present invention to provide a composition for PCR which can be obtained by further adding a blocking oligonucleotide having a predetermined concentration having a base sequence complementary to a primer, It is an object of the present invention to provide a composition for hot start PCR which can suppress the occurrence of primer-dimer formation at temperature or a smeared band of PCR amplification product due to nonspecific reaction to obtain more accurate PCR result.

The present invention provides a composition for hot start PCR comprising a blocking oligonucleotide.

The composition for hot start PCR of the present invention is characterized in that a composition for PCR comprising a reaction buffer solution, MgCl ₂ , four dNTPs and a DNA polymerase is added to a blocking oligonucleotide having a base sequence complementary to the primer, Nucleotides. The composition for PCR may further include a primer, a probe, a template nucleic acid, a fluorescent dye, a reverse transcriptase and the like, if necessary. In the present invention, the term "template nucleic acid" is used to include without limitation DNA, RNA, hybrid of DNA and RNA, and the like.

In the present invention, the blocking oligonucleotide is characterized in that the number of base sequences is smaller than that of a primer having a complementary base sequence. Thus, when the nucleotide sequence of the blocking oligonucleotide is smaller than the nucleotide sequence of the primer, the melting temperature of the primer (or the template nucleic acid) -blocking oligonucleotide bond becomes lower compared to the melting temperature of the primer-template target DNA binding . Therefore, although the primer and the blocking oligonucleotide are bonded with a double helix at a low temperature, the blocking oligonucleotide having a somewhat lower denaturation temperature is disassociated before the annealing temperature of the primer, It has no effect at all and can be usefully used for hot start PCR. The method using such a blocking oligonucleotide can be applied to the probe in the same manner. According to one embodiment of the present invention, the blocking oligonucleotide preferably has a melting temperature lower than the primer by 1 ° C or more, preferably 2 ° C or more, and is preferably at least 25 ° C, .

In addition, the blocking oligonucleotide is characterized in that the hydroxyl group at the 3 'end is substituted with a substituent other than the hydroxyl group. The DNA polymerization proceeds in such a manner that the hydroxyl group at the 3 'end and the phosphate group at the 5' end are combined. Thus, when the hydroxyl group at the 3 'end is substituted with another substituent, the DNA polymerization reaction does not proceed any more. According to one embodiment of the present invention, the above-mentioned substituents include C3-Space represented by Chemical Formula 1, C6-space represented by Chemical Formula 2, C12-space represented by Chemical Formula 3, C18- 5, a phosphate represented by the formula (6), digoxigenin (DIG), thiol, and the like may be used, but the present invention is not limited thereto.

Also, according to one embodiment of the present invention, the first base at the 5 'end of the blocking oligonucleotide forms a complementary bond with the first base at the 3' end of the primer that binds complementarily to the blocking oligonucleotide , It is preferred that one or more bases at the 5'end of the blocking oligonucleotide remain unpaired by forming a complementary bond with the second and subsequent bases of the 3'end of the primer. That is, it is preferable that the base at the 5 'end of the blocking oligonucleotide forms a complementary bond to the base at the 3' end of the primer. If at least one base at the 5 'end of the blocking oligonucleotide remains unbound, DNA polymerization will occur from the 3' end of the primer to the first base at the 5 'end of the blocking oligonucleotide, As a result, the length of the primer can be further increased. When such a primer having an increased length is present in the reaction solution, it is not preferable because a non-specific amplification product resulting from primer-dimer formation, which has been a problem in the prior art, may be further increased.

The blocking oligonucleotide may be specifically bound to either a forward primer or a reverse primer for a pair of primers having a complementary sequence or may be specifically bound to both the forward primer and the back primer .

In the present invention, the reaction buffer solution is preferably 10 mM TrisHCl, 40 mM KCl, pH 9.0. The four dNTPs represent dATP, dTTP, dGTP and dCTP. The DNA polymerase can be any known DNA polymerase without limitation. Among them, a polymerase having 5 '-> 3' exonuclease activity, a polymerase having 3 '-> 5' exonuclease activity Enzymes and 5 '-> 3' exonuclease activity and 3 '-> 5' exonuclease activity can be used alone, or the DNA polymerase can be used in combination. Examples of the polymerase having the 5 '-> 3' exonuclease activity include Taq DNA polymerase and examples of the polymerase having 3 '-> 5' exonuclease activity include Pfu DNA polymerase or TLA DNA polymerase Examples of polymerases without enzyme, 5 '-> 3' exonuclease activity and 3 '-> 5' exonuclease activity include, but are not limited to, Top DNA polymerase. The DNA polymerase can be used in the PCR composition in a concentration of 0.1 to 10 U (unit), preferably 0.5 to 2 U, most preferably 1 U.

Meanwhile, the composition for hot-start PCR of the present invention may further include a non-reactive dye substance for convenience of experiment, prevention of contamination by PCR reaction product, stabilization of DNA polymerase and dNTP and improvement of reactivity have. The non-reactive dye material should be selected from materials that do not affect the PCR reaction. Examples of materials that satisfy these conditions include bromophenol blue, xylene cyanole, bromocresol red, cresol red and the like. The non-reactive dye material may be contained in an amount of 0.0001 to 0.01% by weight, preferably 0.001 to 0.005% by weight, and more preferably 0.001 to 0.003% by weight based on the total composition . When the non-reactive dye material is added in an amount of less than 0.0001% by weight, there is a problem that the concentration of the dye is low during agarose gel electrophoresis for analysis after the PCR reaction and it is difficult to visually observe the movement of the sample. When the reactive dye material is added in an amount exceeding 0.01% by weight, a high concentration of the water-soluble dye may act as a reaction inhibitor during the PCR reaction. In addition, there is a problem that after the sedimentation of the agarose gel, the movement of the sample during electrophoresis may be interrupted.

In addition, the composition for hot start PCR of the present invention may contain a reverse transcriptase for synthesizing cDNA as required. Thus, in the case of reverse transcription-PCR containing reverse transcriptase, it is preferable to add a blocking oligonucleotide to the sense primer of the amplified RNA in the composition for hot start PCR of the present invention.

DNA polymerase is an enzyme that binds to the 3 'terminal part of a partial double helix structure with a single end at the 5' end to cause a polymerization reaction. In the present invention, blocking oligos were designed in consideration of the characteristics of such DMA polymerase. In the present invention, by designing the 5'end of the blocking oligonucleotide to be complementary to the 3'end of the primer, it is possible to prevent the primer from further reacting nonspecifically and to prevent the primer from hybridizing to another nucleic acid to elongate the base. In addition, the 3'end of the blocking oligonucleotide was made shorter than the 5'end of the primer so that the DNA polymerase was adhered well, and the 3'hydroxyl group was blocked with another substituent to prevent the blocking oligo from stretching. Thus, by simply adding the blocking oligonucleotide to the reaction product, the DNA polymerase is attached to the double helix of the primer and the blocking oligonucleotide and can no longer perform a nonspecific reaction, so that the extension reaction of the primer can be prevented at room temperature do.

The blocking oligonucleotide used in the hot start PCR composition of the present invention is an oligonucleotide having a nucleotide sequence complementary to a primer or a probe. The oligonucleotide has a shorter length than the primer / probe and has a primer / So that the DNA polymerase recognizes and binds as a substrate, but DNA polymerization does not occur because the 3 'end is blocked. Therefore, it is possible to prevent non-specific PCR products from occurring by suppressing nonspecific polymerization of primers, which may be a problem in a conventional PCR reaction. Therefore, when the composition for hot-start PCR of the present invention is used, unlike the hot start PCR method using an antibody such as a conventionally used antibody, chemical modification, or pyrophosphate, the primer and target template DNA double- Since the polymerase is released immediately after the temperature is lowered, the PCR reaction can be performed immediately without consuming the incubation time for activation.

As for the mechanism of action of the blocking oligonucleotide, forward and reverse primers serve as essential elements in the PCR reaction. The blocking oligonucleotide is an oligonucleotide composed of a complementary base sequence having one to fewer nucleotides than the forward primer and the reverse primer, so that the specificity for the primer is high. Therefore, when the forward / reverse primer and the blocking oligonucleotide are mixed, the blocking oligonucleotide binds to the forward / reverse primer to prevent the forward / reverse primer from acting, thereby preventing primer dimer formation and non-specific reaction . When the temperature reaches the optimum temperature, that is, the melting temperature of the blocking oligomer, the binding between the primer and the blocking oligonucleotide is broken, so that the primer binds to the target template nucleic acid, so that the PCR reaction can be accurately and effectively performed. That is, since the blocking nucleotides are produced in a relatively shorter length than the primer / probe, when the primer reaches the proper temperature at which the primer binds to the template nucleic acid, the blocking nucleotide dissociates from the primer / probe and therefore does not affect when the primer binds to the template (See FIG. 1).

The hot start PCR method using the blocking oligonucleotide of the present invention can be usefully applied in the following cases, but is not limited thereto.

Ⅰ. When there is a drag of the PCR amplification product due to a nonspecific reaction in monoplex PCR reaction.

Ⅱ. When multiple amplification products are generated by nonspecific reactions in a PCR reaction.

Ⅲ. When the efficiency of PCR reaction is lowered by the primer dimer.

IV. When you want to increase PCR efficiency for specific targets with low amplification.

Ⅴ. To identify one or more amplification products (Multiplex PCR).

VI. When you want to improve the efficiency of PCR reaction for specific target in fields such as quarantine and disease diagnosis.

VII. When performing real-time PCR reactions that require specificity.

VIII. When primers with different Tm values are used specifically.

Ⅸ. When a nonspecific amplification product occurs in a One-Step RT-PCR reaction.

In addition, the composition for hot start PCR of the present invention has the following advantages in comparison with various PCR compositions reported in the past.

1) Since the reaction can proceed according to the general PCR method, long-term pretreatment at a high temperature is not required.

2) It is more economical than conventional PCR composition.

3) Since it prevents the generation of amplification product by priming of low string expression, it can be applied to multiplex PCR reaction using various samples at the same time.

In addition, the present invention provides a hot start PCR method using the hot start PCR composition.

The hot start PCR method is characterized in that a hot start PCR reaction is performed after addition of a blocking oligonucleotide as described above to a composition for ordinary hot start PCR. According to one embodiment of the present invention, the composition for hot-start PCR of the present invention can be used in addition to conventional PCR, such as multiplex PCR, real-time PCR, real-time quantitative PCR, real-time RT / May be used without limitation. "Multiplex PCR" as used herein means to simultaneously amplify one or more multiple DNA targets in one polymerase chain reaction (PCR) mixture.

It is generally difficult to design multiplex PCR conditions that simultaneously amplify two or more target sequences because it is desirable to design an optimal PCR reaction so that both of the two or more target sites can be amplified without nonspecific byproducts . In this case, annealing is required to be performed at a sufficiently high temperature in order to obtain a perfect template DNA-primer match, and it is also necessary to design such that annealing does not occur between two or more pairs of primers. In the case of multiplex PCR, the PCR efficiency is lowered by nonspecific reaction and primer dimer when there is no hot start function, and it is difficult to obtain accurate results by nonspecific amplification products. Therefore, hot start method is widely applied, . The hot start PCR method of the present invention is ideal for optimization of multiplex DNA amplification by adding a blocking oligonucleotide having a base sequence complementary to each target primer to improve the specificity of amplification.

In one embodiment of the invention, the amplified products from each of the target nucleotide sequences have been designed differently for later analysis, so that the amplification products of the multiplex target nucleotide sequence can be easily analyzed through size discrimination. Size fractionation comparisons may be performed through a variety of known methods, i.e., electrophoresis through a polyacrylamide gel matrix or an agarose gel matrix. By using the blocking oligonucleotides of the present invention, background problems as well as non-specificity problems of conventional multiplex PCR methods can be eliminated.

The advantage of multiplex PCR is that many diseases can be analyzed simultaneously in the same reaction. Theoretically, there are no limitations on the number of analytes that can be analyzed at the same time, but in practice, about 20 are the upper limit because they depend on the size difference required for analysis and how the amplified product can be analyzed. The method of the present invention can be applied to diagnosis of genetic and infectious diseases, sex determination, genetic association analysis, and research on crime science.

As described above, in contrast to the conventional hot start PCR method using an antibody, chemical modification, or pyrophosphate, the composition for hot start PCR of the present invention has a blocking oligonucleotide dissociated below the melting temperature of the primer Therefore, it is advantageous in that more accurate PCR result can be obtained by suppressing primer-dimer formation at a low temperature or a drag phenomenon of a PCR amplification product due to nonspecific reaction.

1 is a schematic diagram showing a nonspecific reaction inhibitory effect by a blocking oligonucleotide.
FIG. 2 shows non-specific reaction inhibitory effect according to the length of the blocking oligonucleotide. As a positive control, A and H were confirmed to inhibit nonspecific reaction by hot start function using a product having a hot start function. C, G, and I to M were determined as 18 ° C, 26 ° C, 34 ° C, 37.4 ° C, 40.9 ° C, 46.1 ° C, 48.6 ° C and 50.5 ° C, respectively, Lane 1 to lane 3 show the results obtained by amplifying three different target bases, and lane M indicates the case where the DNA size is differentiated This is a 100 bp DNA Ladder that can be used.
FIG. 3 is a graph showing a non-specific inhibitory effect of blocking oligonucleotides according to the concentration used; A is a result of using a product having no hot start function as a negative control, and B is a positive control, And lane 1 to lane 3 represent the results obtained by adding three oligonucleotides used in Fig. 2, i.e., 5 pmole, 10 pmole, 15 pmole, 20 pmole, 25 pmole and 30 pmole of C to H, And lane M represents a 100 bp DNA ladder that can distinguish DNA size.
FIG. 4 shows the results of confirming the nonspecific reaction inhibitory effect by adding an oligonucleotide blocking the forward or reverse primer, wherein A is a result of using a product having no hot start function as a negative control, and B is a positive control, C and D are the result of addition of a blocking oligonucleotide having a nucleotide sequence complementary to that of the forward primer, and E and F are the results obtained by adding a blocking oligonucleotide having a complementary nucleotide sequence of the reverse primer G is the result of addition of a blocking oligonucleotide having a nucleotide sequence complementary to the forward and reverse primers, lane 1 to lane 3 are the results of amplifying the three target bases used in FIG. 2, This is a 100 bp DNA Ladder that can distinguish DNA size.
FIG. 5 is a graph showing the results of using a product having a hot start function as a positive control group and B to G in the case of using amines, phosphates, C3- lane 1 to lane 3 represent the results of amplifying the three target bases used in Fig. 2, lane M represents the DNA size, A distinguishable 100 bp DNA Ladder is shown.
Fig. 6 shows the effect of inhibiting nonspecific reaction depending on presence or absence of a blocking oligonucleotide in a product which does not have a hot-start function in case (A) and (B) Lane 1 to lane 3 show the results of amplifying three different target bases, and lane M shows a 100 bp DNA Ladder that can distinguish DNA size.
FIG. 7 shows the result of multiplex PCR reaction according to addition of blocking oligonucleotide. It was confirmed that PCR reaction inhibition was confirmed by adding a blocking oligonucleotide to a product having a hot start function, and addition of a blocking oligonucleotide to a product without a hot start function Lane 1 and lane 2 show the cases in which the blocking oligonucleotide was not added and the case in which the blocking oligonucleotide was added, respectively.
Figure 8 shows the nonspecific inhibitory effect of blocking oligonucleotide addition in the One-Step RT / PCR reaction. Lane 1 is the result of RT / PCR product with hot start function, lane 2 is hot start function Lane 3 is the result of adding a blocking oligonucleotide to a product without hot start capability, and lane M represents a 100 bp DNA ladder to distinguish DNA size.
FIG. 9A shows primer dimers and nonspecific inhibitory effects upon addition of a blocking oligonucleotide in a real-time PCR reaction. In the PCR Premix solution containing Taq DNA polymerase, a fluorescent material (Sybrgreen I) and a base complementary to the primer (Experimental group) with addition of a blocking oligonucleotide having a sequence and a fluorescent primer solution containing only Taq DNA polymerase (control group), fluorescence measurement curves obtained by real-time PCR reaction , The horizontal axis represents the PCR reaction cycle, the vertical axis represents the measured fluorescence value according to the reaction cycle, the line I represents the fluorescence detection curve for the control group, and the line II represents the fluorescence detection curve for the experimental group. 9 (B) is a graph showing the result of forming a melting curve by using a fluorescence measurement curve, wherein the horizontal axis represents the temperature change and the vertical axis represents the measured fluorescence value according to the temperature increase, and line I represents the result of the melting curve creation And line Ⅱ shows the result of creating the melting curve for the experimental group.

Hereinafter, the present invention will be described in more detail by way of examples.

However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1. Inhibitory effect of nonspecific amplification product formation on blocking oligonucleotide Tm value

In order to confirm the inhibitory effect of nonspecific amplification products depending on the length of blocking oligonucleotides having a nucleotide sequence complementary to a primer, which is a necessary element of the PCR reaction, PCR products were compared after addition of blocking oligonucleotides of various lengths. For this, primers were designed and synthesized as shown in Table 1, and human genomic DNA (10 ng), each primer (10 pmoles) and a primer (2 pm to 15 pm) were added to a PCR premix (PreMix) The PCR reaction was carried out by adding blocking oligonucleotides (10 pmoles each) having various lengths with a low Tm value. PCR was performed 30 times at 95 ° C for 20 seconds, at 55 ° C for 40 seconds, and at 72 ° C for 60 seconds. The pre-denaturation and final elongation were 5 minutes at 94 ° C, 5 minutes at 72 ° C Respectively. The product obtained in the PCR reaction was electrophoresed on an agarose gel together with a DNA molecular weight marker, stained with ethidium bromide, and a DNA band amplified by a PCR reaction was photographed with a Polaroid camera. AccuPower Hotstart PCR Premix with hot start function, Bioneer Co., Ltd.), Hot start functionless PCR primer (AccuPower PCR Premix PCR) was used as a negative control, The PCR reaction was performed by adding blocking oligonucleotides. The results are shown in Fig.

2, lanes 1, 2 and 3 show results using primer pairs each having a base sequence of P55 / P63 (447 bp), P55 / P73 (1,082 bp) and P55 / P83 to be. A is the result of using a hot start PCR premix with a hot start function as a positive control and B is a result of using a PCR premix without a hot start function as a negative control and C, D, E, F, G, I, J, K, L, and M are the result of performing the PCR reaction by sequentially adding the instant oligonucleotides to the negative control group in order of decreasing length (i.e., in the sequence of SEQ ID NO: 5 to SEQ ID NO: 14). Lane M represents a 100 bp DNA Ladder (Bioneer Co., Ltd.) capable of distinguishing DNA size.

domain size Tm value order SEQ ID NO: P55 23mer 55 ° C CTC TTC CTA CAG TAC TCC CCT GC One P63 24mer 63.3 DEG C GGC CAC TGA CAA CCA CCC TTA ACC 2 P73 23mer 60.3 DEG C CAA GTG GCT CCT GAC CTG GAG TC 3 P83 25mer 60.1 DEG C GTC CTG CTT GCT TAC CTC GCT TAG T 4 P55
Blocking oligo
3 'Amine modification 5mer 18 ℃ GCA GG 5 7mer 26 ℃ GCA GGG G 6 10mer 34 ℃ GCA GGG GAG T 7 15mer 37.4 DEG C GCA GGG GAG TAC TGT 8 17mer 40.9 DEG C GCA GGG GAG TAC TGT AG 9 18mer 46.1 DEG C GCA GGG GAG TAC TGT AGG 10 19mer 48.6 ° C GCA GGG GAG TAC TGT AGGA 11 20mer 50.5 DEG C GCA GGG GAG TAC TGT AGG AA 12 21mer 51.9 DEG C GCA GGG GAG TAC TGT AGG AAG 13 22mer 53.9 ° C GCA GGG GAG TAC TGT AGG AAGE 14

As a result, when a blocking oligonucleotide having a nucleotide sequence complementary to that of the primer is added at the time of performing the PCR reaction, the activity of the DNA polymerase is blocked, and the primers for low stringency generated by mixing the components required for gene amplification at room temperature And to thereby reduce the production of nonspecific amplification products (FIG. 2).

Example 2. Nonspecific amplification product formation inhibition effect depending on the concentration of blocking oligonucleotide

The template-specific primer of Example 1 and the 20-mer P55 blocking oligonucleotide of SEQ ID NO: 12 were used to confirm the appropriate concentration of the blocking oligonucleotide. The PCR reaction was carried out in the same manner as in Example 1, except that the template-specific primer was used at a concentration of 20 pmoles and the blocking oligonucleotide was used at a concentration of 5 pmoles to 30 pmoles. The results are shown in Fig.

3, lanes 1, 2 and 3 are the result of using primer pairs having the nucleotide sequences of P55 / P63 (447 bp), P55 / P73 (1,082 bp) and P55 / P83 (1,296bp) . A is the result of using a PCR primer with no hot start function as a negative control, B is the result of using a hot start PCR premix with a hot start function as a positive control, and C, D, E and F are results of using the negative control The blocking oligosaccharides were added in the order of low concentration and high concentration to perform PCR reaction.

As a result, in the PCR reaction, nonspecific reaction and resulting smeared band of the PCR amplification product were observed when the PCR reaction without the hot start function as the negative control group was performed, whereas the hot start It was confirmed that PCR amplification products with more accurate and clean PCR products were produced when performing functional PCR reaction. Also, when blocking oligonucleotides at various concentrations are added to the negative control group, the same effect as that of the hot start PCR reaction can be obtained. When the blocking oligonucleotide is used at the same or lower concentration than the template DNA target primer, the activity of the polymerase is blocked, And to suppress the nonspecific reaction effect (FIG. 3).

Example 3. Forward and / or reverse Primer and Inhibitory Effect of Blocking Oligonucleotides Having Complementary Nucleotide Sequence on Nonspecific Amplification Products

In principle, the PCR reaction can not occur unless primers of any one of the forward and reverse primers are present. Therefore, the effect on the nonspecific reaction was confirmed after the addition of the blocking oligonucleotide blocking any one or more of the forward or reverse primers of the target primer. To this end, PCR reaction (A) without hot start function as a negative control, PCR reaction (B) with hot start function as a positive control, reverse blocking oligonucleotide added to the negative control (C and D) (E and F) added with blocking oligonucleotides, or both forward and reverse blocking oligonucleotides added) G) was subjected to PCR reaction in the same manner as in Example 2. At this time, the primer and the blocking oligonucleotide were used at the same concentration of 10 pmole.

As a result, non-specific amplification products were confirmed in the negative control group without hot start function (A), whereas non-specific amplification products could not be confirmed in the positive control group with hot start function (B). It is also possible to suppress the nonspecific amplification reaction by adding either one instant blocking nucleotide in the forward direction (C and D) or the reverse direction (E and F), or adding both instant blocking nucleotides (G) It was confirmed that the DNA banding phenomenon caused by the product did not occur (Fig. 4).

Example 4. Nonspecific reaction inhibition effect by various kinds of modified blocking oligonucleotides

The hot start effect of the blocking oligonucleotide was confirmed after applying various substituents to the 3 'end of the blocking oligonucleotide. The substituents used are shown in Table 2 below, and the PCR reaction was carried out in the same manner as in Example 2. The results are shown in Fig.

In Fig. 5, lanes 1, 2 and 3 show results using primer pairs each having a base sequence of P55 / P63 (447 bp), P55 / P73 (1,082 bp) and P55 / P83 to be. A, B, C, D, E, F, and G were negative control, and the PCR primes lacking the hot start function were used as the positive control. This is the result of PCR reaction using an instant blocking oligonucleotide. Lane M is a 100 bp DNA Ladder that can distinguish DNA size.

number Types of deformation constitutional formula One 3 'amine

2 3 'phosphate

3 3 'C3-Space

4 3 'C6-Space

5 3 'C12-Space

6 3 'C18-Space

As a result, it was confirmed that a nonspecific amplification reaction can be inhibited even when a chemical substance capable of blocking a DNA polymerase is introduced at the 3 'side of a blocking oligonucleotide having a nucleotide sequence complementary to the primer (FIG. 5) .

Example 5. Nonspecific reaction inhibition effect of blocking oligonucleotide under severe conditions that may cause nonspecific reaction

The result (Ⅱ) of the hot start PCR reaction using the blocking oligonucleotide and the result (Ⅰ) of the general PCR reaction without the blocking oligonucleotide as the control group were compared. For this, PCR was carried out on the hot-start PCR reaction mixture containing the blocking oligonucleotide prepared in Example 2 under the same conditions as in Example 2, and a part of the PCR reaction was carried out at 37 ° C for 1 hour The PCR reaction was carried out under the same conditions as in Example 2. The results are shown in FIG. The nucleotide sequence pairs of the template-specific oligonucleotides used are shown in Table 3.

name primer order SEQ ID NO: ABCA3 (463) Forward primer GCC CAT CTT ACA TCC TCT CTC 15 Rear primer CCA GCA CCT AAT CAC AGT CAG 16 F-blocking oligo GAG AGA GGA TGT AAG 17 ABCB1 (413) Forward primer TTC AGA ATG GCA GAG TCA AGG 18 Rear primer TTA GCA AGG CAG TCA GTT ACA G 19 F-blocking oligo CCT TGA CTC TGC CAT 20 SLC29A2 (357) Forward primer TTC ATC ATC ATC AGG AGC AGA G 21 Rear primer TCC TTC CAA GAG CCT CAA TTA G 22 F-blocking oligo CTC TGC TCC TGA TGA T 23

As a result, in the PCR reaction using the instant hot start PCR reaction mixture, a clean amplification product was produced with no nonspecific PCR amplification product as compared with the control (Fig. 6A). In addition, the instant hot start PCR reaction mixture using the control and blocking oligonucleotides was reacted at 37 ° C for 1 hour and the PCR reaction was carried out. As a result, a nonspecific amplification product was observed in the control group irrespective of the reaction at 37 ° C for 1 hour , The amplification efficiency was decreased regardless of the harsh temperature condition (reaction at 37 ° C for 1 hour) in the PCR reaction using the blocking oligonucleotide, and the DNA band caused by the nonspecific amplification product did not occur 6, B). From the above results, it can be seen that, when an instant hot start PCR reaction comprising a blocking oligonucleotide is performed, generation of nonspecific amplification products can be effectively suppressed under normal temperature conditions as well as harsh conditions in which nonspecific amplification products are frequently generated Respectively.

Example 6. Hot start PCR How to Multiplex PCR Application to

In order to confirm whether or not the composition for hot start of the present invention can be applied to multiplex PCR, six primer pairs for six target nucleotide sequences and a blocking oligonucleotide for the primers were added, followed by primer annealing , Primer extension and denaturation. The nucleotide sequences of the primers and the blocking oligonucleotides used in this Example are shown in Table 4. An instant hot start PCR reaction with blocking oligonucleotides was performed in the PCR premix without hot start, and a hot start PCR premix was used as a control. Human genomic DNA (100 ng and 10 ng) was used as a template. The results are shown in Fig.

In FIG. 7, lane 1 shows the results obtained without blocking oligonucleotides, and lane 2 shows the results of PCR in which 10 pmoles of the blocking oligonucleotide was added at the same concentration as that of the template target primer.

name primer order SEQ ID NO: ABCA3 (463) Forward primer GCC CAT CTT ACA TCC TCT CTC 24 Rear primer CCA GCA CCT AAT CAC AGT CAG 25 F-blocking oligo GAG AGA GGA TGT AAG 26 SLC29A2 (357) Forward primer TTC ATC ATC ATC AGG AGC AGA G 27 Rear primer TCC TTC CAA GAG CCT CAA TTA G 28 F-blocking oligo CTC TGC TCC TGA TGA T 29 ABCC10 (288) Forward primer CCC ATA GGC TCA ACA CGA TCC 30 Rear primer TGG GGA AGT GTG GAG AGG TAG 31 F-blocking oligo GAT CGT GTT GAG CCT AT 32 ABCGl (258) Forward primer GAC CGA CGA CAC AGA GAC TC 33 Rear primer CTA AGG AGC GAC TGG ACT GAG 34 F-blocking oligo GAG TCT CTG TGT CG 35 ABCC5 (205) Forward primer GGA CAG GTG GTG GAG TTT GAC 36 Rear primer AAA GGC AAG GTT TCG GTA GGA G 37 F-blocking oligo GTC AAA CTC CAC CAC 38 ATP7B (161) Forward primer TCT GAC CTT CAC CTT GGA TGG 39 Rear primer GGA CAC AAT TAC TGA CGG ACA G 40 F-blocking oligo CCA TCC AAG GTG AAG 41

As a result, it was confirmed that the PCR reaction of the hot start PCR premix was not inhibited even when the blocking oligonucleotide was added. In addition, in the case of a PCR primer without a hot-start function, when a blocking oligonucleotide is added, all target sequences are amplified or background problems are eliminated, in contrast to a case in which some target sequences are not amplified or weakly amplified, It was confirmed that the PCR reaction could be realized (Fig. 7).

Example 7. One - step RT / PCR Inhibition of nonspecific reaction by blocking oligonucleotide in reaction

The inhibitory effect of blocking oligonucleotide on nonspecific reaction was confirmed in one-step RT / PCR reaction. For this purpose, 100 ng of human total RNA was used as the template RNA, and the target gene was a GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor). The target primer and the blocking oligonucleotide used in Table 5 were used. As a control group, AccuPower Hotstast RT / PCR Premix (Bioneer) and hot-start AccuPower RT / PCR Premix (Bioneer) were used. Hot-start AccuPower RT / The PCR reaction was performed by adding a blocking oligonucleotide having a nucleotide sequence complementary to the forward primer to the PCR primer. The results are shown in Fig.

In Figure 8, lane M is the DNA size marker, lane 1 is the result using the AccuPower Hotstart RT / PCR premix, lane 2 is the result using the AccuPower RT / PCR premix and lane 3 is the instant Lt; / RTI > addition of oligonucleotides.

name primer Length Tm (占 폚) order SEQ ID NO: CM-CSF 502 Forward primer 20mer 55 TGT TCG TGC ACA TTT CGT GA 42 Rear primer 20mer 54 GCT TCT GAT AGG TCC TGG GC 43 F-blocking oligo 17mer 40.8 GCT TCT GAT AGG TCC TG 44 CM-CSF 501 Forward primer 20mer 54 GCC CAG GAC CTA TCA GAA GC 45 Rear primer 20mer 54 ACA CCC TCT GGG TCT CAG GT 46 F-blocking oligo 17mer 48.7 TCA CGA AAT GTG CAC GA 47

As a result, it was confirmed that a nonspecific reaction similar to the hot start function was suppressed by adding a blocking oligonucleotide to an AccuPower RT / PCR premix without a hot start function (FIG. 8).

Example 8. Real time PCR By blocking oligonucleotides in the reaction primer Dimer Inhibitory effect

In order to confirm the hot start effect by the PCR composition containing the blocking oligonucleotide in the real-time PCR detection, 2x PCR Premix solution (10 mM TrisHCl pH 9.0, 50 mM KCl, 2.0 mM MgCl ₂ , , 1 U Taq DNA polymerase, 0.01% Tween 20, and a stabilizer) was added at a concentration of 0.3 X per reaction, and 20 pmoles of blocking agent (Greenstar (TM), Bioneer) A composition to which the oligonucleotide was added was prepared. As a control, the fluorescent substance was added to the 2x PCR Premix solution so as to be 0.25x per 20 μl reaction. The fluorescent substance is a fluorescent substance which can be easily analyzed without measuring a fluorescent probe having a specific base sequence by measuring the amount of fluorescence generated by intercalation between double-stranded DNAs generated during template DNA amplification. As the template DNA, 100 ng of total RNA extracted from human cells was added to AccuPower CycleScript RT PreMix (Bioneer Co., Ltd.), reacted at 42 ° C for 1 hour, and then the reverse transcriptase-inactivated cDNA 5 Were used, and the primers and blocking oligonucleotides shown in Table 6 were used at a concentration of 20 pmoles per 20 [mu] l reaction.

name primer Length Tm (占 폚) order SEQ ID NO: GM501 qPCR Forward primer 20mer 55.2 GAC GTC CGC ATC TTG AAT TG 48 Rear primer 20mer 55.2 TTC CCA CGA TTA GGA GCA CA 49 F-blocking oligo 17mer 45.8 CAA TTC AAG ATG CGG AC 50

The real-time PCR reaction was performed using an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer, Inc.). The reaction conditions were a denaturation step at 94 ° C for 5 minutes, a denaturation step at 95 ° C for 10 seconds , The annealing step at 60 캜 for 15 seconds, and the simultaneous reaction of the elongation step were carried out in a total of 45 reaction cycles as one reaction cycle. Thereafter, the dissociation step was performed to prepare a melting curve for the amplification product, thereby confirming PCR reactivity and specificity for the PCR amplification product. The results are shown in Fig.

As a result, the threshold cycle (Ct) for line I and line II was found to be 23.89 C (t) and 29.5 C (t), respectively. The fact that C (t) was drawn on line I rather than on line II showed that only one fluorescent peak was produced in line II containing blocking oligonucleotides as shown in the melting curve, while only the correct amplification product was produced, while blocking oligonucleotides This is because two fluorescence peaks appear, indicating that two amplification products were generated. This means that in addition to the desired PCR amplification product, a smaller size primer dimer amplification product or a nonspecific amplification product was generated. Also, the melting temperature (Tm) for the PCR amplification product was confirmed to be 85.5 ° C in both the experimental group (line II) and the control group (line I), and the melting temperature (Tm) for the primer dimer or non- 75.5 캜.

From the above results, in contrast to the case where the primer dimer is generated in addition to the amplification product in the case of the control group, the instant hot-start PCR method used in the present invention shows a low temperature and a nonspecific reaction during the PCR reaction Inhibited the PCR reaction effectively and restrained the production of nonspecific PCR amplification products, which is an advantage of the hot start method, and confirmed the accurate results.

Attach an electronic file to a sequence list

Claims

Characterized in that it comprises a blocking oligonucleotide having a nucleotide sequence complementary to a primer and having a hydroxyl group at the 3 'end blocked in a composition for PCR comprising a reaction buffer solution, MgCl ₂ , four kinds of dNTPs and a DNA polymerase. Composition for start PCR.

The method according to claim 1,
Wherein the blocking oligonucleotide has a smaller number of base sequences than a primer having a complementary base sequence.

The method according to claim 1,
Wherein the blocking oligonucleotide has a lower melting temperature than the primer having a complementary base sequence.

The method of claim 3,
Wherein the blocking oligonucleotide has a melting temperature lower than that of the primer having a complementary base sequence by at least 1 占 폚.

The method of claim 4,
Wherein the blocking oligonucleotide has a melting temperature of at least < RTI ID = 0.0 > 25 C. < / RTI >

The method according to claim 1,
Wherein the blocking oligonucleotide has a hydroxyl group at the 3 'terminal thereof substituted with a substituent other than the hydroxyl group.

The method of claim 6,
The substituent may be selected from the group consisting of a C3-Space represented by Chemical Formula 1, a C6-space represented by Chemical Formula 2, a C12-space represented by Chemical Formula 3, a C18-Space represented by Chemical Formula 4, an amine represented by Chemical Formula 5, DIG (Digoxigenin), and thiol. &Lt; RTI ID = 0.0 > 11. < / RTI >
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

The method according to claim 1,
Wherein the base at the 5 'end of the blocking oligonucleotide forms a complementary bond to the base at the 3' end of the primer.

The method according to claim 1,
Wherein the blocking oligonucleotide specifically binds to at least one of a forward primer and a back primer.

The method according to claim 1,
Wherein the DNA polymerase is selected from the group consisting of a polymerase having a 5'->3'exonuclease activity, a polymerase having a 3'->5'exonuclease activity and a 5'->3'exonuclease activity and a 3'- Gt; 5 ' exonuclease activity. &Lt; / RTI >

The method according to claim 1,
Lt; RTI ID = 0.0 > 1, < / RTI > further comprising a dye material that is non-reactive with the template nucleic acid.

The method of claim 11,
Wherein the non-reactive dye material is selected from the group consisting of bromophenol blue, xylenecyanol, bromocresol red, and cresol red.

The method according to claim 1,
Lt; RTI ID = 0.0 > 1, < / RTI > further comprising a reverse transcriptase.

A hot start PCR method using the composition for hot start PCR according to claim 1.

15. The method of claim 14,
Wherein said PCR is selected from the group consisting of multiplex PCR, real-time PCR, real-time quantitative PCR, real-time RT / PCR and real-time quantitative RT / PCR.