WO2012115464A2

WO2012115464A2 - Composition for hot-start pcr comprising blocking oligonucleotide

Info

Publication number: WO2012115464A2
Application number: PCT/KR2012/001387
Authority: WO
Inventors: Jun Hee Lee; So Ra Choi; Nam Il Kim; Han Oh Park
Original assignee: Bioneer Corp
Current assignee: Bioneer Corp
Priority date: 2011-02-25
Filing date: 2012-02-23
Publication date: 2012-08-30
Anticipated expiration: 2013-08-25
Also published as: KR20120097793A; WO2012115464A3; KR101424192B1

Abstract

The present invention relates to a composition for hot-start PCR, more precisely a composition for hot-start PCR characteristically prepared by adding the blocking oligonucleotide having the hydroxyl group at 3'-end blocked and the nucleotide sequence complementary to the primer to the conventional PCR composition containing reaction buffer, MgCl₂, 4 kinds of dNTP, and DNA polymerase. The composition for hot-start PCR of the present invention can give more authentic PCR results by inhibiting smear band of PCR amplification product caused by non-specific reaction or primer-dimer formation at low temperature since the blocking oligonucleotide included therein can be dissociated at the lower temperature than melting temperature of the primer.

Description

COMPOSITION FOR HOT-START PCR COMPRISING BLOCKING OLIGONUCLEOTIDE

The present invention relates to a composition for hot-start PCR comprising blocking oligonucleotide (referred as "blocking oligo" hereinafter), more precisely, a composition for hot-start PCR prepared by adding a required amount of blocking oligonucleotide having the 3'-end blocked and the nucleotide sequence complementary to the primer to the conventional PCR composition.

Specificity of PCR is determined by high stringency of a primer binding to a target gene sequence. All the ingredients necessary for gene amplification are mixed at room temperature before predenaturation. Thus, during this mixing, low stringency priming occurs. The polymerase retains its enzyme activity at a low temperature, so when priming occurs, a PCR product might be generated. Therefore, low stringency priming is another critical cause of non-specific amplification along with complexity of target DNA sequence and low reaction temperature. Non-specific amplification consumes primers and other ingredients limited in concentrations while PCR cycles repeat, suggesting that non-specific reaction itself acts like a competitive inhibitor. Non-specific amplification causes a serious problem particularly in detection of a target DNA with a low copy number, in amplification of a DNA sample at a low concentration, and in multiplex PCR using different primers at a time.

As an effort to overcome the said problem of PCR, "hot-start PCR" has been developed. Hot-start PCR is a kind of PCR to obtain purer PCR product, which allows high temperature mixing of each reactant, so that PCR specificity can be increased by preventing low stringency priming frequently occurring at room temperature and oligomerization of non-specific primer.

The simplest way to perform hot-start PCR is to open the hot reaction tube and add necessary ingredients therein. However, this method might exposure the reaction mixture on contamination, form aerosol, and cause evaporation. Another way to perform hot-start PCR is to generate a physical barrier between necessary ingredients, for example between a primer and a template. As an electro-physical barrier, paraffin wax has been generally used. Precisely, a reaction mixture is covered with paraffin wax, and when the wax is hardened, reagents (starting reagents) are loaded thereon, to which mineral oil is added. Then, the temperature of PCR machine is raised to 70℃ - 90℃. Then, the wax is dissolved and the reaction mixture and starting reagents are mixed, leading to PCR. As explained, dissolution of the wax and mixing of the ingredients precisely occur at high temperature. So, according to this method, high stringency priming is facilitated, simultaneous amplification of different DNA samples is also facilitated, and long-term shelf-life of the reaction mixture is secured owing to the wax and oil layer. However, the said method is troublesome and takes a long time (DAquila et al., Nucleic Acids Res., 19:3749, 1991).

In addition, mineral oil used as an evaporation barrier can contaminate PCR samples, which are presented as DNA-not-containing bands, making quantitative PCR dada analysis difficult. Therefore, to improve this method, it has been proposed to use paraffin beads alone for hot-start PCR. Paraffin beads form a solid layer at up to 55℃. So, a primer is not mixed well with a template DNA at room temperature and only when the reaction temperature is higher than meting point of paraffin, a primer is mixed with a template DNA, suggesting that it might increase PCR specificity. Paraffin exists on the bottom of the micro-centrifuge tube, so sample collection is easy and it costs less. Therefore, paraffin beads are expected to be a promising candidate for improving hot-start PCR (Wainwright, L.A. et al., Biotechniques, 14:34-36, 1993).

Another hot-start PCR method is characterized by using petroleum jelly such as AmpliGrease instead of paraffin wax. This method is similar to the above method using wax and oil. Precisely, a reaction mixture is separated into two layers, bottom mix and top mix. Petroleum jelly is added between the two mixes to prevent the two mixes from being mixed at room temperature. Petroleum jelly begins to be dissolved at a lower temperature (approximate melting point: 50℃) than the melting point of wax and is not hardened again by cooling, which is the difference from the conventional method using wax (Horton, R.M. et al., Biotechniques, 16;42-43, 1994). However, this method has disadvantages when the amount of sample is large. That is, when a sample is used massively, bottom mix and top mix are not mixed well because of difference in density between the two mixes. So, this method is only effective in the reaction with a small volume of sample.

Other methods such as using reaction beads prepared by coating a reaction mixture dried over trehalose solution with wax (Kaijalainen, S. et al., Nucleic Acids Res., 21:2959-2960, 1993), or adding different organic solvents (PEG, DMSO, Glycerol, etc) used as a PCR accelerator to a reaction mixture to increase hot-start PCR efficiency (Pomp, D. et al., Biotechniques, 10:58-59, 1991) have been proposed.

The most widely used hot-start method is to induce hot-start PCR by regulating DNA polymerase activity by using anti-DNA polymerase antibody. At a low reaction temperature (20℃∼40℃), anti-DNA polymerase antibody is conjugated with DNA polymerase to inactivate the functions of DNA polymerase, while anti-DNA polymerase antibody is disrupted at a high reaction temperature (70℃∼80℃) so that activated DNA polymerase is added to PCR, suggesting that the production of primer-dimer or non-specific product decreases and accordingly PCR yield or sensitivity increases (Sharkey D.J. et al., Bio/Technology, 12:506-509, 1994). The said primer-dimer or non-specific reaction is induced continuously from the stage of mixing PCR reactants to the stage of DNA degeneration. The said reaction also occurs in the middle of PCR reaction, which is from the stage of DNA degeneration to the stage of primer annealing and from the stage of primer annealing to the stage of activation of DNA polymerase as a whole.

It was reported that the novel DNA polymerase (AmpliTaqGold, Perkin-Elmer, USA) has been applied to human platelet alloantigen typing. At this time, the polymerase is only activated by heat, so that hot-start PCR can be performed and in fact non-specific amplification can be prevented (Chen, D.F. et al., Vox Sang 72:192-196, 1997).

Another method to induce hot-start PCR is to use pyrophosphate and pyrophosphatase to inhibit Mg²⁺ reaction at a low temperature. The mechanism of pyrophosphatase and pyrophosphate for hot-start PCR is as follows: Pyrophosphate blocks Mg²⁺ during PCR. Mg²⁺ is required for the activation of DNA polymerase. Thus, when Mg²⁺ is blocked, DNA polymerase activity is reduced. Pyrophosphate has high affinity to Mg²⁺. So, when pyrophosphate is added to PCR reaction mixture, Mg²⁺ required for PCR is arrested by pyrophosphate and accordingly PCR is stopped, that is the reaction by DNA polymerase does not proceed any more. As a result, even under the condition of low stringency priming, non-specific amplification product is not generated. The reaction inhibited by the added pyrophosphate can be recovered by adding a required amount of pyrophosphatase to the PCR mixture. This enzyme plays a role in slow separation of Mg²⁺ from pyrophosphate. At this time, pyrophosphatase eliminates the bond between pyrophosphate and Mg²⁺ to separate Mg²⁺ ions. As a result, PCR reaction inhibited by pyrophosphate can be progressed again. Pyrophosphatase is stable even at a high temperature (70℃ or up). Pyrophosphatase and DNA polymerase exhibit their enzyme activities at equally high temperature. Therefore, Mg²⁺ ions fallen apart from pyrophosphate by pyrophosphatase are utilized by DNA polymerase for PCR reaction and accordingly the generation of non-specific amplification product is inhibited and instead only target product can be simply amplified (Korean Patent No. 10-0292883).

In addition, a new method using DNA aptamer binding to DNA polymerase has been developed (US Patent No. 6,183,967). The mechanism of this method is as follows: DNA aptamer is used as a template for DNA polymerase and as a result it is conjugated with DNA polymerase. The conjugated DNA polymerase interrupts polymerization, which means polymerization is actually stopped until DNA aptamer is denaturated. When reaction temperature is raised, DNA aptamer is denaturated and accordingly DNA polymerase is activated, leading to the re-start of polymerization. However, even though this method is advantageous in prompt PCR reaction, there is still a problem to solve, which is non-specific reaction caused by long aptamer.

PCR method using DNA polymerase inhibitor composed of nucleic acid sequence forming partial double bond, and playing a role in inhibition of DNA polymerase activation by binding DNA polymerase at a specific low temperature is also reported (US Patent Publication No. 2007/0212704).

As explained hereinbefore, studies have been undergoing about hot-start PCR. Even though the necessity of hot-start PCR has been well recognized, it is hardly utilized for experiments owing to its economical or practical limits. DNA polymerase, antibody, and pyrophosphatase are all proteins, so that it takes time and costs to supply them stably. The conventional methods are fundamentally all limited in inhibition of primer extension by primer-dimer formation. Therefore, it is still limited in inhibition of non-specific amplification product generation by the extended primer.

The present invention has been designed to overcome the problems of the prior arts explained hereinbefore. Thus, it is an object of the present invention to provide a composition for hot-start PCR prepared by adding a required amount of blocking oligonucleotide having the 3'-end blocked and the nucleotide sequence complementary to the primer to the conventional PCR composition, in order to inhibit smeared band of PCR product formed by non-specific reaction or primer-dimer formation at a low temperature to give more accurate PCR results.

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

The composition for hot-start PCR of the present invention characteristically comprises a blocking oligonucleotide in addition to the conventional PCR composition containing reaction buffer, MgCl₂, 4 kinds of dNTP and DNA polymerase, wherein the nucleotide sequence of the blocking oligonucleotide is complementary to a primer, and a hydroxyl group at 3'-end of the blocking oligonucelotide is blocked. The composition for hot-start PCR of the invention can additionally include primer, probe, template nucleic acid, fluorescent dye, and reverse transcriptase if necessary. In this invention, the said "template nucleic acid" can include DNA, RNA, and DNA/RNA hybrid without limitation.

In this invention, the said blocking oligonucleotide is characterized by less number of nucleotides than the complementary nucleotide sequence of the primer. In such case, that is when the nucleotide sequence of the blocking oligonucleotide is shorter than that of the primer, the melting temperature of primer (or template nucleic acid)-blocking oligonucleotide bond is lower than the melting temperature of primer-template target DNA bond. Therefore, primer is conjugated with blocking oligonucleotide by double helical bond at a low temperature, but such blocking oligonucleotide having a rather low denaturation temperature is dissociated before reaching annealing temperature of primer, suggesting that it cannot form double helical bond any longer, and accordingly it cannot play any role in PCR any longer, which is advantageous for hot-start PCR. The method using blocking oligonucleotide can also be applied to the probe by the same mechanism. In a preferred embodiment of the present invention, the said blocking oligonucleotide has at least 1℃ lower melting temperature, and preferably at least 2℃ lower than that of the primer. The melting temperature of the blocking oligonucleotide is preferably at least 25℃, and more preferably at least 50℃.

The blocking oligonucleotide herein is characterized by substitution of hydroxyl group of 3'-end with a substituent other than hydroxyl group. DNA polymerization is induced by the binding between hydroxyl group of 3'-end and phosphate group of 5'-end. So, when hydroxyl group of 3'-end is substituted with another substituent, DNA polymerization is no more induced. In a preferred embodiment of the present invention, the substituent is exemplified by C3-Space, C6-Space, C12-Space, C18-Space, amine, phosphate, each of which is represented by Chemical Formula 1∼6, DIG (Digosigenin) or thiol, but not always limited thereto.

[Chemical Formula 1]

[Chemical Formula 2]

[Chemical Formula 3]

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

In a preferred embodiment of the present invention, the first nucleotide at 5'-end of the blocking oligonucleotide forms complementary bond with the first nucleotide at 3'-end of the primer or with the second nucleotide or after at 3'-end of the primer, so as not to leave one or more nucleotides at 5'-end of the blocking oligonucleotide unpaired. That is, it is preferred for all the nucleotides at 5'-end of the blocking oligonucleotide are paired complementarily with the nucleotides at 3'-end of the primer. If one or more nucleotides at 5'-end of the blocking oligonucleotide are left unpaired, DNA polymerization occurs from the 3'end of the primer to the first nucleotide of 5'-end of the blocking oligonucleotide. As a result, the length of the primer is additionally extended. If such extended primer is included in the reaction mixture, non-specific amplification products generated by primer-dimer formation increase, which has been a problem in the prior art.

The said blocking oligonucleotide can be bound to either forward primer or reverse primer of a primer set having complementary sequence, or to both forward primer and reverse primer.

The reaction buffer herein is preferably 10 mM Tris-HCl, 40 mM KCl, pH 9.0. Four kinds of dNTP herein are dATP, dTTP, dGTP and dCTP. Any DNA polymerase known to those in the art can be used in this invention without limitation and particularly the polymerase having the activity of 5'->3' exonuclease, the polymerase having the activity of 3'->5' exonuclease, and the polymerase having none of the activities of 5'->3' exonuclease and 3'->5' exonuclease can be used independently or together. The polymerase having the activity of 5'->3' exonuclease is exemplified by Taq DNA polymerase. The polymerase having the activity of 3'->5' exonuclease is exemplified by Pfu DNA polymerase or TLA DNA polymerase. The polymerase having none of the activities of 5'->3' exonuclease and 3'->5' exonuclease is exemplified by Top DNA polymerase, but not always limited thereto. The content of DNA polymerase in the PCR composition is 0.1 - 10 U (unit), preferably 0.5 - 2 U, and more preferably 1 U.

The composition for hot-start PCR of the present invention can additionally include a dye which is non-reactive to nucleic acid, for the convenience of experiment, for the prevention of contamination by PCR product, for the stabilization of DNA polymerase and dNTP, and for the improvement of reactivity. The non-reactive dye is selected among those dyes which do not affect PCR reaction, which is exemplified by soluble dye such as bromophenol blue, xylene cyanole, bromocresol red, and cresol red. The preferable content of such non-reactive dye in the whole composition is 0.0001-0.01 weight% and more preferably 0.001-0.005 weight% and most preferably 0.001-0.003 weight%. If the content of the non-reactive dye is less than 0.0001 weight%, which means the content of the dye is too low to analyze PCR product by electrophoresis on agarose gel. That is, it is very difficult to observe the sample movement by the naked eye. If the content of the non-reactive dye is higher than 0.01 weight%, such high content of a soluble dye will act as a reaction inhibitor during PCR. In addition, such high concentration interrupts the sample movement during electrophoresis.

The composition for hot-start PCR of the present invention can also include reverse transcriptase for the synthesis of cDNA, if necessary. In the case of RT-PCR including reverse transcriptase, it is preferred to add the blocking oligonucleotide for the sense primer of the amplified RNA to the composition for hot-start PCR of the present invention.

DNA polymerase is the enzyme inducing polymerization by binding to 3'-end of the partial double helical structure having single-stranded 5'-end. In this invention, the blocking oligonucleotide has been designed based on the consideration of such characteristics of DNA polymerase. Precisely, in this invention, it was designed that 5'-end of the blocking oligonucleotide was to be paired with 3'-end of the primer complementarily to prevent non-specific reaction of the primer and the extension of nucleotides by hybridization of the primer with another nucleic acid. Also, 3'-end of the blocking oligonucleotide was designed to be shorter than 5'-end of the primer so that DNA polymerase could be easily conjugated thereto and at the same time hydroxyl group of 3'-end was substituted with another kind of substituent to prevent extension of the blocking oligonucleotide. So, simply the addition of the blocking oligonucleotide to the reaction mixture can prevent non-specific reaction by making DNA polymerase to be attached to double helix of the primer and the blocking oligonucleotide, indicating the inhibition of extension of the primer at room temperature as well.

The blocking oligonucleotide used in the composition for hot-start PCR of the present invention is the oligonucleotide having the nucleotide sequence complementary to the primer or the probe, which is shorter than the said primer or the probe. Thus, it forms double helix with primer/probe at a low temperature, so that DNA polymerase can recognize it as a template to be bound. But, once 3'-end is blocked, DNA polymerization is interrupted. Therefore, the generation of non-specific PCR products can be prevented by inhibiting non-specific polymerization of the primer, which used to be a problem of the conventional PCR. So, unlike the conventional methods such as the method using antibody, the method using chemical modification, or the conventional hot-start PCR using pyrophosphate, the method using the composition for hot-start PCR of the present invention has the advantage of efficient PCR reaction without wasting incubation time to activate the polymerase because the polymerase can be activated at the low temperature under the melting temperature of double helix of target template DNA and primer.

The mechanism of the blocking oligonucleotide is as follows: Forward primer and reverse primer are essential for PCR. The blocking oligonucleotide is a kind of oligonucleotide comprising a complementary nucleotide sequence containing at least one less nucleotides than forward or reverse primer, so that it has high specificity to primer. Therefore, once forward/reverse primers are mixed with the blocking oligonucleotide, the blocking oligonucleotide is bound to the forward/reverse primers, making the primers not work properly, suggesting that the primers are prevented from being involved in the formation of primer dimer or in non-specific reaction. When the reaction temperature reaches proper temperature, which is the melting temperature of the blocking oligonucleotide, the bond between the primer and the blocking oligonucleotide is broken, and accordingly the primer is conjugated to the target template nucleic acid, leading to the accurate and efficient PCR reaction. That is, since the blocking oligonucleotide is constructed shorter than the primer/probe, when the temperature reaches proper temperature where the primer can be attached to the template nucleic acid, the blocking nucleotide becomes fallen apart from the primer/probe with losing its effect on the primer/template binding (see Figure 1).

The hot-start PCR using the blocking oligonucleotide of the present invention can be applied to the following cases, but not always limited thereto.

1. In the case of monoplex PCR, when there is a smeared band of PCR product induced by non-specific reaction.

2. In the case of PCR, when multiple amplification products are generated by non-specific reaction.

3. In the case when PCR efficiency is decreased by primer dimer.

4. In the case when there is an attempt to increase PCR efficiency with a specific target having low amplification rate.

5. In the case when one or more amplification products (multiplex PCR) are needed to be confirmed.

6. In the case when there is an attempt to increase PCR efficiency with a specific target in relation to the field of quarantine and disease diagnosis.

7. In the case when real-time PCR requiring specificity is performed.

8. In the case when the primers having different Tm values are used.

9. In the case of One-Step RT-PCR, when non-specific amplification products are generated.

The composition for hot-start PCR of the present invention has following advantages, compared with the conventional PCR compositions.

1) The reaction can be performed according to the general PCR schedule, so long-time pre-treatment at high temperature is not required.

2) It is more economic than the conventional PCR compositions.

3) It can be applied to multiplex PCR using a variety of samples simultaneously because the generation of amplification products by low stringency priming is prevented with it.

The present invention also provides a method for hot-start PCR using the composition for hot-start PCR.

The method for hot-start PCR of the present invention is characterized by performing PCR after adding the blocking oligonucleotide to the conventional composition for hot-start PCR, as described hereinbefore. In a preferred embodiment of the present invention, the composition for hot-start PCR of the present invention can be applied without limitation to any random nucleic acid amplification method including multiplex PCR, real-time PCR, real-time quantitative PCR, real-time RT/PCR, and real-time quantitative RT/PCR, in addition to the conventional PCR. The "multiplex PCR" used in this invention indicates the simultaneous amplification of multiple DNA targets in one PCR reaction mixture.

In general, it is very difficult to design multiplex PCR condition that can amplify two or more target sequences simultaneously. Because it is importantly required to design the optimum PCR condition to amplify two or more target sequences without generating non-specific byproducts from all of the sequences used. At this time, to make perfect template DNA-primer match, it is necessary to perform annealing at a temperature high enough with no annealing among two or more primer pairs. In the case of multiplex PCR, PCR efficiency decreases by non-specific reaction and primer-dimer in the absence of hot-start function. It is also very difficult to obtain accurate results because of non-specific amplification products. So, hot-start method has been applied thereto. However, the application is still limited. The method for hot-start PCR of the present invention is ideal for multiplex DNA amplification since specificity of amplification has been improved by adding the blocking oligonucleotide having the nucleotide sequence complementary to each target primer.

In a preferred embodiment of the present invention, the products amplified from each target nucleotide sequence were designed to have different sizes for further analysis. So, the amplification products of multiplex target nucleotide sequences can be analyzed easily by size differentiation. Size comparison can be performed by various methods well-informed, such as electrophoresis using polyacrylamide gel matrix or agarose gel matrix. The blocking oligonucleotide of the present invention can be effectively used for solving the problem of non-specificity of the conventional multiplex PCR and for eliminating the problem of background.

The advantage of multiplex PCR is that different kinds of disease can be analyzed simultaneously by one reaction. Theoretically, there is no limit in numbers of the targets for simultaneous analysis, but in fact approximately 20 targets are the most. This is because that this method depends on size difference required for analysis and the method for analysis of amplification products. The method of the present invention can be applied to diagnosis of genetic and contagious diseases, sex determination, genetic linkage analysis, and climinalistics study.

As explained hereinbefore, unlike the conventional hot-start PCR method using antibody, chemical modification, or pyrophosphate, the method using the blocking oligonucleotide of the present invention can give more accurate PCR results by inhibiting smeared bands of PCR amplification products formed by non-specific reaction or primer-dimer formation at a low temperature because the blocking oligonucleotide is dissociated at the temperature under the melting temperature of primer.

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Figure 1 is a diagram illustrating the inhibition of non-specific reaction by the blocking oligonucleotide.

Figure 2 is a set of photographs illustrating the non-specific reaction inhibition effect according to the length of the blocking oligonucleotide, in which A and H show the inhibition of non-specific reaction by hot-start function of the positive control; B shows the non-specific reaction induced when hot-start function free negative control was used; C∼G and I∼M illustrate the reactions with the blocking oligonucleotide having different Tm of respectively 18℃, 26℃, 34℃, 37.4℃, 40.9℃, 46.1℃, 48.6℃, 50.5℃, 51.9℃ and 53.9℃; lane 1∼lane 3 illustrate the results of amplifications of three different target nucleotides; and lane M illustrates 100 bp DNA ladder for DNA size differentiation.

Figure 3 is a photograph illustrating the non-specific reaction inhibition effect according to the concentration of the blocking oligonucleotide, in which A illustrates the result of reaction with the negative control having no hot-start function; B illustrates the result of reaction with the positive control having hot-start function; C∼H illustrate the results of reaction added with instant oligonucleotides of different concentrations of 5 pmole, 10 pmole, 15 pmole, 20 pmole, 25 pmole, and 30 pmole respectively; lane 1∼lane 3 illustrate the results of amplifications of three different target nucleotides used in Figure 2; and lane M illustrates 100 bp DNA ladder for DNA size differentiation.

Figure 4 is a photograph illustrating the non-specific reaction inhibition effect of the reaction added with the oligonucleotide blocking at least one of forward primer and reverse primer, in which A illustrates the result of reaction with the negative control having no hot-start function; B illustrates the result of reaction with the positive control having hot-start function; C and D illustrate the results of reactions added with the blocking oligonucleotide having the nucleotide sequence complementary to forward primer; E and F illustrate the results of reactions added with the blocking oligonucleotide having the nucleotide sequence complementary to reverse primer; G illustrates the result of reaction added with the blocking oligonucleotide having the nucleotide sequence complementary to forward primer or reverse primer; lane 1∼lane 3 illustrate the results of amplifications of three different target nucleotides used in Figure 2; and lane M illustrates 100 bp DNA ladder for DNA size differentiation.

Figure 5 is a photograph illustrating the non-specific reaction inhibition effect according to the various substitutions of the blocking oligonucleotide, in which A illustrates the result of reaction with the positive control having hot-start function; B∼G illustrate the results of reaction added with the blocking oligonucleotide wherein the hydroxyl group is substituted with amine, phosphate, C3-Space, C6-Space, C12-Space, or C18 Space; lane 1∼lane 3 illustrate the results of amplifications of three different target nucleotides used in Figure 2; and lane M illustrates 100 bp DNA ladder for DNA size differentiation.

Figure 6 is a set of photographs illustrating the non-specific reaction inhibition effect in the case of the reaction with the composition having no hot-start function induced at 37℃ for 1 hour which was the severe condition inducing non-specific reaction (A) and in the case of the reaction not induced under the severe condition (B) in the presence or absence of the blocking oligonucleotide, in which lane 1∼lane 3 illustrate the results of amplifications of three different target nucleotides used in Figure 2; and lane M illustrates 100 bp DNA ladder for DNA size differentiation.

Figure 7 is a photograph illustrating the result of multiplex PCR according to the addition of the blocking oligonucleotide, in which multiplex PCR was performed with the composition containing the blocking oligonucleotide and having or not having hot-start function. Lane 1 illustrates the reaction without the blocking oligonucleotide and lane 2 illustrates the reaction with the blocking oligonucleotide.

Figure 8 is a photograph illustrating the non-specific reaction inhibition effect in One-Step RT/PCR according to the addition of the blocking oligonucleotide, in which lane 1 illustrates the result of reaction using RT/PCR composition having hot-start function; lane 2 illustrates the result of reaction using RT/PCR composition not having hot-start function; lane 3 illustrates the result of reaction performed with the composition not having hot-start function but added with the blocking oligonucleotide; and lane M illustrates 100 bp DNA ladder for DNA size differentiation.

Figure 9A is a graph illustrating the inhibition effect on non-specific reaction and primer/dimer formation in real-time PCR by the addition of the blocking oligonucleotide. Particularly, the blocking oligonucleotide having the nucleotide sequence complementary to the primer and fluorescent material (Sybergreen I) were added to PCR Premix containing Taq DNA polymerase (experimental group). For the control, fluorescent material alone was added to PCR Premix containing Taq DNA polymerase. Real-time PCR was performed to produce curves presenting the result of fluorescence measurement, which are shown in Figure 9A. In this graph, horizontal axis indicates the PCR cycle, and vertical axis indicates the fluorescence value measured according to the reaction cycle. Line 1 is the fluorescence curve of the control, and line II is the fluorescence curve of the experimental group. Figure 9B is a graph illustrating the melting curve made based on the fluorescence curve, in which horizontal axis indicates the changes of temperature and vertical axis indicates the fluorescence value according to the raise of the temperature. Line I indicates the melting curve of the control and line II indicates the melting curve of the experimental group.

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Inhibition effect on the generation of non-specific amplification product according to Tm value of the blocking oligonucleotide

To investigate how the length of the blocking oligonucleotide having the nucleotide sequence complementary to the primer that is an essential element for PCR could affect the inhibition of the generation of non-specific amplification product, blocking oligonucleotides in different sizes were added for the reaction and the PCR products were compared. Precisely, primers were designed and synthesized as shown in Table 1. Human genomic DNA (10 ng), each primer (10 pmole), and each blocking oligonucleotide in different size having Tm value that was 2℃∼15℃ lower than that of the target primer (each 10 pmole) were added to PCR Premix (Bioneer, Korea), followed by PCR. PCR condition was as follows: 95℃ for 20 seconds, 55℃ for 40 seconds, and at 72℃ for 60 seconds, 30 cycles. Predenaturation and final extension were performed respectively at 94℃ for 5 minutes and at 72℃ for 5 minutes. The PCR products were electrophoresed on agarose gel with DNA marker, followed by staining with EtBr (Ethidium Bromide). DNA bands amplified by PCR were photographed by Polaroid camera. Hot-start PCR Premix (AccuPower HotStart PCR Premix, Bioneer) having hot-start function was used for the positive control, and PCR Premix (AccuPower PCR Premix PCR) having no hot-start function was used for the negative control. The blocking oligonucleotide was added to each PCR Premix, followed by PCR. The results are shown in Figure 2.

In Figure 2,

lanes

1, 2, and 3 illustrate the results of reaction using the primer pairs having the nucleotide sequences of P55/P63 (447 bp), P55/P73 (1,082 bp), and P55/P83 (1,296 bp) respectively. A illustrates the result of the positive control using hot-start PCR Premix having hot-start function and B illustrates the result of the negative control using PCR Premix without hot-start function. C, D, E, F, G, I, J, K, L, and M illustrate the results of PCR with the negative control added with the instant oligonucleotides at the order of shorter to longer (that is starting with the sequence represented by SEQ. ID. NO: 5 upto the sequence represented by SEQ. ID. NO: 14) stepwise. Lane M illustrates 100 bp DNA Ladder (Bioneer) for DNA size differentiation.

Table 1

Region	Sixe	Tm	Sequence	SEQ. ID. NO:
P55	23mer	55℃	CTC TTC CTA CAG TAC TCC CCT GC	1
P63	24mer	63.3℃	GGC CAC TGA CAA CCA CCC TTA ACC	2
P73	23mer	60.3℃	CAA GTG GCT CCT GAC CTG GAG TC	3
P83	25mer	60.1℃	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℃	GCA GGG GAG TAC TGT	8
	17mer	40.9℃	GCA GGG GAG TAC TGT AG	9
	18mer	46.1℃	GCA GGG GAG TAC TGT AGG	10
	19mer	48.6℃	GCA GGG GAG TAC TGT AGG A	11
	20mer	50.5℃	GCA GGG GAG TAC TGT AGG AA	12
	21mer	51.9℃	GCA GGG GAG TAC TGT AGG AAG	13
	22mer	53.9℃	GCA GGG GAG TAC TGT AGG AAG A	14

As a result, it was confirmed that the addition of the blocking oligonucleotide having the nucleotide sequence complementary to the primer for PCR resulted in the interruption of DNA polymerase activity, the inhibition of low stringency priming caused when necessary elements for gene amplification are mixed at room temperature, and the inhibition of the generation of non-specific amplification products (Figure 2).

Example 2: Inhibition effect on the generation of non-specific amplification product according to the concentration of the blocking oligonucleotide

To determine the proper concentration of the blocking oligonucleotide, the template-specific primer of Example 1 and P55 blocking oligonucleotide represented by SEQ. ID. NO: 12 (20mer) were used. PCR was performed by the same manner as described in Example 1 except that the template-specific primer was used at the concentration of 20 pmol and the blocking oligonucleotide was used at the concentration of 5 pmole∼30 pmole. The results are shown in Figure 3.

In Figure 3,

lanes

1, 2, and 3 illustrate the results of reactions using the primer sets having the nucleotide sequences of P55/P63 (447 bp), P55/P73 (1,082 bp), and P55/P83 (1,296 bp) of Table 1 respectively. A illustrates the result of the negative control using PCR Premix without hot-start function, and B illustrates the result of the positive control using hot-start PCR Premix having hot-start function. C, D, E, and F illustrate the results of PCR with the negative control added with the blocking oligonucleotide at different concentrations, from lower concentration to higher concentration, stepwise.

As a result, when PCR was performed with PCR Premix without hot-start function, like the negative control, non-specific reaction and accordingly smeared band of PCR product were observed. On the other hand, when PCR was performed with hot-start PCR Premix having hot-start function, like the positive control, more accurate and clearer PCR products were confirmed. When the negative control was added with the blocking oligonucleotide at different concentrations, same effect was observed as the one from hot-start PCR. When the blocking oligonucleotide was added at the concentration as same as or lower than the concentration of template DNA target primer, polymerase activity was blocked and primer-dimer formation and non-specific reaction were inhibited effectively (Figure 3).

Example 3: Inhibition effect on the generation of non-specific amplification product by the blocking oligonucleotide having the nucleotide sequence complementary to forward and/or reverse primer

Basically, PCR reaction cannot be induced without either forward primer or reverse primer or both. Thus, the blocking oligonucleotide blocking either or both of forward primer and reverse primer of the target primer set was added, followed by observation of the effect on non-specific reaction. Precisely, PCR was performed by the same manner as described in Example 2 respectively with the negative control without hot-start function (A), with the positive control with hot-start function (B), with the negative control added with forward primer blocking oligonucleotide (C and D), with the negative control added with reverse primer blocking oligonucleotide (E and F), and with the negative control added with forward primer and reverse primer blocking oligonucleotide (G). At this time, the concentrations of the primer and the blocking oligonucleotide were equally 10 pmole.

As a result, in the negative control without hot-start function, non-specific amplification products were confirmed (A). However, in the positive control with hot-start function, non-specific amplification products were not observed (B). When one of instant oligonucleotides blocking forward (C and D) and reverse (E and F) primers was added or when the instant oligonucleotide blocking both primers was added (G), non-specific amplification reaction was inhibited and thus smeared DNA band caused by such non-specific amplification product was not observed (Figure 4).

Example 4: Inhibition effect on non-specific reaction by various types of modified blocking oligonucleotides

Different types of substituents were introduced in 3'-end of the blocking oligonucleotide, followed by investigation of hot-start effect of the blocking oligonucleotide. The substituents used in this invention are shown in Table 2. PCR was performed by the same manner as described in Example 2. The results are shown in Figure 5.

In Figure 5,

lanes

1, 2, and 3 illustrate the results of reactions using the primer sets having the nucleotide sequences of P55/P63 (447 bp), P55/P73 (1,082 bp), and P55/P83 (1,296 bp) of Table 1 respectively. A illustrates the result of the positive control using hot-start PCR Premix having hot-start function. B, C, D, E, F, and G illustrate the results of PCR with the negative control added with each instant blocking oligonucleotide shown in Table 2 in that order. Lane M illustrates 100 bp DNA ladder for DNA size differentiation.

Table 2

No.	Types of modification	Structural formula
1	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 the addition of any chemical material that can block DNA polymerase to 3'-end of the blocking oligonucleotide having the nucleotide sequence complementary to the primer could inhibit non-specific amplification (Figure 5).

Example 5: Inhibition effect on non-specific reaction by the blocking oligonucleotide under the severe condition that can induce non-specific reaction

The result of hot-start PCR using the blocking oligonucleotide (II) was compared with the result of general PCR not including the blocking oligonucleotide (I). To do so, the hot-start PCR mixture including the blocking oligonucleotide prepared in Example 2 proceeded to PCR by the same manner as described in Example 2. Another PCR was performed by the same manner as described in Example 2 after pre-reaction was induced at 37℃ for 1 hour. Nucleotide sequence pairs of the template specific oligonucleotide used herein are shown in Table 3.

Table 3

Name	Primer	Sequence	SEQ. ID. NO:
ABCA3(463)	Forward primer	GCC CAT CTT ACA TCC TCT CTC	15
	Reverse 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
	Reverse primer	TTA GCA AGG CAG TCA GTT ACA G	19
	F-blocking ologo	CCT TGA CTC TGC CAT	20
SLC29A2(357)	Forward primer	TTC ATC ATC ATC AGG AGC AGA G	21
	Reverse primer	TCC TTC CAA GAG CCT CAA TTA G	22
	F-blocking oligo	CTC TGC TCC TGA TGA T	23

As a result, clear amplification products without non-specific amplification products were generated in the PCR using instant hot-start PCR mixture (Figure 6A). When the instant hot-start PCR mixture containing the blocking oligonucleotide was pre-reacted at 37℃ for 1 hour before PCR, amplification efficiency was reduced but smeared DNA band caused by non-specific amplification products was not observed regardless of the severe condition (37℃, 1 hour reaction), while non-specific amplification products were generated from PCR with the control regardless of the reaction for 1 hour at 37℃ (Figure 6B). From the above results, it was confirmed that when PCR was performed with the instant hot-start PCR mixture containing the blocking oligonucleotide, the generation of non-specific amplification products could be effectively inhibited regardless of whether the reaction was performed at normal temperature or under severe condition inducing non-specific reaction.

Example 6: Application of hot-start PCR to multiplex PCR

To confirm whether or not the composition for hot-start PCR of the present invention could be applied to multiplex PCR, 6 primer sets against 6 target nucleotide sequences and the blocking oligonucleotides for the primers were added, followed by investigation of the effect on primer annealing, primer extension, and denaturation. Nucleotide sequences of the primers and the blocking oligonucleotides used herein are shown in Table 4. Instant hot-start PCR was performed with PCR Premix not having hot-start function but with blocking oligonucleotide. Hot-start PCR Premix was used as the control. As a template, human genomic DNA (100 ng and 10 ng) was used. The results are shown in Figure 7.

In Figure 7, lane 1 illustrates the result of PCR without blocking oligonucleotide, and lane 2 illustrates the result of PCR added with the blocking oligonucleotide at the same concentration as that of the template target primer, which was 10 pmole.

Table 4

Name	Primer	Sequence	SEQ. ID. NO:
ABCA3(463)	Forward primer	GCC CAT CTT ACA TCC TCT CTC	24
	Reverse 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
	Reverse 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
	Reverse primer	TGG GGA AGT GTG GAG AGG TAG	31
	F-blocking oligo	GAT CGT GTT GAG CCT AT	32
ABCG1(258)	Forward primer	GAC CGA CGA CAC AGA GAC TC	33
	Reverse 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
	Reverse 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
	Reverse 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 PCR was not inhibited even in PCR using hot-start PCR Premix with the addition of the blocking oligonucleotide. In the case of PCR using PCR Premix not having hot-start function, nucleotide sequence amplification was partially blocked or weakened, but when the blocking oligonucleotide was added thereto, the entire nucleotide sequence was amplified or background was eliminated, suggesting that multiplex PCR reaction was successfully completed (Figure 7).

Example 7: Inhibition effect on non-specific reaction in One-Step RT/PCR by the blocking oligonucleotide

Inhibition effect on non-specific reaction in One-Step RT/PCR by the blocking oligonucleotide was investigated. Precisely, 100 ng of human total RNA was used as template RNA, and GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) was used as a target gene. The target primers and the blocking oligonucleotides shown in Table 5 were used herein. For the controls, AccuPower Hotstart RT/PCR Premix (Bioneer) having hot-start function and AccuPower RT/PCR Premix (Bioneer) not having hot-start function were used. The blocking oligonucleotide having the nucleotide sequence complementary to the forward primer was added to the AccuPower RT/PCR not having hot-start function, followed by PCR. The results are shown in Figure 8.

In Figure 8, lane M indicates the DNA size marker, and lane 1 illustrates the result of reaction using AccuPower Hot-start RT/PCR Premix. Lane 2 illustrates the result of reaction using AccuPower RT/PCR and lane 3 illustrates the result of reaction using AccuPower RT/PCR Premix added with instant oligonucleotide.

Table 5

Name	Primer	Size	Tm(℃)	Sequence	SEQ. ID. NO:
CM-CSF 502	Forward primer	20mer	55	TGT TCG TGC ACA TTT CGT GA	42
	Reverse 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
	Reverse 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, the addition of the blocking oligonucleotide to AccuPower RT/PCR not having hot-start function brought similar inhibition effect on non-specific reaction in the reaction using the Premix having hot-start function (Figure 8).

Example 8: Primer-dimer inhibition effect in real-time PCR by the blocking oligonucleotide

To investigate the hot start effect of the PCR composition containing the blocking oligonucleotide in real-time PCR, a fluorescent material (Greenstar™, Bioneer) was added to 2x PCR Premix (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 2.0 mM MgCl₂, 250 μM each of 4 kinds of dNTP, 1U Taq DNA polymerase, 0.01% Tween 20 and a stabilizer) at the concentration of 0.3X per 20 ㎕ reaction, leading to the preparation of the composition added with the blocking oligonucleotide at the same concentration as the forward primer, 20 pmol. For the control, the said fluorescent material was added to 2x PCR Premix at the concentration of 0.25x per 20 ㎕ reaction. The said fluorescent material facilitates the analysis of specific nucleotide sequence simply without using a fluorescent probe by measuring the fluorescence generated by intercalation in between double strands of DNA during template DNA amplification. 100 ng of total RNA extracted from human cells was added to AccuPower CycleScript RT Premix (Bioneer), followed by reaction at 42℃ for 1 hour. As a result, cDNA was obtained. Reverse transcriptase was inactivated by heating at 95℃ for 5 minutes. 5 ㎕ of the cDNA was used as template DNA. The primers and the blocking oligonucleotides shown in Table 6 were used at the concentration of 20 pmol per 20 ㎕ reaction.

Table 6

Name	Primer	Size	Tm(℃)	Sequence	SEQ. ID. NO:
GM501 qPCR	Forward primer	20mer	55.2	GAC GTC CGC ATC TTG AAT TG	48
	Reverse 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

Real-time PCR was performed using Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer) as follows; predenaturation at 94℃ for 5 minutes, denaturation at 95℃ for 10 seconds, annealing and extension at 60℃ for 15 seconds, 45 cycles from denaturation to extension. Then, dissociation step was carried out to make melting curve of the amplification product, from which PCR reactivity and specificity of the PCR amplification product was confirmed. The results are shown in Figure 9.

As a result, threshold cycle (Ct) of line I was 23.89, and threshold cycle (Ct) of line II was 29.5 (Figure 9). As shown in melting curve, Ct of line I was reduced compared with line II, which means that only one fluorescence peak was observed in line II containing the blocking oligonucleotide with suggesting that accurate amplification product was produced, while two fluorescence peaks were observed in line I not including the blocking oligonucleotide with suggesting that two kinds of amplification products were generated. Two fluorescence peaks indicate that primer-dimer product or non-specific amplification product which was smaller than the target PCR product was produced. Melting temperature (Tm) of the accurate PCR product was confirmed as follows: Tm of the experimental group (line II) and Tm of the control (line I) were all 85.5℃, and Tm of primer-dimer or non-specific amplification product was 75.5℃.

The above results suggest that low specificity was observed in the control owing to the generation of primer-dimer in addition to the amplification product. On the contrary, the instant hot-start PCR used in the present invention could effectively recover PCR reaction with inhibiting non-specific reaction induced at low temperature during real-time PCR, suggesting that the method was effective in inhibiting the generation of non-specific PCR product to give more accurate results.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

A composition for hot-start PCR comprising a blocking oligonucleotide to the conventional PCR composition containing reaction buffer, MgCl₂, 4 kinds of dNTP and DNA polymerase, wherein the nucleotide sequence of the blocking oligonucleotide is complementary to a primer, and a hydroxyl group at 3'-end of the blocking oligonucelotide is blocked.
The composition for hot-start PCR according to claim 1, wherein the blocking oligonucleotide has the nucleotide sequence shorter than that of the complementary primer.
The composition for hot-start PCR according to claim 1, wherein the blocking oligonucleotide has the melting temperature lower than that of the complementary primer.
The composition for hot-start PCR according to claim 3, wherein the blocking oligonucleotide has the melting temperature at least 1℃ lower than that of the complementary primer.
The composition for hot-start PCR according to claim 4, wherein the blocking oligonucleotide has the melting temperature of 25℃ or higher.
The composition for hot-start PCR according to claim 1, wherein the hydroxyl group at 3'-end of the blocking oligonucleotide is substituted with another kind of substituent other than hydroxyl group.
The composition for hot-start PCR according to claim 6, wherein the substituent is selected from the group consisting of C3-Space, C6-Space, C12-Space, C18-Space, amine, phosphate, each of which is represented by Chemical Formula 1∼6, DIG, and thiol.

[Chemical Formula 1]

[Chemical Formula 2]

[Chemical Formula 3]

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]
The composition for hot-start PCR according to claim 1, wherein all the nucleotides at 5'-end of the blocking oligonucleotide are paired complementarily with the nucleotides at 3'-end of the primer.
The composition for hot-start PCR according to claim 1, wherein the blocking oligonucleotide is bound to one or both of a forward primer and a reverse primer.
The composition for hot-start PCR according to claim 1, wherein the DNA polymerase is selected from the group consisting of the polymerase having the activity of 5'->3' exonuclease, the polymerase having the activity of 3'->5' exonuclease, and the polymerase having none of the activities of 5'->3' exonuclease and 3'->5' exonuclease.
The composition for hot-start PCR according to claim 1, wherein the composition further comprises a dye which is non-reactive to the target nucleic acid.
The composition for hot-start PCR according to claim 11, wherein the non-reactive dye is selected from the group consisting of bromophenol blue, xylene cyanole, bromocresol red, and cresol red.
The composition for hot-start PCR according to claim 1, wherein the composition further comprises a reverse transcriptase.
A method of hot-start PCR using the composition for hot-start PCR according to claim 1.
The hot-start PCR method according to claim 14, wherein the 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.