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WO2012115464A2 - Composition pour pcr à démarrage à chaud comprenant un oligonucléotide de blocage - Google Patents

Composition pour pcr à démarrage à chaud comprenant un oligonucléotide de blocage Download PDF

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WO2012115464A2
WO2012115464A2 PCT/KR2012/001387 KR2012001387W WO2012115464A2 WO 2012115464 A2 WO2012115464 A2 WO 2012115464A2 KR 2012001387 W KR2012001387 W KR 2012001387W WO 2012115464 A2 WO2012115464 A2 WO 2012115464A2
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pcr
hot
composition
primer
blocking oligonucleotide
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WO2012115464A3 (fr
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Jun Hee Lee
So Ra Choi
Nam Il Kim
Han Oh Park
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Bioneer Corp
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Bioneer Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • 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.
  • blocking oligo blocking oligonucleotide
  • PCR 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.
  • 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°C - 90°C. Then, the wax is dissolved and the reaction mixture and starting reagents are mixed, leading to PCR.
  • paraffin beads form a solid layer at up to 55°C. 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°C) 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).
  • 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.
  • reaction beads prepared by coating a reaction mixture dried over trehalose solution with wax
  • organic solvents PEG, DMSO, Glycerol, etc
  • the most widely used hot-start method is to induce hot-start PCR by regulating DNA polymerase activity by using anti-DNA polymerase antibody.
  • 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°C ⁇ 80°C) 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.
  • Another method to induce hot-start PCR is to use pyrophosphate and pyrophosphatase to inhibit Mg 2+ reaction at a low temperature.
  • the mechanism of pyrophosphatase and pyrophosphate for hot-start PCR is as follows: Pyrophosphate blocks Mg 2+ during PCR. Mg 2+ is required for the activation of DNA polymerase. Thus, when Mg 2+ is blocked, DNA polymerase activity is reduced. Pyrophosphate has high affinity to Mg 2+ . So, when pyrophosphate is added to PCR reaction mixture, Mg 2+ required for PCR is arrested by pyrophosphate and accordingly PCR is stopped, that is the reaction by DNA polymerase does not proceed any more.
  • 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 Mg 2+ from pyrophosphate.
  • pyrophosphatase eliminates the bond between pyrophosphate and Mg 2+ to separate Mg 2+ ions.
  • PCR reaction inhibited by pyrophosphate can be progressed again.
  • Pyrophosphatase is stable even at a high temperature (70°C or up). Pyrophosphatase and DNA polymerase exhibit their enzyme activities at equally high temperature.
  • Mg 2+ 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).
  • the present invention has been designed to overcome the problems of the prior arts explained hereinbefore.
  • 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, MgCl 2 , 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.
  • the said "template nucleic acid” can include DNA, RNA, and DNA/RNA hybrid without limitation.
  • 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.
  • 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.
  • the said blocking oligonucleotide has at least 1°C lower melting temperature, and preferably at least 2°C lower than that of the primer.
  • the melting temperature of the blocking oligonucleotide is preferably at least 25°C, and more preferably at least 50°C.
  • 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.
  • 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.
  • 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.
  • DNA polymerization occurs from the 3'end of the primer to the first nucleotide of 5'-end of the blocking oligonucleotide.
  • 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.
  • dNTP 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%.
  • 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.
  • composition for hot-start PCR of the present invention can also include reverse transcriptase for the synthesis of cDNA, if necessary.
  • reverse transcriptase for the synthesis of cDNA
  • DNA polymerase is the enzyme inducing polymerization by binding to 3'-end of the partial double helical structure having single-stranded 5'-end.
  • 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.
  • 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.
  • 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.
  • the reaction temperature reaches proper temperature, which is the melting temperature of the blocking oligonucleotide
  • 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.
  • composition for hot-start PCR of the present invention has following advantages, compared with the conventional PCR compositions.
  • reaction can be performed according to the general PCR schedule, so long-time pre-treatment at high temperature is not required.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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°C, 26°C, 34°C, 37.4°C, 40.9°C, 46.1°C, 48.6°C, 50.5°C, 51.9°C and 53.9°C; 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°C 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.
  • 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).
  • 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.
  • horizontal axis indicates the PCR cycle
  • vertical axis indicates the fluorescence value measured according to the reaction cycle.
  • Line 1 is the fluorescence curve of the control
  • 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.
  • Example 1 Inhibition effect on the generation of non-specific amplification product according to Tm value of the blocking oligonucleotide
  • 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.
  • 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°C ⁇ 15°C 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°C for 20 seconds, 55°C for 40 seconds, and at 72°C for 60 seconds, 30 cycles. Predenaturation and final extension were performed respectively at 94°C for 5 minutes and at 72°C 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
  • 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.
  • 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.
  • Example 2 Inhibition effect on the generation of non-specific amplification product according to the 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.
  • 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
  • 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.
  • 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
  • PCR reaction cannot be induced without either forward primer or reverse primer or both.
  • 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.
  • 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).
  • the concentrations of the primer and the blocking oligonucleotide were equally 10 pmole.
  • Example 4 Inhibition effect on non-specific reaction by various types of modified blocking oligonucleotides
  • 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.
  • 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).
  • 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°C for 1 hour. Nucleotide sequence pairs of the template specific oligonucleotide used herein are shown in Table 3.
  • 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.
  • human genomic DNA 100 ng and 10 ng
  • lane 1 illustrates the result of PCR without blocking oligonucleotide
  • 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.
  • Example 7 Inhibition effect on non-specific reaction in One-Step RT/PCR by the blocking oligonucleotide
  • lane M indicates the DNA size marker
  • 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
  • lane 3 illustrates the result of reaction using AccuPower RT/PCR Premix added with instant oligonucleotide.
  • Example 8 Primer-dimer inhibition effect in real-time PCR by the blocking oligonucleotide
  • a fluorescent material (GreenstarTM, Bioneer) was added to 2x PCR Premix (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 2.0 mM MgCl 2 , 250 ⁇ M 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 ⁇ l reaction, leading to the preparation of the composition added with the blocking oligonucleotide at the same concentration as the forward primer, 20 pmol.
  • 2x PCR Premix 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 2.0 mM MgCl 2 , 250 ⁇ M each of 4 kinds of dNTP, 1U Taq DNA polymerase, 0.01% Tween 20 and a stabilizer
  • the said fluorescent material was added to 2x PCR Premix at the concentration of 0.25x per 20 ⁇ l 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°C for 1 hour.
  • cDNA was obtained.
  • Reverse transcriptase was inactivated by heating at 95°C for 5 minutes. 5 ⁇ l 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 ⁇ l reaction.
  • Real-time PCR was performed using Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer) as follows; predenaturation at 94°C for 5 minutes, denaturation at 95°C for 10 seconds, annealing and extension at 60°C 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.
  • threshold cycle (Ct) of line I was 23.89
  • threshold cycle (Ct) of line II was 29.5 ( Figure 9).
  • 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.
  • Tm Melting temperature

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Abstract

La présente invention concerne une composition pour PCR à démarrage à chaud, plus précisément une composition pour PCR à démarrage à chaud caractéristiquement préparée par adjonction de l'oligonucléotide de blocage dont le groupe hydroxyle à l'extrémité 3' est bloqué et dont la séquence nucléotidique est complémentaire à l'amorce à la composition de PCR classique contenant un tampon réactionnel, MgCl2, 4 sortes de dNTP, et une polymérase d'ADN. La composition pour PCR à démarrage à chaud de la présente invention peut donner des résultats de PCR plus authentiques par l'inhibition de la trainée du produit d'amplification PCR provoquée par une réaction non spécifique ou la formation de dimères d'amorces à basse température puisque l'oligonucléotide de blocage contenu dans celle-ci peut être dissocié à une température plus basse que la température de fusion de l'amorce.
PCT/KR2012/001387 2011-02-25 2012-02-23 Composition pour pcr à démarrage à chaud comprenant un oligonucléotide de blocage Ceased WO2012115464A2 (fr)

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WO2015063154A1 (fr) * 2013-10-31 2015-05-07 Multiplicom N.V. Amorces de blocage dans des essais à base de pcr multiplexe
WO2017121843A1 (fr) * 2016-01-15 2017-07-20 Thermo Fisher Scientific Baltics Uab Anticorps se liant à des polymérases d'adn thermophiles
WO2022034303A1 (fr) * 2020-08-11 2022-02-17 Oxford University Innovation Limited Oligonucléotide switch
WO2022084214A1 (fr) * 2020-10-20 2022-04-28 Friz Biochem Gmbh Plaque de microtitration fonctionnalisée, solution de réaction et kit de préparation d'une solution de réaction
US11560553B2 (en) 2016-01-15 2023-01-24 Thermo Fisher Scientific Baltics Uab Thermophilic DNA polymerase mutants
US11618891B2 (en) 2017-06-26 2023-04-04 Thermo Fisher Scientific Baltics Uab Thermophilic DNA polymerase mutants

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KR102124058B1 (ko) 2013-09-16 2020-06-17 삼성전자주식회사 폴리뉴클레오티드 및 그의 용도
KR101897490B1 (ko) 2016-12-02 2018-09-12 건국대학교 산학협력단 폴리에틸렌글리콜-접합된 나노 사이즈의 산화그래핀을 포함하는 pcr용 조성물
KR102141312B1 (ko) 2019-04-19 2020-08-04 주식회사 제노헬릭스 짧은 RNA-primed 제노 센서 모듈 증폭 기반 짧은 RNA 탐지 기법
KR20250000081A (ko) 2023-06-23 2025-01-02 고려대학교 산학협력단 Taq DNA 중합효소가 발현된 대장균을 이용한 핫 스타트 PCR

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015063154A1 (fr) * 2013-10-31 2015-05-07 Multiplicom N.V. Amorces de blocage dans des essais à base de pcr multiplexe
WO2017121843A1 (fr) * 2016-01-15 2017-07-20 Thermo Fisher Scientific Baltics Uab Anticorps se liant à des polymérases d'adn thermophiles
US10273530B2 (en) 2016-01-15 2019-04-30 Thermo Fisher Scientific Baltics Uab Antibodies that bind thermophilic DNA polymerases
US11560553B2 (en) 2016-01-15 2023-01-24 Thermo Fisher Scientific Baltics Uab Thermophilic DNA polymerase mutants
US11618891B2 (en) 2017-06-26 2023-04-04 Thermo Fisher Scientific Baltics Uab Thermophilic DNA polymerase mutants
WO2022034303A1 (fr) * 2020-08-11 2022-02-17 Oxford University Innovation Limited Oligonucléotide switch
WO2022084214A1 (fr) * 2020-10-20 2022-04-28 Friz Biochem Gmbh Plaque de microtitration fonctionnalisée, solution de réaction et kit de préparation d'une solution de réaction

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