A rapid PCR buffer, its kit, and method

Abstract

This invention discloses a rapid PCR buffer, its kit, and method, relating to the field of PCR amplification technology. The invention uses Bis-Tris as a buffer pair to prepare the PCR buffer. Using this PCR buffer for rapid PCR amplification, it can still achieve high amplification efficiency even with enzyme concentrations as low as 0.15625 U / 25 μl. It has the characteristics of high amplification efficiency, short reaction time, and high amplification stability, and can effectively shorten the DNA sample detection time.

Description

Technical Field

[0001] This invention relates to the field of PCR amplification technology, and more specifically, to a rapid PCR buffer, its kit, and its method. Background Technology

[0002] PCR, short for Polymerase Chain Reaction, is a molecular biology technique used to amplify specific DNA fragments. Its research has a profound impact on various fields of biology and is closely related to medicine, agriculture, and bioengineering. For example, PCR technology involves new approaches to breeding in agriculture, the detection and treatment of genetic diseases in medicine, emerging industries based on genetic engineering, and forensic evidence identification and paternity testing.

[0003] "Rapid PCR" (or "rapid cycling") generally refers to completing 30 cycles within 10-60 minutes. The actual time for each cycle is longer than the total time typically allocated to denaturation, annealing, and extension, because time is needed for the temperature to rise between each of these stages. Over the years, systems have become faster, and the kinetics of denaturation, annealing, and extension have become more clearly defined. Many rapid PCR instruments have been invented on the market, and while some can effectively shorten reaction times, further improvements in rapid PCR technology are still needed to significantly increase reaction rates.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid PCR buffer, its kit, and its method.

[0006] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a rapid PCR amplification kit, which includes a PCR buffer and a rapid Taq enzyme; wherein the buffer pair used in the PCR buffer is Bis-Tris.

[0007] Secondly, embodiments of the present invention provide a method for rapid PCR amplification, which includes PCR amplification using the rapid Taq enzyme and PCR buffer in the kit described in the foregoing embodiments.

[0008] Thirdly, embodiments of the present invention provide a rapid PCR buffer comprising the following components: buffer pair Bis-Tris, salt ions, surfactant, and enhancer.

[0009] The present invention has the following beneficial effects: This invention uses Bis-Tris as a buffer pair to prepare PCR buffer. Using this PCR buffer for rapid PCR amplification, it can still achieve high amplification efficiency even with enzyme concentrations as low as 0.15625 U / 25 μl. It has the characteristics of high amplification efficiency, short reaction time, and high amplification stability, which can effectively shorten the DNA sample detection time. Attached Figure Description

[0010] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 To verify the test results of the Bis Tris buffer solution at different pH values ​​in Example 1; Figure 2 To verify the different concentrations of Bis Tris buffer solution (pH 8.8) in Example 1; Figure 3 To verify the test results of different types of ammonium salts in Example 2; Figure 4 To verify the test results of different types of potassium salts in Example 3; Figure 5 To verify the amplification results in Example 4 with and without surfactant; Figure 6 To verify the difference in amplification results between BSA and acetyl BSA under the same dosage conditions in Example 5; Figure 7 To verify the test results of the different Taq enzymes and buffer solutions provided in Example 7 of this invention; Figure 8 Results of cross-testing of AK Taq and 5×Fast buffer with SpeedSTAR™ HS kit. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0013] This invention provides a rapid PCR amplification kit, comprising: a PCR buffer and a rapid Taq enzyme; wherein the buffer pair used in the PCR buffer is Bis-Tris.

[0014] This invention uses Bis-Tris as a buffer pair to prepare PCR buffer. Using this PCR buffer for rapid PCR amplification, it can still achieve high amplification efficiency even with enzyme concentrations as low as 0.15625 U. It has the characteristics of high amplification efficiency, short reaction time, and high amplification stability, which can effectively shorten the DNA sample detection time.

[0015] Rapid Taq polymerase refers to a DNA polymerase that has been genetically engineered to achieve rapid amplification. Its extension rate can be arbitrarily selected in the range of 1 to 20 sec / kb, such as 2.5 sec / kb, 10 sec / kb, or 15 sec / kb.

[0016] In a preferred embodiment, the rapid Taq enzyme is selected from any one of the following: AK Taq enzyme, TaKaRa's SpeedSTAR™ HS DNA Polymerase, Thermo Fisher's Platinum™ Taq DNA polymerase, and Novizan 2 × Rapid Taq Master Mix Taq enzyme. AK Taq enzyme includes Phibron AK Taq DNA polymerase, catalog number: MD099.

[0017] In a preferred embodiment, the pH value of the PCR buffer can be arbitrarily selected within the range of 8.0 to 9.2, for example, any one or any two of 8.0, 8.2, 8.4, 8.6, 8.8, 9.0 and 9.2.

[0018] In a preferred embodiment, the final concentration of Bis-Tris in the PCR buffer can be arbitrarily selected within the range of 15 to 150 mM, for example, within the range of 18.75 to 150 mM, specifically any one or any two of the following: 15 mM, 18.75 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM. Within this range, effective amplification is possible.

[0019] In a preferred embodiment, the PCR buffer further includes at least one of the following components: salt ions, surfactant, and enhancer.

[0020] In a preferred embodiment, the salt ion includes at least one of ammonium salt and potassium salt.

[0021] Preferably, in the PCR buffer, when the salt ion includes an ammonium salt, the concentration of the ammonium salt in the PCR buffer can be arbitrarily selected within the range of 20-60 mM, for example, any one or any two of 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, and 60 mM. The ionization of hydrogen ions by ammonium ions exhibits reversible competition, which can enhance the binding force between specifically binding primers and templates (by providing hydrogen ions). Competitively capturing hydrogen ions can reduce non-specific binding forces, improve the rigor of annealing conditions, further reduce non-specific amplification during the reaction process, and significantly improve the specificity and sensitivity of the PCR reaction. Preferably, the ammonium salt is selected from at least one of (NH4)2SO4, NH4Cl, and CH3COONH4, more preferably NH4Cl.

[0022] When the salt ion includes a potassium salt, the final concentration of the potassium salt in the PCR buffer can be arbitrarily selected within the range of 5 to 45 mM, for example, any one or any two of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, and 45 mM. Potassium ions bind to the phosphate groups on the template and primers to neutralize the excess negative charge on the DNA, thereby improving the template-primer binding affinity and further increasing amplification efficiency. Preferably, the potassium salt is selected from either KCl or K₂SO₄, more preferably KCl.

[0023] In a preferred embodiment, when an enhancer is included, the final concentration of the enhancer in the PCR buffer can be arbitrarily selected within the range of 0.1 to 5 mg / mL, specifically within any one or any two of the following: 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, and 5 mg / mL. Preferably, the enhancer is selected from at least one of BSA and acetylated BSA, more preferably BSA.

[0024] In a preferred embodiment, when a surfactant is included, the volume fraction of the surfactant in the PCR buffer can be arbitrarily selected within the range of 0.001% to 0.05%, for example, any one or any two of 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, and 0.05%. Preferably, the surfactant is selected from Tween 20 and CA630, more preferably Tween 20.

[0025] In a preferred embodiment, the kit includes: a dNTP mixture, primer pairs, and Mg. 2+ And at least one of them in water.

[0026] This invention provides a method for rapid PCR amplification, which includes PCR amplification using the rapid Taq enzyme and PCR buffer from the kit described in any of the foregoing embodiments or implementations.

[0027] In a preferred embodiment, the amount of rapid Taq enzyme in the PCR amplification reaction system is ≥0.15625 U / 25 μl. In an optional embodiment, the amount of rapid Taq enzyme can be 0.15625 U to 1.25 U / 25 μl.

[0028] In a preferred embodiment, the PCR amplification step includes: preparing a DNA template, primer pairs, the PCR buffer, dNTPs, and Mg. 2+ And the rapid Taq enzyme mixture is amplified based on PCR reaction conditions.

[0029] Mg 2+ It is essential for Taq DNA polymerase activity, Mg 2+ Besides affecting enzyme activity and fidelity, Mg concentration also influences primer annealing, the melting temperature of the template and PCR product, and the formation of specific primer dimers. 2+ At too low a concentration, enzyme activity is significantly reduced; at too high a concentration, the enzyme can catalyze non-specific amplification. Testing has shown that a lower concentration of Mg is required in this system. 2+ The concentration was as low as 1 mM. This is because excessively high MgCl2 concentrations may cause the pyrophosphate ions precipitated from dNTPs during the reaction to react with the MgCl2, producing a byproduct magnesium pyrophosphate (a milky white precipitate). This precipitate clumps together and cannot dissolve. Agarose gel electrophoresis of the precipitate showed a bright appearance, indicating the presence of the product. In a preferred embodiment, the MgCl2... 2+ The effective concentration can be arbitrarily selected within the range of 0.1 to 3 mM, for example, any one or any two of 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM and 3 mM.

[0030] Rapid PCR amplification is an amplification reaction with a total reaction time of less than 1 hour. In some implementations, the total amplification reaction time is less than 50 min, less than 40 min, less than 30 min, less than 20 min, or less than 10 min.

[0031] In a preferred embodiment, the reaction conditions for the rapid PCR amplification are selected from either condition 1 or condition 2: Condition 1 includes: 93~97℃, 2min15s~2min45s; (95~99℃, 0.5~1.5s; 45~65℃, 4~6s; 70~74℃, 4~6s)×30; 70~74℃, 4~6min; 11~13℃, ∞; Condition 2 includes: (95~99℃, 0.5~1.5s; 58~62℃, 14~16s)×30; 70~74℃, 4~6min; 11~13℃, ∞.

[0032] In addition, embodiments of the present invention also provide a rapid PCR buffer comprising the following components: buffer pair Bis-Tris, salt ions, surfactant, and enhancer.

[0033] It is understood that the components and concentrations of the rapid PCR buffer in this embodiment can be the same as those described in any of the corresponding embodiments or embodiments mentioned above, and will not be repeated here.

[0034] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0035] Example 1 This embodiment provides a fast PCR buffer (5×Fast buffer), the components of which are shown in Table 1.

[0036] Table 1 Components of 5×Fast buffer

[0037] A rapid PCR amplification method is provided, which uses the aforementioned 5×Fast buffer and rapid Taq enzyme at 5 U / μl AKTaq enzyme (Feipeng AK Taq DNA polymerase, catalog number: MD099) for rapid PCR amplification. The specific steps are as follows.

[0038] Reaction system: as shown in Table 1.

[0039] The reaction conditions were: 95℃, 2 min 30 s; (98℃, 1 s; 55℃, 5 s; 72℃, 5 s) × 30; 72℃, 5 min; 12℃, ∞.

[0040] All results from the examples are consistent with this rapid three-step procedure. Alternatively, a two-step reaction condition can be selected: (98℃, 1s; 60℃, 15s) × 30; 72℃, 5min; 12℃, ∞. Both reaction methods are rapid PCR procedures, and there is no significant difference in their effectiveness; therefore, no two-step results are presented.

[0041] Verification Example 1 Multiple rapid PCR buffers were prepared, largely the same as in Example 1, differing only in the buffer pairs and pH values, as detailed in Table 2. Rapid PCR was then performed with four different enzyme concentrations (1.25 U / 25 μl, 0.625 U / 25 μl, 0.3125 U / 25 μl, and 0.15625 U / 25 μl) to verify the effect of different buffer pairs and pH values ​​on amplification efficiency. The results are shown in the table below. Figure 1 .

[0042] Table 2. Amplification results under different pH conditions with different buffer pairs.

[0043] The results show that only Bis Tris exhibited significant amplification of the target band in all four enzyme concentrations (1.25 U / 25 μl, 0.625 U / 25 μl, 0.3125 U / 25 μl, and 0.15625 U / 25 μl), and the amplification results were relatively stable. This indicates that Bis Tris can promote amplification at low enzyme concentrations (0.15625 U / 25 μl) in rapid PCR, meaning that a buffer pair with strong buffering capacity is needed in rapid PCR to preserve enzyme activity within a short period of time.

[0044] Furthermore, four sets of rapid PCR buffers were prepared, one of which was the same as in Example 1, and the other three sets were largely the same as in Example 1, except for the Bis Tris concentration. The Bis Tris concentrations of the four sets were 150 mM, 85 mM, 37.5 mM, and 18.75 mM, respectively, and were tested. The results are as follows: Figure 2 As shown, the amplification bands of four enzyme amounts were most obvious when the final concentration of Bis Tris was 85 mM, and specific enzyme amounts could be effectively amplified at other concentrations.

[0045] Verification Example 2 Three sets of rapid PCR buffers were prepared. One set was the same as in Example 1, and the other two sets were largely the same as in Example 1, except for the ammonium salts. The ammonium salts for the three sets were 45 mM (NH4)2SO4, 45 mM NH4Cl, and 45 mM CH3COONH4, respectively. Rapid PCR was then performed at four different enzyme concentrations (1.25 U / 25 μl, 0.625 U / 25 μl, 0.3125 U / 25 μl, and 0.15625 U / 25 μl) to verify the effect of different ammonium salts on the amplification effect. The results are as follows: Figure 3 As shown.

[0046] The results show that, compared with the different ammonium salts tested in each group, the amplification results were best when using NH4Cl with a final concentration of 45mM.

[0047] Experiments were conducted at different concentrations, and (NH4)2SO4, NH4Cl, or CH3COONH4 were all effectively amplified at concentrations of 20 mM, 40 mM, and 60 mM.

[0048] Verification Example 3 Two sets of rapid PCR buffers were prepared. One set was the same as in Example 1, and the other set was largely the same as in Example 1, except for the potassium salt. The two sets were 45mM NH4Cl + 20mM K2SO4 and 45mM NH4Cl + 20mM KCl, respectively. Rapid PCR was then performed at four different enzyme concentrations (1.25U / 25μl, 0.625U / 25μl, 0.3125U / 25μl, and 0.15625U / 25μl) to verify the effect of different potassium salts on amplification efficiency. The results are as follows: Figure 4 As shown.

[0049] The results show that the target bands were very bright when using KCl at a final concentration of 20 mM, which is superior to K2SO4; adding 20 mM KCl to a solution containing 45 mM NH4Cl can further improve the amplification efficiency.

[0050] Experiments were conducted at different concentrations, and KCl or K2SO4 could be effectively amplified at concentrations of 5 mM, 30 mM, and 45 mM.

[0051] Verification Example 4 Three sets of rapid PCR buffers were prepared, one of which was the same as in Example 1, and the other two were largely the same as in Example 1, except for the different surfactants. The surfactants in the three sets were 0.02% Tween and 0.02% CA630, respectively. Rapid PCR was then performed under four enzyme concentrations (1.25U / 25μl system, 0.625U / 25μl system, 0.3125U / 25μl system, and 0.15625U / 25μl system) to verify the effect of different surfactants on the amplification effect.

[0052] Amplification results as follows Figure 5 As shown, the amplification effect is better under the conditions of 0.02% Tween 20 and 0.02% CA630.

[0053] Experiments were conducted at different concentrations, and Tween 20 or CA630 were effectively amplified at concentrations of 0.001%, 0.01%, 0.03%, and 0.05%.

[0054] Verification Example 5 Two sets of rapid PCR buffers were prepared. One set was the same as in Example 1, and the other set was largely the same as in Example 1, except for the different enhancers. The enhancers for the two sets were 2 mg / ml BSA and 2 mg / ml acetylated BSA, respectively. Rapid PCR was then performed under four enzyme concentrations (1.25 U, 0.625 U, 0.3125 U, and 0.15625 U / 25 μl system) to verify the effect of different enhancers on the amplification effect.

[0055] Amplification results as follows Figure 6 As shown, the amplification efficiency is significantly improved when using BSA, and BSA can be replaced by acetylated BSA. Under the same dosage conditions, the amplification efficiency of acetylated BSA is more significantly improved.

[0056] Experiments were conducted at different concentrations, and BSA or acetylated BSA could be effectively amplified at concentrations of 0.1 mg / mL, 0.5 mg / mL, 3.5 mg / mL and 5 mg / mL.

[0057] Verification Example 6 The PCR buffer (5×Fast buffer) provided in Example 1 was combined with TaKaRa's SpeedSTAR™ HSDNA Polymerase and compared with TaKaRa's SpeedSTAR™ HSDNA Polymerase + original 10×Buffer I. The amplification results are as follows: Figure 7 As shown.

[0058] The results show that using 5×Fast buffer instead of the original 10×Buffer I from TaKaRa's SpeedSTAR™ HS DNA Polymerase achieves the same amplification efficiency as the original kit (e.g., ...). Figure 7 (As shown).

[0059] Verification Example 7 The 5×Fast buffer from Example 1 was used to prepare a 2×Fast PCR Mix without primers and probes. Stability was assessed at -20°C, 4°C, and 37°C. The reaction system is as follows: Table 3. Preparation system of 2×Fast PCR Mix

[0060] The results show that... Figure 8 As shown.

[0061] After being kept at 4℃ for 20 days, subjected to 20 freeze-thaw cycles, and kept at 37℃ for 7 days, the target band amplified by this 2×Fast PCR Mix showed no significant difference from the control group stored at -20℃. The stability was accelerated by being kept at 37℃, indicating that the Mix has a shelf life of at least one year.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A rapid PCR amplification kit, characterized in that, It includes a PCR buffer and a rapid Taq enzyme; wherein the buffer pair used in the PCR buffer is Bis-Tris, the PCR buffer also includes salt ions, the salt ions include ammonium salt and potassium salt, the ammonium salt is NH4Cl, the potassium salt is KCl, the pH value of the PCR buffer is 8.0~9.2, and the final concentration of Bis-Tris in the PCR buffer is 15~150mM.

2. The rapid PCR amplification kit according to claim 1, characterized in that, The rapid Taq enzyme is selected from any one of the following: AK Taq enzyme from Phytoray, SpeedSTAR™ HS DNA polymerase from TaKaRa, Platinum™ Taq DNA polymerase from Thermo Fisher, and Taq enzyme from Novizan 2 × Rapid Taq Master Mix.

3. The rapid PCR amplification kit according to claim 1, characterized in that, The PCR buffer also includes at least one surfactant and an enhancer.

4. The rapid PCR amplification kit according to claim 1, characterized in that, The concentration of ammonium salt in the PCR buffer is 20-60 mM; the final concentration of potassium salt in the PCR buffer is 5-45 mM.

5. The rapid PCR amplification kit according to claim 3, characterized in that, When a surfactant is included, the volume fraction of the surfactant in the PCR buffer is 0.001% to 0.05%.

6. The rapid PCR amplification kit according to claim 5, characterized in that, The surfactant is selected from either Tween 20 or CA630.

7. The rapid PCR amplification kit according to claim 6, characterized in that, The surfactant is Tween 20.

8. The rapid PCR amplification kit according to claim 3, characterized in that, When an enhancer is included, the final concentration of the enhancer in the PCR buffer is 0.1~5 mg / mL.

9. The rapid PCR amplification kit according to claim 3, characterized in that, The reinforcing agent is selected from at least one of BSA and acetylated BSA.

10. The rapid PCR amplification kit according to any one of claims 1 to 9, characterized in that, The kit includes: dNTP mixture, primer pairs, and Mg. 2+ And at least one of them in water.

11. A method for rapid PCR amplification, characterized in that, It includes PCR amplification using the rapid Taq enzyme and PCR buffer in the kit as described in any one of claims 1 to 10.

12. The method for rapid PCR amplification according to claim 11, characterized in that, In the PCR amplification reaction system, the amount of the rapid Taq enzyme is ≥0.15625U / 25μl.

13. The method for rapid PCR amplification according to claim 11, characterized in that, The PCR amplification steps include: preparing the DNA template, primer pair, PCR buffer, dNTPs, and Mg. 2+ And the rapid Taq enzyme mixture is amplified based on PCR reaction conditions.

14. The method for rapid PCR amplification according to claim 13, characterized in that, The Mg 2+ The effective concentration is 0.1~3mM.

15. The method for rapid PCR amplification according to any one of claims 11 to 14, characterized in that, The total time for rapid PCR amplification reaction is less than 1 hour.

16. The method for rapid PCR amplification according to any one of claims 11 to 14, characterized in that, The reaction conditions for the rapid PCR amplification are selected from either condition 1 or condition 2: Condition 1 includes: 93~97℃, 2min15s~2min45s; 95~99℃, 0.5~1.5s, 45~65℃, 4~6s, 70~74℃, 4~6s, 30 cycles; 70~74℃, 4~6min; storage at 11~13℃. Condition 2 includes: 95~99℃, 0.5~1.5s, 58~62℃, 14~16s, 30 cycles; 70~74℃, 4~6min; storage at 11~13℃.

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