Anhydride-modified DNA polymerase, preparation method thereof, PCR reaction reagent and application thereof

Abstract

The application belongs to the technical field of molecular biology, and discloses an acid anhydride modified DNA polymerase, a preparation method thereof, a PCR reaction reagent and application. The acid anhydride modified DNA polymerase is obtained by subjecting the DNA polymerase to an acid anhydride modification reaction with an acid anhydride compound, and the DNA polymerase comprises an amino acid fragment with a sequence shown in SEQ ID NO:1. The acid anhydride modified DNA polymerase has good anti-inhibition effect and can be well applied to extract-free PCR.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology, and particularly relates to an acid anhydride modified DNA polymerase, a preparation method thereof, a PCR reaction reagent and application thereof. BACKGROUND

[0002] Polymerase chain reaction (PCR) is a specific and sensitive in vitro nucleic acid amplification technique, which is widely used in food safety, drug development, agricultural science and biomedical fields. At present, in order to comply with the trend of rapid amplification and rapid detection, researchers further put forward the extraction-free PCR, which directly amplifies after simple pretreatment of the sample. However, there may be inhibitory substances in the sample matrix, nucleic acid treatment reagent and reaction system used in the extraction-free PCR, and such substances will inhibit the activity of the DNA polymerase. The DNA polymerase is a key substance in PCR, and its amplification performance, inhibition resistance and thermal stability will greatly affect the final amplification effect of PCR. The inhibition may cause the amplification efficiency to decrease, and even may cause the amplification to fail.

[0003] Therefore, it is of great significance to obtain a DNA polymerase with good inhibition resistance for the research and development of extraction-free and rapid PCR. SUMMARY

[0004] The first object of the present application is to provide an acid anhydride modified DNA polymerase. The acid anhydride modified DNA polymerase has good inhibition resistance and can be well applied to extraction-free PCR.

[0005] The second object of the present application is to provide a preparation method of the acid anhydride modified DNA polymerase.

[0006] The third object of the present application is to provide a PCR reaction reagent.

[0007] The fourth object of the present application is to provide a PCR reaction kit.

[0008] The fifth object of the present application is to provide application of the acid anhydride modified DNA polymerase and / or the PCR reaction reagent in in vitro nucleic acid amplification.

[0009] Specifically, the acid anhydride modified DNA polymerase provided by the present application is obtained by subjecting the DNA polymerase to an acid anhydride modification reaction with an acid anhydride compound, and the amino acid sequence of the DNA polymerase is shown in SEQ ID NO: 1 or a variant sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1.

[0010] Further, the acid anhydride compound includes one or more of acetic anhydride, hexanoic anhydride, cyclohexanoic anhydride, citraconic anhydride, maleic anhydride and succinic anhydride.

[0011] Further, the acid anhydride compound is selected from at least one of citraconic anhydride, maleic anhydride and succinic anhydride.

[0012] The preparation method of the acid anhydride modified DNA polymerase provided by the present application comprises: mixing the DNA polymerase with an acid anhydride compound, and performing an acid anhydride modification reaction to obtain the acid anhydride modified DNA polymerase.

[0013] Further, the acid anhydride compound is added in an amount of 0.01-0.1 mol / mg based on the total mass of the DNA polymerase.

[0014] Further, the acid anhydride modification reaction is performed at a temperature of 40-45℃, a pH of 9-9.5 and for a time of 2-6h.

[0015] The PCR reaction provided by the present application actually includes the acid anhydride modified DNA polymerase.

[0016] Further, the PCR reaction reagent includes dNTPs, Tris-HCl buffer with a pH of 7.5-8.5, Mg 2+ and KCl.

[0017] Further, the PCR reaction reagent includes 0.001-0.01 μg / μL of the acid anhydride modified DNA polymerase, 0.1-0.5 mM of dNTPs, 10-20 mM of Tris-HCl buffer with a pH of 7.5-8.5, 1-5 mM of Mg 2+ and 25-50 mM of KCl.

[0018] The present application also provides the acid anhydride modified DNA polymerase and / or the PCR reaction reagent for use in in vitro nucleic acid amplification.

[0019] Advantages:

[0020] The acid anhydride modified DNA polymerase provided by the present application is obtained by performing an acid anhydride modification reaction on a DNA polymerase with a specific structure using an acid anhydride compound. The acid anhydride modification seals the enzyme activity of the DNA polymerase, improves its stability, and reduces the inhibitory effect of inhibitory substances on the DNA polymerase. High temperature conditions can reduce the adverse effects of PCR inhibitors on the amplification reaction. The acid anhydride modified DNA polymerase has good thermal stability and can maintain high enzyme activity at high temperatures. Therefore, the amide bond can be broken and the acid anhydride compound can be removed by high temperature conditions, thereby gradually releasing the enzyme activity and achieving good amplification effect. The acid anhydride modified DNA polymerase can be well applied to extract-free PCR. DETAILED DESCRIPTION

[0021] The present application provides an acid anhydride modified DNA polymerase, which is obtained by subjecting a DNA polymerase to an acid anhydride modification reaction with an acid anhydride compound.

[0022] In the present application, the DNA polymerase specifically includes an amino acid fragment having a sequence as shown in SEQ ID NO: 1. In this case, the DNA polymerase has excellent thermal stability and can maintain good enzyme activity under high temperature conditions.

[0023] In the present application, the acid anhydride compound is a compound formed by removing one molecule of water from an oxygen-containing acid, which can react with the amino group on the amino acid to form an amide bond to achieve modification of the DNA polymerase.

[0024] In some specific embodiments, specific examples of the acid anhydride compound include, but are not limited to, one or more of acetic anhydride, hexanoic anhydride, cyclohexanoic anhydride, citraconic anhydride, maleic anhydride, and succinic anhydride. In some more preferred modes, the acid anhydride compound is preferably at least one selected from citraconic anhydride, maleic anhydride, and succinic anhydride.

[0025] Based on the purpose of obtaining the acid anhydride modified DNA polymerase described above, the present application also provides a preparation method of the acid anhydride modified DNA polymerase, which comprises mixing the DNA polymerase with an acid anhydride compound and subjecting to an acid anhydride modification reaction to obtain the acid anhydride modified DNA polymerase.

[0026] In the present application, the amount of the acid anhydride compound added is preferably 0.01-0.1 mol / mg, such as 0.01 mol / mg, 0.03 mol / mg, 0.05 mol / mg, 0.08 mol / mg, 0.1 mol / mg, or any value therebetween, based on the total mass of the DNA polymerase. In this case, the acid anhydride compound can achieve good acid anhydride modification effect while not affecting the enzyme activity of the DNA polymerase.

[0027] In the present application, the conditions of the acid anhydride modification reaction include a temperature of preferably 40-45℃, such as 40℃, 40.5℃, 41℃, 42℃, 43℃, 45℃, or any value therebetween; a pH of preferably 9-9.5, such as 9, 9.1, 9.2, 9.3, 9.4, 9.5, or any value therebetween; and a time of preferably 2-6h, such as 2h, 2.5h, 3h, 4h, 5h, 6h, or any value therebetween.

[0028] Based on the good anti-inhibition effect of the acid anhydride modified DNA polymerase, the application further provides a PCR reaction reagent. The PCR reaction reagent comprises the acid anhydride modified DNA polymerase.

[0029] In the application, the PCR reaction reagent is a kind of reagent commonly used in PCR, and a person skilled in the art can make adaptive selection according to actual needs, and the application does not particularly limit it.

[0030] In some specific embodiments, the PCR reaction reagent specifically comprises dNTPs, Tris-HCl buffer (pH = 7.5-8.5), Mg 2+ , and KCl. In some more preferred modes, in the PCR reaction reagent, the concentration of the acid anhydride modified DNA polymerase is preferably 0.001-0.01 μg / μL, such as 0.001 μg / μL, 0.003 μg / μL, 0.005 μg / μL, 0.008 μg / μL, 0.01 μg / μL or any value therebetween; the concentration of dNTPs is preferably 0.1-0.5 mM, such as 0.1 mM, 0.13 mM, 0.15 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM or any value therebetween; the concentration of Tris-HCl buffer (pH = 7.5-8.5) is preferably 10-20 mM, such as 10 mM, 11 mM, 13 mM, 15 mM, 18 mM, 20 mM or any value therebetween; the concentration of Mg 2+ is preferably 1-5 mM, such as 1 mM, 1.3 mM, 1.8 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM or any value therebetween; and the concentration of KCl is preferably 25-50 mM, such as 25 mM, 28 mM, 30 mM, 34 mM, 38 mM, 40 mM, 45 mM, 50 mM or any value therebetween.

[0031] Based on the good anti-inhibition effect of the acid anhydride modified DNA polymerase, the application further provides application of the acid anhydride modified DNA polymerase and / or the PCR reaction reagent in in vitro nucleic acid amplification.

[0032] The amino acid and nucleotide sequences involved in the application are shown in Table 1.

[0033]

[0034]

[0035]

[0036] Embodiments of the present application are described in detail below, examples of which are intended to explain the present application and are not to be understood as limiting the present application. When a specific technique or condition is not mentioned in the embodiments, the technique or condition described in the literature in the art or according to the product manual is used. When a reagent or instrument is not mentioned by the manufacturer, it is a conventional product that can be obtained on the market.

[0037] Example 1

[0038] This example is used to illustrate a DNA polymerase whose amino acid sequence is shown as SEQ ID NO: 1 and the preparation thereof, which specifically comprises:

[0039] (1) The coding gene (nucleotide sequence shown as SEQ ID NO: 2) of the DNA polymerase obtained after E. coli optimization is added with Nde I and Xho I enzyme sites at both ends and sent to a chemical company for synthesis, which is recombined into a His-tagged vector pET24a(+) using Nde I and Xho I enzymes to obtain a recombinant plasmid, and the recombinant plasmid is amplified in a large amount using competent E. coli DH5α and a plasmid extraction kit (Merck, product number PLN350) to obtain a large amount of recombinant plasmid.

[0040] (2) The recombinant plasmid is transformed into competent E. coli BL21(DE3), and the transformed cells are inoculated into LB liquid medium containing 50 mg / L kanamycin sulfate and cultured at 37°C and 200 rpm overnight to obtain a single colony that can grow, which is an enzyme-producing engineering strain.

[0041] (3) The enzyme-producing engineering strain is inoculated into fresh LB liquid medium containing 50 mg / L kanamycin sulfate at an inoculation amount of 1%, and cultured at 37°C and 200 rpm until the OD 600 of the culture solution is 0.8, then IPTG is added at a final concentration of 0.1 mM, and the culture is cultured at 18°C and 200 rpm for 16 h to obtain an induced expression culture solution; the induced expression culture solution is centrifuged at 4°C and 10,000 rpm for 10 min to collect cell slurry; 1 g of the cell slurry is resuspended in 5 mL of Tris-HCl buffer (pH=7.5) with a concentration of 50 mM, and the cells are broken by ultrasonic treatment in an ice bath for 10 min at a working time of 4 s and an intermittent time of 4 s, and then centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant is collected to obtain a crude enzyme solution.

[0042] (4) The crude enzyme solution is purified using a Ni-NTA affinity chromatography column (Shanghai Sangon, product number C597593) and referring to the instruction manual, and the eluate is collected to obtain a pure enzyme solution, and the pure enzyme solution is freeze-dried at -80°C for 12 h to obtain the DNA polymerase.

[0043] Example 2

[0044] This example is used to illustrate the preparation of anhydride modified DNA polymerase, which specifically comprises:

[0045] S1, according to the final concentration of 5mg / L, the pure enzyme solution provided by Example 1 is mixed with Tris-HCl buffer (40mM, pH=9) to obtain the enzyme solution to be treated;

[0046] S2, according to the final concentration of 0.1mol / L, citraconic anhydride is added to the enzyme solution to be treated, mixed uniformly, and the anhydride modification reaction is carried out at 42℃, 100rpm for 4h, and then dialysis buffer is used for dialysis, the concentrated solution is frozen and dried at-80℃ for 12h to obtain the anhydride modified DNA polymerase.

[0047] Among them, the dialysis buffer includes 0.1mM of ethylenediaminetetraacetic acid disodium, 50%(v / v) of glycerol and 50mM of Tris-HCl buffer (pH=9).

[0048] Example 3

[0049] This example uses the method provided in Example 2 to prepare anhydride modified DNA polymerase, the difference is that in step S2, the molar concentration of citraconic anhydride added is 0.01mol / L, and other conditions are the same, to obtain the anhydride modified DNA polymerase.

[0050] Example 4

[0051] This example uses the method provided in Example 2 to prepare anhydride modified DNA polymerase, the difference is that in step S2, the molar concentration of citraconic anhydride added is 0.05mol / L, and other conditions are the same, to obtain the anhydride modified DNA polymerase.

[0052] Example 5

[0053] This example uses the method provided in Example 2 to prepare anhydride modified DNA polymerase, the difference is that in step S2, the molar concentration of citraconic anhydride added is 0.5mol / L, and other conditions are the same, to obtain the anhydride modified DNA polymerase.

[0054] Example 6

[0055] This example uses the method provided in Example 2 to prepare anhydride modified DNA polymerase, the difference is that in step S2, maleic anhydride is used instead of citraconic anhydride at an equimolar concentration, and other conditions are the same, to obtain the anhydride modified DNA polymerase.

[0056] Example 7

[0057] The anhydride-modified DNA polymerase was prepared by the method provided in Embodiment 2, except that in step S2, succinic anhydride was used instead of citraconic anhydride in an equimolar concentration, and other conditions were the same, to obtain the anhydride-modified DNA polymerase.

[0058] Test Example 1

[0059] This test example was used to illustrate the catalytic activity and thermal stability of the DNA polymerase provided in Embodiment 1 and the anhydride-modified DNA polymerases provided in Embodiments 2-7. The test specifically included:

[0060] (1) The DNA polymerase, the anhydride-modified DNA polymerase, and water were mixed to prepare a test enzyme solution with a concentration of 0.05 μg / μL, and the test enzyme solution was incubated at 90°C for 0 min, 1 min, 5 min, 10 min, 1 h, 5 h, and 10 h. Then, the enzyme catalytic reaction was carried out at 74°C for 30 min using salmon sperm DNA as a template / primer, and the reaction was terminated immediately after the reaction by placing the reaction mixture on ice.

[0061] The reaction system of the enzyme catalytic reaction included 1 ng of salmon sperm DNA, 200 μmol / L of dNTPs, 1 μCi of [α-32P] dCTP, 20 mM of Tris-HCl, 2 mM of MgCl2, 5 mM of (NH4)2SO4, 10 mM of KCl, 0.1 g / L of BSA, and 0.05% of Triton X-100, and the pH was 8.0.

[0062] (2) 5 μL of the reaction product was added to an ion exchange paper disc, and after the spots were dried, they were washed 3 times with 2xSSC buffer (including 300 mM of NaCl and 30 mM of Na3C6H5O7, pH 7.0), and finally washed once with anhydrous ethanol in an ice bath and dried. The radioactivity of the penetrated material was determined by a liquid scintillation counter. One enzyme unit (1 U) was defined as the amount of enzyme that catalyzed the incorporation of 10 nmol of dNTPs into DNA. The results are shown in Table 2.

[0063] Table 2.

[0064]

[0065] As shown by the test results in Table 2, the DNA polymerase provided in Embodiment 1 has excellent enzyme activity and thermal stability. The DNA polymerase is anhydride-modified by the method provided in Embodiments 2-7, which can achieve the desired enzyme activity blocking effect without affecting the enzyme activity, and the enzyme activity of the polymerase can be restored after 5-10 min of heat treatment. At the same time, the anhydride modification can also improve the thermal stability of the DNA polymerase to some extent.

[0066] Test Example 2

[0067] This test example is used to illustrate the anti-inhibition performance of the DNA polymerase provided in Example 1 and the acid anhydride modified DNA polymerase provided in Examples 2-7, and a commercially available DNA polymerase (Beijing Baiolaibo, product number BTN101002) is used as a control. The test specifically includes:

[0068] (1) DNA polymerase, acid anhydride modified DNA polymerase and RNase-free water were mixed to prepare an enzyme solution with a concentration of 0.05 μg / μL.

[0069] (2) 1 mL of the collected sputum was mixed with 0.1 mL of sodium hydroxide solution (0.1 M) to perform liquefaction treatment for 30 min, and 10 mL of Tris-HCl buffer (1 mM, pH = 7.5) was added and mixed uniformly to obtain an interference solution; human genomic DNA and the interference solution were mixed according to a final concentration of 4 mg / L to obtain a DNA sample to be tested.

[0070] (3) The DNA sample to be tested was subjected to PCR amplification using the enzyme solution and the primer system shown in Table 4 to obtain a PCR amplification product with a length of 1 kb. The PCR reaction system includes: 2.5 μL of the DNA sample to be tested, 0.5 μL of dNTPs (10 mM), 2.5 μL of PCR amplification buffer, 2.5 μL of BSA (1 mg / L), 0.5 μL of each of the upper and lower primers (10 nmol / L), 1 μL of the enzyme solution, and ddH2O to make up to 25 μL. The PCR reaction program includes: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s; 58°C annealing for 15 s; 72°C extension for 2 min; 45 cycles; and 72°C extension for 5 min.

[0071] (4) 0.5 μL of the PCR amplification product and DNA Ladder were subjected to gel electrophoresis, the gel electrophoresis bands were analyzed by gray scale using Image J, and the band intensity of the PCR amplification product of the DNA polymerase provided in Example 1 was normalized to obtain the band normalized intensity of the PCR amplification product of Examples 2-7. The results are shown in Table 3.

[0072] Table 3.

[0073] Group Band Normalized Intensity Example 1 1.00 Example 2 7.68 Example 3 6.80 Example 4 7.59 Example 5 8.38 Example 6 6.71 Example 7 5.05 Commercially Available DNA Polymerase 0.93

[0074] As shown by the test results in Table 3, compared with the commercially available DNA polymerase, the DNA polymerase provided in Example 1 has good anti-inhibition performance, and modification of the DNA polymerase with citraconic anhydride, maleic anhydride and succinic anhydride can effectively improve its anti-inhibition performance.

[0075] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that those skilled in the art can make changes, modifications, substitutions and variations to the above-described embodiments without departing from the spirit and scope of the present application.

Claims

1. An anhydride-modified DNA polymerase, characterized in that The anhydride-modified DNA polymerase is obtained by subjecting DNA polymerase to an anhydride modification reaction with an anhydride compound. The amino acid sequence of the DNA polymerase is shown in SEQ ID NO:

1.

2. The anhydride-modified DNA polymerase according to claim 1, characterized in that The acid anhydride compound includes one or more of acetic anhydride, hexanoic anhydride, cyclohexanoic anhydride, citraconic anhydride, maleic anhydride and succinic anhydride.

3. The anhydride-modified DNA polymerase according to claim 1, characterized in that The acid anhydride compound is selected from at least one of citraconic anhydride, maleic anhydride and succinic anhydride.

4. The method for preparing the anhydride-modified DNA polymerase according to any one of claims 1 to 3, wherein: The preparation method comprises: mixing the DNA polymerase with an acid anhydride compound, performing an acid anhydride modification reaction, and obtaining the acid anhydride-modified DNA polymerase; and adding the acid anhydride compound in an amount of 0.05-0.1 mol / mg based on the total mass of the DNA polymerase.

5. The method for preparing anhydride-modified DNA polymerase according to claim 4, wherein The temperature of the acid anhydride modification reaction is 40-45° C., the pH is 9-9.5, and the reaction time is 2-6 hours.

6. A PCR reaction reagent, characterized in that: The PCR reaction reagent comprises the anhydride-modified DNA polymerase according to any one of claims 1 to 3.

7. The PCR reaction reagent according to claim 6, characterized in that The PCR reaction reagents include dNTPs, Tris-HCl buffer with a pH value of 7.5-8.5, Mg 2+ and KCl.

8. The PCR reaction reagent according to claim 6, characterized in that The PCR reaction reagents include 0.001-0.01 μg / μL of anhydride-modified DNA polymerase, 0.1-0.5 mM dNTPs, 10-20 mM Tris-HCl buffer with a pH value of 7.5-8.5, 1-5 mM MgCl2 2+ and 25–50 mM KCl.

9. Use of the anhydride-modified DNA polymerase according to any one of claims 1 to 3 and / or the PCR reaction reagent according to any one of claims 6 to 8 in in vitro amplification of nucleic acids for non-therapeutic or non-diagnostic purposes.

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