METHOD FOR RAPIDLY PREPARING SANGER SEQUENCING TEMPLATE

Abstract

A method for rapidly preparing a Sanger sequencing template, comprising: performing denaturing treatment on a sample containing circular DNAs; performing rolling circle amplification of the denaturing product; and treating the rolling circle amplification product with alkaline phosphatase to remove residual dNTP.

Claims

1. A method for rapidly preparing a Sanger sequencing template, comprising the steps of: performing denaturing treatment on a sample containing circular DNAs; performing rolling circle amplification of the denaturing product; and treating a rolling circle amplification product with alkaline phosphatase to remove residual dNTPs.

2. The method of claim 1, wherein the samples are mixed with random primers for the rolling circle replication before starting the denaturing treatment.

3. The method of claim 1, wherein the denaturing treatment comprises heating the samples to 95° C. and keeping for 3 minutes, and then cooling to 4° C. and keeping.

4. The method of claim 1, wherein a system of the denaturing treatment comprises EDTA.

5. The method of claim 1, wherein a reaction system of the rolling circle amplification comprises bovine serum albumin.

6. The method of claim 1, wherein the reaction system of the rolling circle amplification comprises KCl.

7. The method of claim 1, wherein the rolling circle amplification comprises an isothermal amplification at a temperature of 30° C. for 3 hours.

8. The method of claim 1, wherein the alkaline phosphatase treatment comprises treating a rolling circle amplification product at 37° C., in the presence of 0.01 to 0.1 U/uL.

9. The method of claim 1, wherein the entire preparation process according to the method is completed within 4 hours.

10. A Sanger sequencing method, comprising: preparing a sample to be sequenced by the method of claim 1, and directly performing Sanger sequencing on a template sample prepared by the method.

11. The method of claim 4, wherein the amount of EDTA is 0.04 mM.

12. The method of claim 5, wherein the amount of bovine serum albumin is 0.1 mg/mL.

13. The method of claim 6, wherein the amount of KCl is 75 mM.

14. The method of claim 8, wherein the method is performed for 30 minutes.

15. The method of claim 8, wherein the amount of shrimp alkaline phosphatase is 0.05 U/uL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows a flow chart of sequencing template preparation of the present invention.

[0017] FIG. 2 shows a comparison of typical sequencing results of pUC57 in Example 1.

[0018] FIG. 3 shows a comparison of typical sequencing results of pET in Example 1.

[0019] FIG. 4 shows a comparison of typical sequencing results of pTGE5 in Example 1.

[0020] FIG. 5 shows a comparison of typical sequencing results of pcDNA3.1 in Example 1.

DETAILED DESCRIPTION OF EMBODIMENTS

[0021] In order to further understand the method described in the present invention, the present invention will be further described below with reference to the accompanying drawings and examples.

Example 1

[0022] This example compares the sequencing effects of preparing a Sanger sequencing template within four hours by using three different rolling circle amplification methods for preparing a sequencing template:

A. NEB phi29 DNA polymerase reagent (Cat #M0269S, this reagent provides some supporting reagents and experimental protocols for RCA reactions) (hereinafter referred to as “NEB reagent (phi29 DNA polymerase)”)
B. RCA experimental protocol cited from literature (Frank B. Dean, John R. Nelson, Theresa L. Giesler, and Roger S. Lasken. 2001. Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification. Genome Research. 1095-1099) (hereinafter referred to as the “methods in the literature”)
C. Optimized RCA solution provided by the present application (hereinafter referred to as “the present application”)

[0023] The test samples were 32 bacterial solutions transformed with 4 different types of plasmids. The OD600 value of each bacterial solution was measured with a spectrophotometer, and the results are shown in Table 1.

[0024] Three different sequencing template preparation processes were used to prepare sequencing templates, as described below:

1) Step 1 (Denaturation Process): Performing Denaturation and Random Primer Annealing on a Bacterial Solution Template

[0025] The experimental samples were bacterial solutions transformed with plasmids as described above. The system configurations of denaturation reactions for three different sequencing template preparation methods are listed in the following table respectively:

TABLE-US-00001 NEB reagent (phi29 DNA polymerase) Component Volume (ul) Bacterial solution 1 10x reaction buffer 1 (500 mM Tris-HCl, 100 mM MgCl.sub.2, 100 mM (NH.sub.4).sub.2SO.sub.4, 40 mMDTT, pH 7.5, 25° C.) 7 bp random primer (100 uM) 2.5 ddH.sub.2O 4.3 Total volume 8.8

TABLE-US-00002 Methods in the literature Component Volume (ul) Bacterial solution 2 TE buffer (10 mM Tris-HCl, 8 1 mM EDTA, pH 8.0, 25° C.) Total volume 10

TABLE-US-00003 Method of the present application Component Volume (ul) Bacterial solution 1 Denaturing buffer 2 (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0, 25° C.) 7 bp random primer (120 uM) 2 Total volume 5

[0026] Three different sequencing template preparation methods used the same experimental operation flow of denaturation reaction, as shown in the following table:

TABLE-US-00004 Temperature Time (min) 95° C. 3 4° C. Keeping
2) Step 2 (Amplification Process): Performing Isothermal Rolling Circle Amplification on Target Plasmid after Denaturation and Annealing

[0027] Using the reaction product from Step 1 as an experimental sample, the isothermal rolling circle amplification was performed according to three different sequencing template preparation methods. The system configuration of the amplification reaction in each method is shown in the following table:

[0028] The system configurations of amplification reactions in three different sequencing template preparation methods:

TABLE-US-00005 NEB reagent (phi29 DNA polymerase) Component Volume (ul) Reaction product from Step 1 8.8 dNTP (10 mM) 0.5 BSA (10 mg/mL) 0.2 Phi29 DNA polymerase (10 U/uL) 0.5 Total volume 10

TABLE-US-00006 Methods in the literature Component Volume (ul) Reaction product from Step 1 10 10x amplification buffer 2 (500 mM Tris-HCl, 50 mM MgCl.sub.2, 750 mM KCl, 1 mM DTT, pH 8.2, 25° C.) 7 bp random primer (100 uM) 1 dNTP (2.5 mM) 0.8 Phi29 DNA polymerase (10 U/ul) 0.5 Yeast pyrophosphatase (0.1 U/ul) 0.3 ddH.sub.2O 5.4 Total volume 20

TABLE-US-00007 Method of the present application Component Volume (ul) Reaction product from Step 1 5 10x reaction buffer (500 mM 1 Tris-HCl, 50 mM MgCl.sub.2, 750 mM KCl, 50 mM DTT, pH 8.2, 25° C.) BSA (10 mg/mL) 0.1 dNTP (2.5 mM) 1.6 Phi29 (10 U/uL) 1 ddH.sub.2O 1.3 Total volume 10

[0029] Three different sequencing template preparation methods used the same experimental operation flow of isothermal amplification, as shown in the following table:

TABLE-US-00008 Temperature Time 30° C. 3 hours 65° C. 10 minutes 4° C. Keeping

3) Step 3 (Digestion Process)

[0030] The method provided by the present application further comprises a shrimp alkaline phosphatase digestion treatment step after the isothermal amplification. The experimental sample in this step was the amplification product from Step 2.

[0031] The system configuration of the shrimp alkaline phosphatase treatment reaction is as follows:

TABLE-US-00009 Component Volume (ul) Amplification product from Step 2 10 10x Cut Smart Buffer 2 (520 mM potassium acetate, 200 mM Tris-acetic acid, 120 mM magnesium acetate, 1,000 μg/ml BSA, pH 7.9, 25° C.) Shrimp alkaline phosphatase 1 (rSAP) (1 U/uL) ddH.sub.2O 7 Total volume 20

[0032] The experimental operation flow of the shrimp alkaline phosphatase treatment reaction is as follows:

TABLE-US-00010 Temperature Time (min) 37° C. 30 65° C. 5 4° C. Keeping

4) On-Line Sequencing

[0033] 1 ul of an equal-concentration sequencing template prepared by each of the three different preparation methods described above within 4 hours was taken for Sanger sequencing, and compared with the sequencing result from standard shaking culture and extraction of plasmids (which took 14 hours). We found that for different plasmid types, the sequencing results of the template prepared by NEB reagent have the phenomena of doublet signals (i.e. fluorescence signals being superposed) and peak shape overlap (i.e. the peak shapes being not separate); the method reported in the literature has the phenomena of no signal, weak sequencing signals (i.e. the peak height being too low) and high background peak (i.e., the background signal value being too high); and only the RCA method optimized in the present application is consistent with the sequencing effect obtained by standard shaking culture plus extraction of plasmids (i.e. the main signal peak being clear and independent, and the background peak value being low).

[0034] This example utilizes the optimized method provided by the present application to successfully complete the preparation of a sequencing template within 4 hours, with a sequencing success rate of 100%. Meanwhile, we also compared the differences between the NEB reagent product method, the method reported in the literature and the method of the present invention in the template prepared within the same 4 hours. The sequencing success rate of the method of the present invention is much higher as compared with the other two methods (see Table 1), and is consistent with the sequencing effect obtained by standard shaking culture and extraction of plasmids (which takes 14 hours). It generally takes 16 hours to achieve the same effect in the case of using an unoptimized RCA to prepare a Sanger sequencing template. It indicates that the present method has great advantages in the rapid preparation of plasmid sequencing templates (that is, the sequencing template preparation time is shortened to within 4 hours on the premise that the sequencing success rate remains unchanged).

TABLE-US-00011 TABLE 1 Comparison of sequencing success rates in Example 1 Sequencing success rate Method Method of Sample NEB in the the present Sample type number reagent literature application pUC57 series bacterial 8 62.5%  0% 100% solution pET series bacterial 8 12.5%  0% 100% solution pTGE5 series bacterial 8 .sup. 75% 75% 100% solution pcDNA3.1 series bacterial 8  100% 50% 100% solution