METHOD FOR QUICKLY HOMOGENIZING CIRCULAR DNA SAMPLES

Abstract

The present application provides a method for quickly homogenizing circular DNA samples, comprising performing rolling circle amplification on circular DNAs in the samples, so that the concentration of the circular DNAs in the samples are homogenized.

Claims

1. A method for homogenizing a plurality of samples comprising circular DNAs, comprising: performing rolling circle amplification on circular DNAs in the samples, so that the concentrations of the circular DNAs in the samples are homogenized.

2. The method of claim 1, wherein the method further comprises performing denaturing treatment on each sample before performing the rolling circle amplification.

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

4. The method of claim 2, wherein the denaturing treatment comprises heating the samples to 95 C. and keeping for 3 minutes, and then cooling to 4 C. and keeping.

5. The method of claim 2, wherein a system of the denaturing treatment comprises EDTA.

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

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

8. The method of claim 1, wherein the method comprises neither a step of quantifying an initial amount of the circular DNAs in the samples, nor a step of calculating a sample amount to be pipetted based on the measured amount of the circular DNAs in the samples.

9. A method for preparing a DNA library from a plurality of samples comprising circular DNAs, comprising treating the samples using the method of claim 1.

10. A next-generation sequencing method, comprising: treating a plurality of samples to be sequenced using the method of claim 1 to prepare a sequencing library, and performing a next-generation sequencing on the sequencing library.

11. The method of claim 5, wherein the system of the denaturing treatment comprises about 0.05 mM EDTA.

12. The method of 6, wherein the reaction system of the rolling circle amplification comprises about 0.1 mg/mL bovine serum albumin.

13. The method of claim 7, wherein the reaction system of the rolling circle amplification comprises about 75 mM KCl.

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

15. The method of claim 14, wherein the reaction system of the rolling circle amplification further comprises about 75 mM KCl.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows a flow chart of homogenization of circular DNAs of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

[0020] 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

[0021] This example compares the homogenization effect (i.e. the coefficient of variation quantified by Qubit) of the concentrations of DNA templates prepared by three different rolling circle amplification methods within the same time:

[0022] A. Related procedures for 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))

[0023] 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)

[0024] C. The homogenization method provided by the present application (hereinafter referred to as the present application)

[0025] The test samples were 16 bacterial solutions used in actual production which were transformed with plasmids. The OD600 value of each bacterial solution was measured with a spectrophotometer, and the results are shown in Table 1.

[0026] Three different sequencing template preparation processes were used to prepare sequencing templates, as described below:

[0027] 1) Step 1 (Denaturation Process): Performing Denaturation and Random Primer Annealing on a Bacterial Solution Template

[0028] 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 Component Volume (l) NEB reagent (phi29 DNA polymerase) 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 Methods in the literature Bacterial solution 2 TE buffer (10 mM Tris-HCl, 8 1 mM EDTA, pH 8.0, 25 C.) Total volume 10 The present application Bacterial solution 2 Denaturing buffer 5 (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0, 25 C.) 7 bp random primer (175 uM) 3 Total volume 10

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

TABLE-US-00002 Temperature Time (min) 95 C. 3 4 C. Keeping

[0030] 2) Step 2 (Amplification Process): Performing Isothermal Rolling Circle Amplification on Target Plasmid after Denaturation and Annealing

[0031] 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:

TABLE-US-00003 Component Volume (l) NEB reagent (phi29 DNA polymerase) Reaction product from Step 1 8.8 dNTP (10 mM) 0.5 BSA (10 mg/mL) 0.2 Phi29 DNA polymerase (10 U/l) 0.5 Total volume 10 Methods in the literature 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/l) 0.5 Yeast pyrophosphatase (0.1 U/l) 0.3 ddH.sub.2O 5.4 Total volume 20 The present application Reaction product from Step 1 10 10x reaction buffer (500 mM 2 Tris-HCl, 50 mM MgCl.sub.2, 750 mM KCl, 50 mM DTT, pH 8.2, 25 C.) BSA (10 mg/mL) 0.2 dNTP (3 mM) 2.4 Phi29 DNA polymerase (5 U/l) 3 ddH.sub.2O 2.4 Total volume 20

[0032] Three different sequencing template preparation methods used the same experimental operation flow of isothermal amplification, as shown in the following table:

TABLE-US-00004 Temperature Time 30 C. 4 hours 65 C. 10 minutes 4 C. Keeping

[0033] 3) RCA Product Treatment and Quantification

[0034] The RCA product has a high-order structure; therefore, in this example, all the RCA products were enzymatically digested with BamH1-HF to make the quantification more accurate. The NEB reagent (phi29 DNA polymerase) was configured by a digestion reaction system, and the methods in the literature and the method provided in the present application were configured by another enzymatic digestion reaction system, as described in the following table:

TABLE-US-00005 Component Volume (l) NEB reagent (phi29 DNA polymerase) 10x Cut Smart Buffer (Cat# B7203S, 3 500 mM potassium acetate 200 mM Tris-acetic acid 100 mM magnesium acetate 1,000 ug/ml BSA pH 7.9, 25 C.) RCA product 10 BamH1-HF (2 U/l, Cat# R3136S) 1 ddH.sub.2O 16 Total volume 30 Methods in the literature/The present application 10x Cut Smart Buffer (Cat# B7203S, 3 500 mM potassium acetate 200 mM Tris-acetic acid 100 mM magnesium acetate 1,000 ug/ml BSA pH 7.9, 25 C.) RCA product 20 BamH1-HF (2 U/l, Cat# R3136S) 1 ddH.sub.2O 6 Total volume 30

[0035] The experimental operation flow of digestion reaction in three different sequencing template preparation methods is consistent:

TABLE-US-00006 Temperature Time 37 C. 1 hour 65 C. 20 minutes 4 C. Keeping

[0036] 4 l of each sample after the above reaction was quantified using a Qubit fluorescence quantifier (Cat #Q33216, Thermo Fisher). The results are shown in Table 2. Based on the raw data in Tables 1 and 2, the coefficients of variation (ratio of standard deviation to mean) of the OD600 measurement of each initial bacterial solution, and of the Qubit values of the DNA templates respectively prepared by using the NEB phi29 DNA polymerase kit, the method in the literature, and the method of the present application in Table 2 were calculated, respectively. The results are shown in Table 3.

[0037] Results

[0038] In this example, the use of the method provided in the present application successfully achieves the homogenization of circular DNA samples. Meanwhile, we compared the differences among the method of NEB reagent product and the method reported in the literature and the method of the present application. The coefficient of variation of the OD600 measurement (see Table 1) before treatment on an initial bacterial solution was 0.408 (see Table 3); the coefficient of variation of the Qubit value (see Table 2 for details) of the DNA template obtained by treating with NEB reagent was 0.174 (see Table 3); and the coefficient of variation of the Qubit value (see Table 2) of the DNA template obtained by treating with the RCA method reported in the literature was 0.12 (see Table 3). The coefficient of variation of the Qubit value (see Table 2) of the DNA template obtained by the method of the present application was 0.086 (see Table 3). It can be seen that the coefficients of variation of the concentrations (see Table 2) of the DNA templates obtained after three treatments are all lower than the coefficient of variation of the OD value of the initial bacterial solution, and the effect of homogenizing the content of circular DNAs in the samples is achieved. In particular, the coefficient of variation of the sample obtained by the method of the present application is significantly lower than that of the sample obtained by the NEB reagent treatment and the method in the literature, indicating that the method provided by the present application further improves the homogenization effect of DNA samples.

TABLE-US-00007 TABLE 1 OD600 measurements of the bacterial solution samples of Example 1 Bacterial Solution OD600 Bacterial solution 1 0.269 Bacterial solution 2 0.38 Bacterial solution 3 0.264 Bacterial solution 4 0.331 Bacterial solution 5 0.308 Bacterial solution 6 0.277 Bacterial solution 7 0.772 Bacterial solution 8 0.305 Bacterial solution 9 0.308 Bacterial solution 10 0.286 Bacterial solution 11 0.291 Bacterial solution 12 0.305 Bacterial solution 13 0.658 Bacterial solution 14 0.255 Bacterial solution 15 0.354 Bacterial solution 16 0.341

TABLE-US-00008 TABLE 2 Qubit measurements of the amplified products obtained in each experimental method of Example 1 RCA product concentration (ng/L, after enzymatic digestion) NEB phi29 DNA polymerase Methods The reagent in the present Bacterial solution (Cat# M0269S) literature invention Bacterial solution 1 39.4 6.1 29 Bacterial solution 2 39 7 35.5 Bacterial solution 3 44.2 5.7 29.7 Bacterial solution 4 42.4 6.75 32.7 Bacterial solution 5 42.8 6.75 33.3 Bacterial solution 6 43 6.6 31.2 Bacterial solution 7 39.4 6.2 30.8 Bacterial solution 8 38.8 7.3 34.4 Bacterial solution 9 18.6 7.55 31.3 Bacterial solution 10 33.6 6.6 33 Bacterial solution 11 47.4 5.7 31.8 Bacterial solution 12 39.4 6.35 32.9 Bacterial solution 13 47 5.4 26.9 Bacterial solution 14 48.1 5.15 26.6 Bacterial solution 15 45 7.5 35.8 Bacterial solution 16 38.2 7.7 33.8

TABLE-US-00009 TABLE 3 Comparison table for coefficients of variation (CV) of the sample concentrations of Example 1. Coefficient of Type of measurement data variation (CV) OD600 value of the initial bacterial solution 0.408 Qubit value of the DNA template prepared by 0.174 homogenization using the NEB phi29 DNA polymerase kit Qubit value of the DNA template prepared by 0.12 homogenization using the method in the literature Qubit value of the DNA template prepared by 0.086 homogenization using the method of the present invention

[0039] In addition, the present invention is easy to use in connection with an automatic pipetting platform. Taking Example 1 as an example, after combining the present invention with an automatic pipetting platform, the time taken to homogenize 10,000 samples using the method of the present invention can be controlled within 5 hours; while the time taken by the conventional homogenization method (i.e. quantification-calculation-pipetting) increases linearly with the increase of the sample amount, and for the homogenization of 10,000 samples to be treated, it takes up to about 200 hours.