Patent Application
20180080092
The present invention relates to the field of molecular biology, and more particularly to one-stop treatment methods and reagents for breaking a nucleic acid by means of a transposase.
Since the pyrophosphate sequencing method invented by Roche, which has opened up the next generation of sequencing, until now, the next generation of sequencing has undergone a period of rapid development. However, with the development of high-throughput sequencing, the sample preparation with high-throughput and low-cost has become a key consideration in the field of sequencing. Sample processing methods and automation devices of various principles have been developed, including: samples fragmentation, terminal treatment of nucleic acid molecules and adapters ligation and the generation of final libraries.
The methods of samples fragmentation mainly include physical methods (such as ultrasound shear) and enzymatic methods (i.e., treatment of non-specific endonuclease). Wherein the physical methods are dominated by Covaris based on patented Adaptive Focused Acoustic (AFA) technology. Under an isothermal condition, the acoustic energy with a wavelength of 1 mm is focused on a sample by a spherical solid state ultrasonic sensor with >400 kHz, using geometric focusing acoustic energy. This method ensures the integrity of nucleic acid samples, and a high recovery rate can be achieved. Covaris's instruments include an economical M-series, a single-tube full-power S-series and higher-throughput E- and L-series. The randomization of fragments based on physical methods is good, but the physical methods depend on a large number of Covaris interrupters, and require subsequent separate terminal treatment, adapter ligation and PCR, and various purification operations. Wherein the enzymatic methods include the NEB Next dsDNA Fragmentase from NEB company. The reagent first cleaves the double stranded DNA to produce a random cleavage site, and then clears the complementary DNA strand by identifying the cleavage site through another enzyme to achieve the purpose of interruption. This reagent can be used for genomic DNA, whole genome amplification products and PCR products, and randomness is also good, but some artificial short fragments insertion and deletion will be generated. And also inevitably need to carry out subsequent separate terminal treatment, adapter ligation and PCR, and various purification operations. In addition, the transposase disrupting kit led by Nextera kit of Epicentra company (acquired by Illumina) has been used to complete the DNA fragmentation and the adapters ligation simultaneously using the transposase, thereby reducing the time of sample processing.
From the simplicity of the various operations, the method of interruption by transposase is far superior to other methods in terms of flux and ease of operation, but this interruption has its own shortcomings: subsequent enzymatic reactions will be inhibited by the transposase embedded in the target sequence, although relevant transposase kit manufacturers provide some reagents for low starting amounts sample (e.g., 5 ng of the starting amount of genomic DNA) transposase treatment to achieve free-purification after interruption. However, for a method with 50 ng starting amount which requires to increase the amount of transposase, it is necessary to remove the transposase by means of column or magnetic beads purification, which undoubtedly increases the cost and process of experimental procedures (
The present invention provides a one-stop treatment method and reagent for breaking a nucleic acid by means of a transposase, which are capable of achieving a one-stop treatment of from a nucleic acid interruption by transposase to a downstream PCR amplification reaction, and column or magnetic beads purification is not required, thus simplifying the experimental operating procedures and reducing experimental costs.
According to a first aspect of the present invention, a one-stop treatment method for breaking a nucleic acid by means of a transposase is provided, comprising the following steps:
randomly interrupting a nucleic acid using a transposase-embedded complex, wherein the transposase-embedded complex comprises a transposase and a first adapter comprising a transposase identification sequence;
adding a first reagent for treatment, so as to break the adsorption effect of the transposase and the target sequence of the nucleic acid;
adding a second reagent for treatment, so as to weaken the influence of the first reagent on the subsequent enzymatic reactions; and
performing a PCR reaction by using a product generated after the second reagent treatment as a template component, so as to obtain a PCR product with an adapter at each end of the interrupted nucleic acid fragment.
In the method of the present invention, the reaction product of the nucleic acid interruption by the transposase is treated with the first reagent to break the adsorption effect of the transposase and the target sequence of the nucleic acids, replacing the conventional column or magnetic beads purification which is complicated process and costly; followed by treatment with a second reagent to weaken the influence of the first reagent on the subsequent enzymatic reaction, ensuring that downstream PCR amplification proceeds smoothly.
As a preferred embodiment of the present invention, the first reagent comprises one or more members of the group consisting of a protease solution, a sodium dodecyl sulfate (SDS) solution and a NT buffer. These solutions allow the transposase to degrade or denature and escape from the target sequence of the nucleic acids. It is to be noted that the first reagent may be one or more members of the group consisting of the above solutions, wherein more of the above solutions may be two or three above solutions, such as the protease solution and the SDS solution, the SDS solution and the NT buffer, the protease solution and the NT buffer, the protease solution, the SDS solution and the NT buffer, wherein the NT buffer can be the NT buffer in S5 series of Truprep kit.
As a preferred embodiment of the present invention, ethylenediaminetetraacetic acid (EDTA) is further added for treatment after the treatment with the first reagent, if the first reagent comprises a protease solution. EDTA inhibits protease activity and thus prevents proteases from degrading enzymes in subsequent PCR reactions.
As a preferred embodiment of the present invention, the second reagent comprises Triton-X100 solution. Triton-X100, whose chemical name octylphenyl polyoxyethylene ether, as a nonionic surfactant, in the role of the present invention is to weaken the influence of the first reagent on the subsequent enzymatic reactions.
As a preferred embodiment of the present invention, the second reagent further comprises a Tween-20 solution if the first reagent comprises an SDS solution. The addition of Tween-20 could further weaken the influence of SDS on the subsequent enzymatic reaction and enhance the PCR effect. It should be noted that Tween-20 may be used as a component of the second reagent in the form of a mixture with Triton-X100; it may also be provided separately in the form of separation from Triton-X100, in which case the second reagent refers to the Triton-X100 solution and the Tween-20 solution. It is to be understood that the first reagent and the second reagent in the present invention are not intended to be limited to a single object or a combination of a plurality of objects. Also, in the present invention, concepts such as “first” and “second”, which are used in any case, should not be construed as having the meaning of order or technique, instead their role in the present invention is to distinguish themselves from other objects.
As a preferred embodiment of the present invention, after adding the second reagent for treatment and before performing the PCR reaction, a second adapter is ligated at a gap by a ligase, wherein the gap is a 9 bp base deletion formed after both ends of the interrupted nucleic acid each ligated to the first adapter. The incorporation of the second adapter makes it possible to obtain a PCR product with different adapter-sequences at both ends, when performing a PCR reaction by using a primer that specifically targets the first adapter and a primer that specifically targets the second adapter, respectively. Thereby the application of the interrupted nucleic acids is not limited by the effect of the presence of transposase identification sequences at both ends. The sequence of the second adapter is not limited and can be any sequence.
As a preferred embodiment of the present invention, the 3′ end of the second adapter is a dideoxynucleotide to prevent the second adapter from ligating to the first adapter.
According to a second aspect of the present invention, a one-stop treatment reagent for breaking a nucleic acid by means of a transposase is provided, the reagent comprises the following components:
a first reagent comprising one or more members of the group consisting of a protease solution, a SDS solution and a NT buffer to break the adsorption effect of the transposase and the target sequence of the nucleic acids; and
a second reagent comprising a Triton-X100 solution to weaken the influence of the first reagent on the subsequent enzymatic reactions.
It is to be noted that the first reagent may be one or more members of the group consisting of a protease solution, a SDS solution and a NT buffer, wherein more of the above solutions may be two or three above solutions, such as the protease solution and the SDS solution, the SDS solution and the NT buffer, the protease solution and the NT buffer, the protease solution, the SDS solution and the NT buffer, wherein the NT buffer can be the NT buffer in S5 series of Truprep kit.
As a preferred embodiment of the present invention, the first reagent further comprises an additional reagent containing EDTA if the first reagent comprises a protease solution. EDTA inhibits protease activity and prevents proteases from degrading enzymes in subsequent PCR reactions. The additional reagents containing EDTA are provided as part of the first reagent separately from the protease solution.
As a preferred embodiment of the present invention, the second reagent further comprises a Tween-20 solution if the first reagent comprises a SDS solution. Tween-20 may be provided as a component of the second reagent in the form of a mixture with Triton-X100, or may be provided separately from the Triton-X100.
As a preferred embodiment of the present invention, the reagent further comprises a transposase and a first adapter comprising a transposase identification sequence for forming a transposase-embedded complex to randomly interrupte the nucleic acids.
As a preferred embodiment of the present invention, the reagent further comprises PCR components for carrying out a PCR reaction using the product generated by the treatment of the second reagent as a template component. Wherein, the PCR components are well known to include DNA polymerase, PCR buffer, dNTPs, Mg2+ solution and primer, etc.
As a preferred embodiment of the present invention, the reagent further comprises a second adapter component for ligation into the gap formed by ligating the first adapter to the interrupted nucleic acid at both ends.
As a preferred embodiment of the present invention, the reagent further comprises a ligase component for ligating a second adapter to the gap formed by ligating the first adapter to the interrupted nucleic acid at both ends.
In the present invention, the nucleic acids to be interrupted may be a genomic DNA, a whole genome amplification product or a PCR product, which may be DNA or cDNA, and may be not limited to the source of the nucleic acids and may be nucleic acid samples derived from an animal, a plant or a microorganism.
In the present invention, the working concentration of the first reagent and the second reagent can be determined empirically by those skilled in the art. In general, in the first reagent, the working concentration of the protease is preferably from 50 to 5000 mAU/mL, more preferably from 75 to 3750 mAU/mL, most preferably 1500 mAU/mL; the working concentration of EDTA is preferably from 1 to 50 mmol/L, more preferably 14 mmol/L; the working concentration of SDS is preferably from 0.01% to 1.5% (by volume), more preferably 1% (by volume); the final concentration of NT buffer can be used according to 1×. In the second reagent, the working concentration of Triton-X100 is preferably from 0.1% to 2% (by volume), more preferably 1% (by volume); the working concentration of Tween-20 is preferably from 0.1% to 2% (by volume), more preferably 0.5% (by volume).
The method of the present invention utilizes the first reagent and the second reagent to treat the product of nucleic acids interrupted by the transposase, instead of the traditional column or magnetic beads purification, to achieve the one-stop treatment from the transposase interruption of the nucleic acids to the downstream PCR amplification. The whole process is carried out in a single tube, simplifying the experimental operating flow and reducing the cost of the experiment, shortening the processing cycle, making high-throughput sample processing possible.
The invention will now be described in further detail by way of specific examples. Unless otherwise specified, the techniques used in the examples below are conventional techniques known to those skilled in the art; the instruments and reagents used are accessable to those skilled in the art through public approaches such as commercial approaches and so on.
Referring to
Referring to
In the transposase embedding stage, the present invention selects two embedding modes: the first one is the transposase embedding the NO. 1 adapter in a double-adapters form to generate a transposase-embedded complex (
In the present invention, a transposase kit of domestic production (S50 series of Truprep kit of Nanjing Nuoweizan Ltd.) was used to carry out the following experiment. The kit contains two doses respectively for 5ng genomic DNA and 50 ng genomic DNA. In this embodiment, the dose for 50 ng genomic DNA was used to carry out an experiment.
In this example, 50 ng of high quality genomic DNA was first interrupted by an embedded transposase complex, followed by treating with protease, SDS, NT or a composition of protease and EDTA to remove the transposase protein bound to DNA; and then directly amplified using PCR primers, with a certain concentration of TritonX-100 is added into the PCR reaction system.
1. Three primer sequences with a 19 bp transposase identification sequence, sequence A, sequence B and sequence C were designed and prepared, for preparation of NO. 1 adapter in the form of double-adapters (referred to herein as the NO. 1 adapter) for embedding, wherein, sequence A+sequence B forms the 5′ end of the NO. 1 adapter in the form of double-adapters, and sequence A+sequence C forms the 3′ end of the NO. 1 adapter in the form of double-adapters:
2. The sequence A, sequence B and sequence C were diluted to 100 μM respectively, sequence A+sequence B combination, sequence A+sequence C combination, fully mixed and centrifuged, and then annealed to form NO. 1 adapter (stored at −20° C.) in a PCR instrument according to the following procedure (Table 1), and used for the preparation of embedded composites.
After the reaction, the two sets of annealed adapters were mixed in equal volume, for embedding the transposase complex.
3. The NO. 1 adapter and the transposase were embedded into a transposase-embedded complex according to the following system (Table 2), after gently blowing 20 times and incubating 1 hour at 30° C., the complex embedding was completed. The complex was stored at −20° C.
4. 50 ng of high quality genome and transposase complex were mixed according to the following system (Table 3), after gently mixing 20 times and incubating for 10 minutes at 55° C., and then cooling to 4° C., genome interruption is completed.
5. The sample processing methods after the interruption comprises the following options. Method 1: adding 1 times of the volume of PBI (a commercial reagent in Qiagen PCR purification kit), after mixing evenly, purifying with 1.3 times of Ampure XP beads, and dissolving with pure water. Method 2: 0.1-5 μL of protease (750 mAU/mL) was added and then added to a final concentration of 1-50 mM EDTA. This example preferred 2 μL of protease and final concentration of 14 mM EDTA, and at the same time 0.1 μL protease plus 1 mM EDTA and 5 μL of protease plus 50 mM EDTA was tested. Method 3: adding 0.01% to 1.5% (by volume) of SDS, preferably 1% (by volume) of SDS in this example, and 0.01% (by volume) and 1.5% (by volume) concentrations were tested separately. Method 4: adding the final concentration of commercial 1×NT buffer (a matching reagent in Truprep kit S5 series). Method 5: adding 0.1-5 μL of protease for treatment, preferably 2 μL of protease in this example, and 0.1 μL and 5 μL protease were tested separately.
6. In the product after the above treatment, 0.1%-2% (by volume) of Triton-X100 was added, preferably 1% (by volume) in this example, while 0.1% (by volume) and 2% (by volume) of Triton-X100 was used to test.
7. PCR amplification was carried out according to the following PCR reaction system (Table 4) and reaction conditions (Table 5). For the experimental group with SDS added, a specific concentration of Tween-20 was added to the PCR system to partially increase the efficiency of the PCR. The working concentration of Tween-20 could be adjusted to different, such as 0.1% -2% (by volume), preferably 0.5% (by volume) in this example, while the working concentrations of 0.1% (by volume) and 2% (by volume) was tested.
8. PCR product detection result of the transposase complex of the NO. 1 adapter in the form of double-adapters is shown in
In this example, 50 ng of high quality genomic DNA was first interrupted by an embedded transposase complex, treated with protease, SDS, NT or protease and EDTA compositions to remove the transposase protein bound to DNA. After the ligation of the gap adapter, PCR primers were used to amplify, and a certain concentration of TritonX-100 was added to the PCR reaction system.
1. A pair of primer sequences, sequences A and B, with a 19 bp transposase identification sequence were designed and used to prepare a NO. 1 adapter in the form of single-adapter for embedding (referred to as the NO. 1 adapter in the present invention).
2. The sequence A and sequence B were diluted to 100 μM respectively, fully mixed and centrifuged, and then annealed to form NO. 1 adapter (stored at −20° C.) in a PCR instrument according to the following procedure (Table 7), and used for the preparation of embedded composites.
3. The NO. 1 adapter and the transposase were embedded into a transposase-embedded complex according to the following system (Table 8), after gently blowing 20 times and incubating 1 hour at 30° C., the complex embedding was completed. The complex was stored at −20° C.
4. 50 ng of high quality genome and transposase complex were mixed according to the following system (Table 9), after gently mixing 20 times and incubating for 10 minutes at 55° C., and then cooling to 4° C., genome interruption is completed.
5. The sample processing methods after the interruption comprises the following options. Method 1: 0.1-5 μL of protease (750 mAU/mL) was added, in this example preferred 2 μL of protease, and at the same time 0.1 μL protease and 5 μL of protease was tested respectively. Method 2: adding the final concentration of commercial 1×NT buffer (a matching reagent in Truprep kit S5 series). Method 3: adding 0.01% to 1.5% (by volume) of SDS, preferably 1% (by volume) of SDS in this example, and 0.01% (by volume) and 1.5% (by volume) concentrations were tested separately. Method 4: 0.1-5 μL of protease (750 mAU/mL) was added and then added to a final concentration of 1-50 mM EDTA. This example preferred 2 μL of protease and final concentration of 14 mM EDTA, and at the same time 0.1 μL protease plus 1 mM EDTA and 5 μL of protease plus 50 mM EDTA was tested. Method 5: adding 1 times of the volume of PBI (a commercial reagent in Qiagen PCR purification kit), after mixing evenly, purifying with 1.3 times of Ampure XP beads, and dissolving with pure water.
6. In the product after the above treatment, 0.1%-2% (by volume) of Triton-X100 was added, preferably 1% (by volume) in this example, while 0.1% (by volume) and 2% (by volume) of Triton-X100 was used to test.
7. The product treated with the above methods was ligated to the annealed gap adapter (the NO. 2 adapter) according to the following system (Table 10), after annealing at 25° C. for 60 minutes, the adapter ligation was completed.
8. PCR amplification was carried out according to the following PCR reaction system (Table 11) and reaction conditions (Table 12). For the experimental group with SDS added, a specific concentration of Tween-20 was added to the PCR system to partially increase the efficiency of the PCR. The working concentration of Tween-20 could be adjusted to different, such as 0.1% -2% (by volume), preferably 0.5% (by volume) in this example, while the working concentrations of 0.1% (by volume) and 2% (by volume) was tested.
9. PCR product detection result of after interruption by single-adapter embedding complex and ligation of the gap adapter is shown in
The foregoing is a further detailed description of the present invention in reference with the specific embodiments, thus it cannot be determined that the specific implementation of the invention is limited to these above illustrations. It will be apparent to one skilled in the art to which the invention pertains that several simple deductions or substitutions may be made without departing from the inventive concept.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2014/088541 | 10/14/2014 | WO | 00 |
