CN112725905A - Digital microfluidic library construction system - Google Patents
Digital microfluidic library construction system Download PDFInfo
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Abstract
The invention constructs a chip through a full-flow full-automatic digital microfluidic library, wherein a single chip comprises a sample of full-flow library construction, and the chip comprises a low-temperature region for keeping the effectiveness of a reagent in a waiting process; comprises a temperature-variable region for providing temperature conditions required by each reaction in library construction; comprising a plurality of reservoir and distribution regions 2 for distributing a washing solution, a solution of magnetic beads and droplets of an elution solution, respectively. The user can realize the whole-flow DNA or RNA library construction only by loading the sample to the chip.
Description
Technical Field
The invention belongs to the technical field of digital microfluidic, and relates to a digital microfluidic library construction system.
Background
Library construction is one of the essential steps of DNA and RNA sequencing, and the library construction process is complex and usually includes many operations such as fragmentation, end repair, linker ligation, magnetic bead washing, library amplification, etc. The library construction process is complicated in steps and long in time consumption, and the existing library construction work mainly depends on manual operation or a large-scale workstation based on a mechanical arm. Fully automated laboratory sample processing platform such as beckmann coulter
4000 can complete liquid processing work such as liquid transferring, gradient dilution, liquid separation, liquid combination and the like, so that scientific research personnel are free from complicated experimental operation of library construction. The Neoprep product of Illumina and the Voltrax product of Oxford Nanopore both adopt the digital microfluidic technology to realize the full-automatic library construction.
Because the library construction process is complex and long in time consumption, the library construction is easy to fail due to misoperation in the manual operation process. The large workstation based on the mechanical arm is large in size and high in cost, and is only suitable for large enterprises, central laboratories, hospitals and other large-scale mechanisms. Currently, a low-cost miniaturized automatic library construction system is lacking in the market. The Neoprene product of Illumina was recalled in 2017 due to insufficient chip reliability, the Voltrax product of Oxford Nanopore was expensive due to the chip fabrication using thin film transistor technology, and the product was only suitable for library construction of Oxford Nanopore's own sequencing equipment.
Therefore, it is necessary to provide a new full-automatic digital microfluidic library construction chip with high integration level, small volume and low cost, which has wide application prospect and great market value in the technical field of DNA or RNA library construction.
Disclosure of Invention
In view of the deficiencies and practical needs of the prior art, the present invention provides a digital microfluidic library construction system. The method comprises the steps that a sample is contained in a single chip, all operations related to the experimental process, including reagent distribution, solution stirring, solution transfer, magnetic bead separation and the like, are automatically completed by the digital microfluidic chip, finally, products are conveyed to an output end for a user to extract, and the user can realize the full-process library construction only by loading the sample to the chip.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a digital microfluidic library construction chip, which comprises a plurality of sample/reagent inlets 1, a plurality of liquid storage and distribution areas 2, a waste liquid area 3, a product outlet 4, a low temperature area 5, a variable temperature area 6 and magnetic bead separation points 7;
the plurality of stock solution and distribution areas 2 can respectively distribute washing solution, magnetic bead solution and eluent liquid drops;
the temperature of the low-temperature zone 5 is set to 0-20 ℃, preferably 4 ℃, and is used for keeping the effectiveness of the reagent in the waiting process;
the temperature-variable region 6 is used for providing temperature conditions required by each reaction in library construction.
Preferably, the chip is filled with biocompatible medium oil.
Preferably, the plurality of sample/reagent inlets 1 are loaded with sample liquid and various reagents including an enzyme, an adaptor reaction reagent and an amplification reaction reagent, respectively;
preferably, the various reagents further comprise any one or a combination of at least two of a probe hybridization reaction reagent, an RNase H digestion reaction reagent, a D Nase I digestion reaction reagent, a first strand cDNA synthesis reaction reagent, a second strand cDNA synthesis reaction reagent and/or a fragmentation reaction reagent;
preferably, the plurality of stock solution and distribution areas 2 are loaded with washing solution, magnetic bead solution and eluent respectively;
preferably, the sample solution and various reagents can be loaded in a manner of injection by a pipette, loading by an independent reagent pack or pre-embedding the reagents in a chip;
preferably, the sample liquid and various reagents are located in a low-temperature zone 5;
preferably, the washing solution is a 70% -95% ethanol solution, preferably an 80% ethanol solution.
In a second aspect, the present invention provides a digital microfluidic library construction system, which includes the digital microfluidic library construction chip of the first aspect.
In the invention, the basic process of the digital microfluidic library construction system is as follows:
(1) loading: loading the chip on the detection device, and loading the sample solution and various reagents in the corresponding inlets according to the volume shown in fig. 2 or fig. 5;
(2) fragmentation, end repair, linker ligation: transferring the mixed solution containing the sample solution and the enzyme to a variable temperature region 6, and controlling the temperature to realize fragmentation, end repair and joint connection reaction;
(3) adaptation and connection reaction: transferring the adaptive ligation reaction reagent to a temperature-variable region 6, fully mixing the adaptive ligation reaction reagent with the product obtained in the third step, and controlling the temperature to realize adaptive ligation reaction;
(4) and (3) magnetic bead binding: fully stirring the product obtained in the step (3) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(5) washing: fully stirring the magnetic beads obtained by separation in the step (4) and the liquid drops of the washing liquid distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(6) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (5) and eluent liquid drops distributed by the liquid storage and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a temperature changing area 6;
(7) cleaning: the stock solution and distribution region 2 distributes an eluent droplet cleaning path and transfers used magnetic beads to the waste solution region 3;
(8) and (3) amplification reaction: transferring the amplification reaction reagent to the temperature-variable region 6 to be fully mixed with the product in the step (6), and controlling the temperature to realize the amplification reaction;
(9) and (3) magnetic bead binding: fully stirring the product obtained in the step (8) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(10) washing: fully stirring the magnetic beads obtained by separation in the step (9) and the liquid drops of the washing liquid distributed by the liquid storage and distribution area 2, and performing magnetic bead separation at a magnetic bead separation point 7;
(11) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (10) and eluent liquid drops distributed by the stock solution and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a product outlet 4;
(12) and (3) extracting a product: the user extracts the product from the product outlet 4 for subsequent experiments.
Preferably, step (1) is preceded by a step of manually preparing the sample liquid, and the step of manually preparing the sample liquid specifically includes: and manually preparing a mixed solution of the sample, the deionized water and the buffer solution, and uniformly mixing by shaking. The reason for this step being manual is that the amount of sample required differs for different kinds of samples.
Preferably, in the step (1), the chip is filled with biocompatible medium oil;
preferably, in step (1), the various reagents include an enzyme, an aptameric ligation reaction reagent, and an amplification reaction reagent;
preferably, the various reagents further include any one or a combination of at least two of a probe hybridization reaction reagent, an RNase H digestion reaction reagent, a DNase I digestion reaction reagent, a first strand cDNA synthesis reaction reagent, a second strand cDNA synthesis reaction reagent, and/or a fragmentation reaction reagent;
preferably, the plurality of stock solution and distribution areas 2 are loaded with washing solution, magnetic bead solution and eluent respectively;
preferably, the sample solution and various reagents can be loaded in a manner of pipette injection, a manner of loading independent reagent packs or a manner of pre-embedding reagents in a chip.
Preferably, the sample liquid and various reagents are located in the low temperature zone 5.
Preferably, the washing solution is a 70% -95% ethanol solution, preferably an 80% ethanol solution.
Preferably, the washing steps of step (5) and step (10) are each repeated 3 times.
Preferably, for the RNA library, the steps of probe hybridization reaction, RNase H digestion reaction and DNase I digestion reaction are further included after the step (1) and before the step (2);
the probe hybridization reaction is to transfer a mixed solution containing a sample, deionized water and the probe hybridization reaction to a temperature-variable region 6, and the temperature is controlled to realize the probe hybridization reaction;
the RNase H digestion reaction is that an RNase H digestion reaction reagent is transferred to the temperature-variable region 6 to be fully mixed with a probe hybridization reaction product, and the temperature is controlled to realize the RNase H digestion reaction;
and the DNase I digestion reaction is realized by transferring a DNase I digestion reaction reagent to the temperature-variable region 6 to be fully mixed with an RNase H digestion reaction product and controlling the temperature.
Preferably, for the RNA library, the steps of a first strand cDNA synthesis reaction and a second strand cDNA synthesis reaction are further included after the fragmentation reaction and before the ligation reaction;
the first strand cDNA synthesis reaction is to transfer a first strand cDNA synthesis reaction reagent to a temperature changing region 6 to be fully mixed with the fragmentation reaction product, and the temperature is controlled to realize the first strand cDNA synthesis reaction;
the second strand cDNA synthesis reaction is realized by transferring a second strand cDNA synthesis reaction reagent to the temperature-variable region 6 to be fully mixed with the first strand cDNA synthesis reaction product and controlling the temperature.
Preferably, the subsequent experiment of step (12) comprises sequencing.
Preferably, the digital microfluidic library construction system can be further matched with an optical detection module;
preferably, the optical detection module is a fluorescence detection module used for quality control in the library construction process.
In a third aspect, the present invention provides a method for constructing a library using the chip of the first aspect, the method comprising:
(1) loading: loading a chip on detection equipment, and loading a sample solution and various reagents into corresponding inlets according to a specified volume;
(2) fragmentation, end repair, linker ligation: transferring the mixed solution containing the sample solution and the enzyme to a variable temperature region 6, and controlling the temperature to realize fragmentation, end repair and joint connection reaction;
(3) adaptation and connection reaction: transferring the adaptive ligation reaction reagent to a temperature-variable region 6, fully mixing the adaptive ligation reaction reagent with the product obtained in the third step, and controlling the temperature to realize adaptive ligation reaction;
(4) and (3) magnetic bead binding: fully stirring the product obtained in the step (3) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(5) washing: fully stirring the magnetic beads obtained by separation in the step (4) and the liquid drops of the washing liquid distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(6) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (5) and eluent liquid drops distributed by the liquid storage and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a temperature changing area 6;
(7) cleaning: the stock solution and distribution region 2 distributes an eluent droplet cleaning path and transfers used magnetic beads to the waste solution region 3;
(8) and (3) amplification reaction: transferring the amplification reaction reagent to the temperature-variable region 6 to be fully mixed with the product in the step (6), and controlling the temperature to realize the amplification reaction;
(9) and (3) magnetic bead binding: fully stirring the product obtained in the step (8) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(10) washing: fully stirring the magnetic beads obtained by separation in the step (9) and the liquid drops of the washing liquid distributed by the liquid storage and distribution area 2, and performing magnetic bead separation at a magnetic bead separation point 7;
(11) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (10) and eluent liquid drops distributed by the stock solution and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a product outlet 4;
(12) and (3) extracting a product: the user extracts the product from the product outlet 4 for subsequent experiments.
Preferably, step (1) is preceded by a step of manually preparing the sample liquid, and the step of manually preparing the sample liquid specifically includes: and manually preparing a mixed solution of the sample, the deionized water and the buffer solution, and uniformly mixing by shaking. The reason for this step being manual is that the amount of sample required differs for different kinds of samples.
Preferably, in the step (1), the chip is filled with biocompatible medium oil;
preferably, in step (1), the various reagents include an enzyme, an aptameric ligation reaction reagent, and an amplification reaction reagent;
preferably, the various reagents further comprise any one or a combination of at least two of a probe hybridization reaction reagent, an RNase H digestion reaction reagent, a D Nase I digestion reaction reagent, a first strand cDNA synthesis reaction reagent, a second strand cDNA synthesis reaction reagent and/or a fragmentation reaction reagent;
preferably, the plurality of stock solution and distribution areas 2 are loaded with washing solution, magnetic bead solution and eluent respectively;
preferably, the sample solution and various reagents can be loaded in a manner of pipette injection, a manner of loading independent reagent packs or a manner of pre-embedding reagents in a chip.
Preferably, the sample liquid and various reagents are located in the low temperature zone 5.
Preferably, the washing solution is a 70% -95% ethanol solution, preferably an 80% ethanol solution.
Preferably, the washing steps of step (5) and step (10) are each repeated 3 times.
Preferably, for the RNA library, the steps of probe hybridization reaction, RNase H digestion reaction and DNase I digestion reaction are further included after the step (1) and before the step (2);
the probe hybridization reaction is to transfer a mixed solution containing a sample, deionized water and the probe hybridization reaction to a temperature-variable region 6, and the temperature is controlled to realize the probe hybridization reaction;
the RNase H digestion reaction is that an RNase H digestion reaction reagent is transferred to the temperature-variable region 6 to be fully mixed with a probe hybridization reaction product, and the temperature is controlled to realize the RNase H digestion reaction;
and the DNase I digestion reaction is realized by transferring a DNase I digestion reaction reagent to the temperature-variable region 6 to be fully mixed with an RNase H digestion reaction product and controlling the temperature.
Preferably, for the RNA library, the steps of a first strand cDNA synthesis reaction and a second strand cDNA synthesis reaction are further included after the fragmentation reaction and before the ligation reaction;
the first strand cDNA synthesis reaction is realized by transferring a first strand cDNA synthesis reaction reagent to a temperature-variable region 6, fully mixing the first strand cDNA synthesis reaction reagent with a fragmentation reaction product, and controlling the temperature;
and the second strand cDNA synthesis reaction is realized by transferring a second strand cDNA synthesis reaction reagent to the temperature-variable region 6 to be fully mixed with a first strand cDNA synthesis reaction product and controlling the temperature.
Preferably, the subsequent experiment of step (12) comprises sequencing.
Preferably, the digital microfluidic library construction method may further comprise a step of performing quality control by an optical detection module;
preferably, the optical detection module is a fluorescence detection module.
Compared with the prior art, the invention has the following beneficial effects:
(1) the full-flow full-automatic digital microfluidic library construction chip provided by the invention replaces manual operation to greatly improve the library construction efficiency and accuracy, the library construction method is short in time consumption, small in equipment size, low in equipment cost and low in digital microfluidic chip cost, and the equipment cost is far lower than that of a workstation based on a mechanical arm;
(2) the chip type totally-enclosed environment has no pollution risk and high flexibility, and is suitable for library construction of various DNA or RNA samples;
(3) the digital microfluidic library construction chip has high expansibility, can realize higher flux through electrode design or cascade, has high integration level, and can realize customized experimental process.
Drawings
Fig. 1 is a schematic diagram of a digital microfluidic DNA library chip, which includes a plurality of sample/reagent inlets 1, a plurality of reservoir and distribution regions 2, a waste liquid region 3, a product outlet 4, a low temperature region 5, a temperature change region 6, and a magnetic bead separation point 7.
FIG. 2 is a schematic diagram of sample and reagent loading positions in DNA library construction.
FIG. 3 is a schematic diagram of the operation flow of digital microfluidic DNA library construction.
FIG. 4 is a schematic diagram of a digital microfluidic RNA library construction chip.
FIG. 5 is a schematic diagram of sample and reagent loading positions in RNA library construction.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1: library construction of DNA samples
Fig. 1 is a schematic diagram of a digital microfluidic DNA library chip, which includes a plurality of sample/reagent inlets 1, a plurality of reservoir and distribution regions 2, a waste liquid region 3, a product outlet 4, a low temperature region 5, a temperature change region 6, and a magnetic bead separation point 7.
The process of constructing the DNA library by using the digital microfluidic DNA library construction chip comprises the following steps:
firstly, manually preparing a sample liquid, wherein the step of manually preparing the sample liquid specifically comprises the following steps: and manually preparing a mixed solution of the sample, the deionized water and the buffer solution, and uniformly mixing by shaking. The reason for this step being manual is that the amount of sample required differs for different kinds of samples.
Then, the totally-enclosed full-automatic chip-type DNA library construction is carried out, and the method comprises the following steps:
(1) loading: loading a chip on the detection equipment, filling the chip with biocompatible medium oil, and loading sample liquid and various reagents at corresponding inlets according to a specified volume according to the figure 2; loading a washing solution, a magnetic bead solution and an eluent in the plurality of stock solution and distribution areas 2 respectively; the sample liquid and various reagents are positioned in a low-temperature area 5, and the temperature of the low-temperature area 5 is set to be 4 ℃ for keeping the effectiveness of the reagents in the waiting process;
(2) fragmentation, end repair, linker ligation: transferring the mixed solution containing the sample solution and the enzyme to a variable temperature region 6, and controlling the temperature to realize fragmentation, end repair and joint connection reaction;
(3) adaptation and connection reaction: transferring the adaptive ligation reaction reagent to a temperature-variable region 6, fully mixing the adaptive ligation reaction reagent with the product obtained in the third step, and controlling the temperature to realize adaptive ligation reaction;
(4) and (3) magnetic bead binding: fully stirring the product obtained in the step (3) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(5) washing: fully stirring the magnetic beads obtained by separation in the step (4) and 80% ethanol solution drops distributed in the liquid storage and distribution area 2, performing magnetic bead separation at a magnetic bead separation point 7, and repeating the process for 3 times;
(6) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (5) and eluent liquid drops distributed by the liquid storage and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a temperature changing area 6;
(7) cleaning: the stock solution and distribution region 2 distributes an eluent droplet cleaning path and transfers used magnetic beads to the waste solution region 3;
(8) and (3) amplification reaction: transferring the amplification reaction reagent to the temperature-variable region 6 to be fully mixed with the product in the step (6), and controlling the temperature to realize the amplification reaction;
(9) and (3) magnetic bead binding: fully stirring the product obtained in the step (8) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(10) washing: fully stirring the magnetic beads obtained by separation in the step (9) and 80% ethanol solution drops distributed in the stock solution and distribution area 2, performing magnetic bead separation at a magnetic bead separation point 7, and repeating the steps for 3 times;
(11) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (10) and eluent liquid drops distributed by the stock solution and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a product outlet 4;
(12) and (3) extracting a product: the product is extracted from the product outlet 4 for sequencing.
Example 2: library construction of RNA samples
Fig. 4 is a schematic diagram of a digital microfluidic RNA library construction chip, which includes a plurality of sample/reagent inlets 1, a plurality of reservoir and distribution regions 2, a waste liquid region 3, a product outlet 4, a low temperature region 5, a temperature change region 6, and a magnetic bead separation point 7.
The process of constructing the RNA library by applying the digital microfluidic RNA library construction chip comprises the following steps:
firstly, manually preparing a sample liquid, wherein the step of manually preparing the sample liquid specifically comprises the following steps: and manually preparing a mixed solution of the sample and the deionized water, and shaking and mixing uniformly. The reason for this step being manual is that the amount of sample required differs for different kinds of samples.
Then, the totally-enclosed full-automatic chip type RNA library construction is carried out, and the method comprises the following steps:
(1) loading: loading a chip on the detection equipment, filling the chip with biocompatible medium oil, and loading sample liquid and various reagents at corresponding inlets according to a specified volume according to the figure 5; loading a washing solution, a magnetic bead solution and an eluent in the plurality of stock solution and distribution areas 2 respectively; the sample liquid and various reagents are positioned in a low-temperature area 5, and the temperature of the low-temperature area 5 is set to be 4 ℃ for keeping the effectiveness of the reagents in the waiting process;
(2) and (3) probe hybridization reaction: transferring the mixed solution containing the sample, deionized water and the probe hybridization reaction to a temperature-variable region 6, and controlling the temperature to realize the probe hybridization reaction;
(3) RNase H digestion reaction: transferring the RNase H digestion reaction reagent to a temperature-variable region 6, fully mixing the RNase H digestion reaction reagent with the product obtained in the step (2), and controlling the temperature to realize the RNase H digestion reaction;
(4) DNase I digestion reaction: transferring the DNase I digestion reaction reagent to a temperature changing zone 6 to be fully mixed with the product obtained in the step (3), and controlling the temperature to realize DNase I digestion reaction;
(5) and (3) magnetic bead binding: fully stirring the product obtained in the step (4) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(6) washing: fully stirring the magnetic beads obtained by separation in the step (5) and 80% ethanol solution drops distributed in the stock solution and distribution area 2, performing magnetic bead separation at a magnetic bead separation point 7, and repeating the steps for 3 times;
(7) elution and fragmentation reactions: fully stirring the magnetic beads obtained by separation in the step (6) and the fragmentation reaction reagent, carrying out magnetic bead separation at a magnetic bead separation point 7, transferring the fragmentation reaction liquid obtained by separation to a variable temperature region 6, and controlling the temperature to realize the fragmentation reaction;
(8) cleaning: the stock solution and distribution region 2 distributes an eluent droplet cleaning path and transfers used magnetic beads to the waste solution region 3;
(9) first strand cDNA synthesis reaction: transferring the first chain cDNA synthesis reaction reagent to a temperature changing area 6 to be fully mixed with the product obtained in the step (7), and controlling the temperature to realize the first chain cDNA synthesis reaction;
(10) second strand cDNA synthesis reaction: transferring the second chain cDNA synthesis reaction reagent to a temperature changing area 6 to be fully mixed with the product of the ninth step, and controlling the temperature to realize the second chain cDNA synthesis reaction;
(11) and (3) connection reaction: transferring the connecting reaction reagent to the temperature changing area 6 to be fully mixed with the product in the step (10), and controlling the temperature to realize the connecting reaction;
(12) and (3) magnetic bead binding: fully stirring the product obtained in the step (11) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and performing magnetic bead separation at a magnetic bead separation point 7;
(13) washing: fully stirring the magnetic beads obtained by separation in the step (12) and 80% ethanol solution drops distributed in the stock solution and distribution area 2, performing magnetic bead separation at a magnetic bead separation point 7, and repeating the steps for 3 times;
(14) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (13) and eluent liquid drops distributed by the stock solution and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a temperature-variable area 6;
(15) cleaning: the stock solution and distribution region 2 distributes an eluent droplet cleaning path and transfers used magnetic beads to the waste solution region 3;
(16) and (3) magnetic bead binding: fully stirring the product obtained in the step (14) and the magnetic bead solution drops distributed by the stock solution and distribution area 2, and carrying out magnetic bead separation at a magnetic bead separation point 7;
(17) washing: fully stirring the magnetic beads obtained by separation in the step (16) and 80% ethanol solution drops distributed in the stock solution and distribution area 2, performing magnetic bead separation at a magnetic bead separation point 7, and repeating the steps for 3 times;
(18) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (17) and eluent liquid drops distributed by the stock solution and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a temperature-variable area 6;
(19) cleaning: the stock solution and distribution region 2 distributes an eluent droplet cleaning path and transfers used magnetic beads to the waste solution region 3;
(20) and (3) amplification reaction: transferring the amplification reaction reagent to the temperature-variable region 6 to be fully mixed with the product obtained in the step (18), and controlling the temperature to realize the amplification reaction;
(21) and (3) magnetic bead binding: fully stirring the product obtained in the step (20) and the magnetic bead solution drops distributed by the liquid storage and distribution area 2, and performing magnetic bead separation at a magnetic bead separation point 7;
(22) washing: fully stirring the magnetic beads obtained by separation in the step (21) and 80% ethanol solution drops distributed in the stock solution and distribution area 2, performing magnetic bead separation at a magnetic bead separation point 7, and repeating the steps for 3 times;
(23) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (22) and eluent liquid drops distributed by the stock solution and distribution area 2, carrying out magnetic bead separation at a magnetic bead separation point 7, and transferring the eluent liquid drops obtained by separation to a product outlet 4;
(24) and (3) extracting a product: the product is extracted from the product outlet 4 for sequencing.
In conclusion, the invention realizes the construction of the DNA or RNA library by the full-automatic digital microfluidic library construction chip in the whole process. The chip comprises a low-temperature region for keeping the effectiveness of the reagent in the waiting process; comprises a temperature-variable region for providing temperature conditions required by each reaction in library construction; comprising a plurality of reservoir and distribution regions 2 for distributing a washing solution, a solution of magnetic beads and droplets of an elution solution, respectively. The chip function comprises all reactions required by library construction, and can be matched with an optical detection module to carry out quality control in the library construction process; higher flux can be achieved by electrode design or cascading; different reagents can be matched to realize different library constructions. The invention contains a sample of the whole process library construction on a single chip, all operations involved in the experimental process including reagent distribution, solution stirring, solution transfer, magnetic bead separation, etc. are automatically completed by the digital microfluidic chip, and finally the product is conveyed to the output end for the user to extract, and the user can realize the whole process library construction only by loading the sample to the chip.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A chip for constructing a digital microfluidic library comprises a plurality of sample/reagent inlets (1), a plurality of liquid storage and distribution areas (2), a waste liquid area (3), a product outlet (4), a low-temperature area (5), a variable-temperature area (6) and magnetic bead separation points (7);
the plurality of stock solution and distribution areas (2) can respectively distribute washing solution, magnetic bead solution and eluent liquid drops;
the low-temperature zone (5) is used for keeping the effectiveness of the reagent in the waiting process;
the temperature-variable region (6) is used for providing temperature conditions required by each reaction in library construction.
2. The digital microfluidic library building chip according to claim 1, wherein the temperature of the low temperature region (5) is set to 0-20 ℃, preferably 4 ℃;
preferably, the chip is filled with biocompatible medium oil;
preferably, the plurality of sample/reagent inlets (1) are loaded with sample liquid and various reagents, respectively, the various reagents including an enzyme, an adaptor-ligation reaction reagent and an amplification reaction reagent;
preferably, the various reagents further include any one or a combination of at least two of a probe hybridization reaction reagent, an RNase H digestion reaction reagent, a DNase I digestion reaction reagent, a first strand cDNA synthesis reaction reagent, a second strand cDNA synthesis reaction reagent, and/or a fragmentation reaction reagent;
preferably, the plurality of stock solution and distribution areas (2) are loaded with washing solution, magnetic bead solution and eluent respectively;
preferably, the sample solution and various reagents can be loaded in a manner of injection by a pipette, loading by an independent reagent pack or pre-embedding the reagents in a chip;
preferably, the sample liquid and various reagents are located in a low temperature zone (5);
preferably, the washing solution is a 70% -95% ethanol solution, preferably an 80% ethanol solution.
3. A digital microfluidic library construction system comprising the digital microfluidic library construction chip of any one of claims 1-2.
4. The digital microfluidic library construction system of claim 4, wherein the system comprises a plurality of cascades of chips that enable high throughput library construction.
5. A method for library construction using the chip of any one of claims 1-2 or the system of any one of claims 3-4, the method comprising:
(1) loading: loading a chip on detection equipment, and loading a sample solution and various reagents into corresponding inlets according to a specified volume;
(2) fragmentation, end repair, linker ligation: transferring the mixed solution containing the sample solution and the enzyme to a temperature changing area (6), and controlling the temperature to realize fragmentation, end repair and joint connection reaction;
(3) adaptation and connection reaction: transferring the adaptive ligation reaction reagent to a temperature-changing area (6), fully mixing the adaptive ligation reaction reagent with the product obtained in the third step, and controlling the temperature to realize adaptive ligation reaction;
(4) and (3) magnetic bead binding: fully stirring the product obtained in the step (3) and the magnetic bead solution drops distributed by the liquid storage and distribution area (2), and carrying out magnetic bead separation at a magnetic bead separation point (7);
(5) washing: fully stirring the magnetic beads obtained by separation in the step (4) and washing liquid drops distributed by the liquid storage and distribution area (2), and performing magnetic bead separation at a magnetic bead separation point (7);
(6) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (5) and eluent liquid drops distributed by the stock solution and distribution area (2), carrying out magnetic bead separation at a magnetic bead separation point (7), and transferring the eluent liquid drops obtained by separation to a temperature-variable area (6);
(7) cleaning: the stock solution and distribution area (2) distributes an eluent liquid drop cleaning path and transfers used magnetic beads to the waste solution area (3);
(8) and (3) amplification reaction: transferring the amplification reaction reagent to a temperature-variable region (6) to be fully mixed with the product in the step (6), and controlling the temperature to realize the amplification reaction;
(9) and (3) magnetic bead binding: fully stirring the product obtained in the step (8) and the magnetic bead solution drops distributed by the liquid storage and distribution area (2), and carrying out magnetic bead separation at a magnetic bead separation point (7);
(10) washing: fully stirring the magnetic beads obtained by separation in the step (9) and washing liquid drops distributed by the liquid storage and distribution area (2), and performing magnetic bead separation at a magnetic bead separation point (7);
(11) and (3) elution: fully stirring the magnetic beads obtained by separation in the step (10) and eluent liquid drops distributed by the stock solution and distribution area (2), carrying out magnetic bead separation at a magnetic bead separation point (7), and transferring the eluent liquid drops obtained by separation to a product outlet (4);
(12) and (3) extracting a product: the user extracts the product from the product outlet (4) for subsequent experiments.
6. The method according to claim 5, wherein step (1) is preferably preceded by a step of manually preparing the sample liquid, the step of manually preparing the sample liquid comprising in particular: manually preparing a mixed solution of a sample, deionized water and a buffer solution, and uniformly mixing by shaking;
preferably, in the step (1), the chip is filled with biocompatible medium oil;
preferably, in step (1), the various reagents include an enzyme, an aptameric ligation reaction reagent, and an amplification reaction reagent;
preferably, the plurality of stock solution and distribution areas (2) are loaded with washing solution, magnetic bead solution and eluent respectively;
preferably, the sample solution and various reagents can be loaded in a manner of injection by a pipette, loading by an independent reagent pack or pre-embedding the reagents in a chip;
preferably, the sample liquid and various reagents are located in a low temperature zone (5);
preferably, the washing solution is 70% -95% ethanol solution, preferably 80% ethanol solution;
preferably, the washing steps of step (5) and step (10) are each repeated 3 times.
7. The method according to claim 6, wherein the reagents further comprise any one or a combination of at least two of a probe hybridization reaction reagent, an RNase H digestion reaction reagent, a DNase I digestion reaction reagent, a first strand cDNA synthesis reaction reagent, a second strand cDNA synthesis reaction reagent, and/or a fragmentation reaction reagent;
preferably, for the RNA library, the steps of probe hybridization reaction, RNase H digestion reaction and DNase I digestion reaction can be further included after the step (1) and before the step (2);
the probe hybridization reaction is realized by transferring a mixed solution containing a sample, deionized water and the probe hybridization reaction to a temperature-variable region (6) and controlling the temperature;
the RNase H digestion reaction is that an RNase H digestion reaction reagent is transferred to a temperature changing zone (6) to be fully mixed with a probe hybridization reaction product, and the temperature is controlled to realize the RNase H digestion reaction;
the DNase I digestion reaction is that a DNase I digestion reaction reagent is transferred to a temperature changing zone (6) to be fully mixed with a product of the RNase H digestion reaction, and the temperature is controlled to realize the DNase I digestion reaction;
preferably, for the RNA library, the steps of a first strand cDNA synthesis reaction and a second strand cDNA synthesis reaction are further included after the fragmentation reaction and before the ligation reaction;
the first strand cDNA synthesis reaction is realized by transferring a first strand cDNA synthesis reaction reagent to a temperature changing region (6), fully mixing the first strand cDNA synthesis reaction reagent with the fragmentation reaction product, and controlling the temperature;
and the second strand cDNA synthesis reaction is realized by transferring a second strand cDNA synthesis reaction reagent to a temperature-variable region (6) to be fully mixed with a first strand cDNA synthesis reaction product and controlling the temperature.
8. The method of claim 5, wherein preferably, the subsequent experiment of step (12) comprises sequencing;
preferably, the method further comprises the step of performing quality control by using an optical detection module;
preferably, the optical detection module is a fluorescence detection module.
9. A DNA library or RNA library prepared according to the method of any one of claims 5-8.
10. Use of the chip according to any one of claims 1-2 or the system according to any one of claims 3-4 for the construction of a DNA library or an RNA library.
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| WO2024056012A1 (en) * | 2022-09-15 | 2024-03-21 | 上海思路迪生物医学科技有限公司 | Automatic ngs library preparation and sub-packaging kit and library construction method |
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