CN114829627B - Method for enriching nucleic acid and constructing sequencing library based on digital microfluidic platform - Google Patents
Method for enriching nucleic acid and constructing sequencing library based on digital microfluidic platform Download PDFInfo
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Abstract
The invention provides a method for enriching nucleic acid based on a digital microfluidic platform and a method for constructing a sequencing library. The digital microfluidic platform comprises a microfluidic chip and a microfluidic device which are detachably connected, and the method for enriching nucleic acid based on the platform comprises the following steps: preparing microfluidic drops on a microfluidic chip, wherein the microfluidic drops contain surfactant, silicone oil and nucleic acid; specifically amplifying a target region of the nucleic acid in the microfluidic droplet based on a specific probe, so as to amplify a product, the target product constituting an enriched nucleic acid. The enriched product can be used for library construction and sequencing.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a method for enriching nucleic acid and constructing a sequencing library based on a digital microfluidic platform.
Background
In recent years, second generation high throughput sequencing technology (NGS) has been widely used in many research and clinical fields, NGS can help identify genetic alterations, including Single Nucleotide Variations (SNVs), copy Number Variations (CNVs), and gene fusions, among others. Compared with conventional NGS sequencing, sequencing after enrichment of the target region is more commonly applied due to its specificity, economy and expansibility. To achieve higher sequencing depth in the coverage area and reduce sequencing costs, we need to design capture techniques with high efficiency and sequencing uniformity, which also makes the target region enrichment technique more challenging.
Multiplex PCR and hybrid capture techniques are two widely used classes of target region enrichment techniques. Enrichment amplification of the multiplex PCR technology is carried out in two rounds of PCR, wherein the first round of PCR mainly aims at designing hundreds of pairs of PCR primer pairs for target areas to simultaneously amplify different target areas, and the primers of the first round of PCR are simultaneously designed with a section of identical sequence for the second round of amplification. The second round of PCR uses the same primer to amplify the product enriched in the first round, and the amplified library can be used for subsequent sequencing. The primers used in the second round of PCR have a partial sequence complementary to the primers used in the first round of PCR, with additional sequences for sequencing. The hybridization capture technology is to hybridize fragments containing target regions by using probes with biotin, then to absorb and recycle the captured probes by using magnetic beads modified with biotin specific binding, and finally to wash out redundant fragments of non-target regions by using a magnetic bead purification mode, thereby obtaining the required target fragments.
However, when the method is used for enriching and banking the target region, the required initial DNA, reagent and the like are determined based on the whole genome sequencing data, so that a great amount of data is wasted, and the use efficiency of the initial DNA and the use efficiency of the reagent in the banking process are reduced. Thus, there remains a need for further improvements in enrichment techniques for target regions.
Disclosure of Invention
The invention aims to solve at least one of the technical problems in the related art to a certain extent, and provides a method for enriching nucleic acid based on a digital microfluidic platform and a method for constructing a sequencing library. The method can realize the enrichment of the nucleic acid sample by only needing a small amount of nucleic acid sample, and a sequencing library is obtained and is applied to the field of sequencing; furthermore, by means of the digital microfluidic platform, manual operations, such as lengthy washing steps, manual operation control to a specific temperature, etc., can be eliminated, and an automated operation can be realized.
Multiplex PCR and hybrid capture techniques are two types of commonly used techniques for enrichment of target regions of nucleic acids, which can be used for enrichment of target regions of nucleic acids. These techniques also have drawbacks such as cumbersome enrichment procedures, involving multiple steps of complex manual operations, which have many drawbacks, are not easy to control temperature, and are unstable during washing, which can result in poor final library results. Taking the hybridization capture technology as an example, in addition to the high cost, most of the hybridization capture steps are the unavoidable complex operation procedures, including multiple washing steps under a certain temperature control and time and effort consuming.
The invention provides a method for enriching nucleic acid based on a digital microfluidic platform and a method for constructing a sequencing library, and the method also realizes the enrichment of nucleic acid by means of the digital microfluidic platform, can complete the enrichment process by using a small amount of nucleic acid samples, can be operated without relying on manual operation, is convenient for controlling the temperature, and can realize the enrichment of a target region of a rapid low-cost automated nucleic acid molecule due to the shortened reaction time caused by the reduced reaction system. Meanwhile, the stability of operation of each batch can be ensured due to the operation by means of the digital microfluidic platform.
Specifically, the invention provides the following technical scheme:
in a first aspect of the present invention, there is provided a method of enriching nucleic acid based on a digital microfluidic platform comprising a microfluidic chip and a microfluidic device, the microfluidic chip and the microfluidic device being detachably connected, the method comprising: preparing microfluidic drops on a microfluidic chip, wherein the microfluidic drops contain a surfactant, silicone oil and nucleic acid; and carrying out specific capture or amplification on a target region of the nucleic acid in the microfluidic droplet based on a specific probe or a specific primer to obtain an enriched nucleic acid product.
The method for enriching the target region of the nucleic acid based on the digital microfluidic platform provided by the invention is used for enriching the target region, solves the problem of complex operation in the enrichment experimental process of the target region, and does not need manual operation, including lengthy washing steps and manual operation at a specific temperature. Thereby reducing the time of the experimental operation part and the resulting errors. And the amount of DNA required is less than that of other techniques, and the cost of reagents required for biochemical reactions is reduced.
According to an embodiment of the present invention, the method for enriching nucleic acid based on a digital microfluidic platform may further include the following technical features:
in some embodiments of the invention, the silicone oil is a low viscosity silicone oil. The low viscosity silicone oil is easier when the electric field controls the movement of the droplets. Generally, silicone oils below 10cps are low viscosity silicone oils.
In some embodiments of the invention, further comprising the step of purifying the enriched nucleic acid product using the microfluidic device.
In some embodiments of the invention, the microfluidic droplet has a volume of no more than 16 microliters.
In some embodiments of the invention, the surfactant is present in the microfluidic droplet at a concentration of no more than 0.5% by mass, preferably 0.01% to 0.1% by mass, more preferably 0.075% by mass. When the content of the surfactant is too high, the biochemical reaction is seriously affected, for example, tween 20 with the mass concentration of more than 1% can seriously affect the biochemical reaction, the tween 20 with the mass concentration of 0.01% -0.1% has lower influence on biochemistry and can reach the surface tension of digital microfluidics, and tween 20 with the mass concentration of 0.075% can obviously increase the surface tension of liquid drops.
In some embodiments of the invention, the surfactant is tween 20.
In some embodiments of the invention, the microfluidic chip further comprises:
a substrate on which an electrode matrix is disposed;
The operation area is positioned on the substrate and comprises a sample adding area, a storage area, a reaction area and a collection area, wherein the sample adding area, the storage area, the reaction area and the collection area are sequentially connected.
The flow of the liquid drops can be controlled through the electrode matrix in the substrate, for example, the movement of the micro-fluid drops with different sizes or different micro-fluid drops with different sizes can be controlled through the switch of the electrode, so that different operations and treatments of the micro-fluid drops can be realized without manual operations.
In some embodiments of the invention, the microfluidic device further comprises: and the temperature control module is arranged opposite to the reaction zone and is used for controlling the temperature of the reaction zone.
In some embodiments of the invention, the microfluidic device further comprises: the magnetic control module is opposite to the collecting area, a magnet is arranged in the magnetic control module, and the magnetic control module controls the lifting of the magnet through a stepping motor. The magnet is arranged in the magnetic control module, a stepping motor can be arranged at the lower part of the magnet, and then the magnet is controlled to move up and down through the stepping motor. For example, when the magnet is above, i.e., near the substrate, it can be used to pick up the magnetic beads; when the magnet is positioned below, namely far away from the substrate, the magnet can be used for releasing the magnetic beads, so that the aim of purifying the reagent is fulfilled.
In some embodiments of the invention, the microfluidic device further comprises a water cooling module disposed opposite the storage region through a metal plate, the water cooling module being configured to control the storage region temperature. The water cooling module is arranged opposite to the storage area, and can be in an opened state all the time to protect the reagent in the storage area from being deactivated. According to an embodiment of the invention, the metal plate is a peltier plate.
In some embodiments of the invention, the low viscosity silicone oil may have a viscosity of 5 or less, for example, 4.6.
In some embodiments of the invention, the microfluidic device further comprises: the fluorescence detection module is connected with the reaction zone and is used for carrying out fluorescence detection on the product of the reaction zone. Because the electrode matrix is arranged on the substrate, the large liquid drops after reaction can be divided into two or three by controlling the switch of the electrode matrix, and the separated liquid drops can be used for fluorescence detection without manual operation.
In some embodiments of the invention, the digital microfluidic platform further comprises: the data processing device is used for converting the reaction result of the microfluidic chip into an electric signal and outputting the electric signal. According to the embodiment of the invention, the data processing device is further provided with a mechanical arm, and the mechanical arm is used for realizing automatic operation of the microfluidic chip.
In some embodiments of the invention, the nucleic acid is genomic DNA, which is 5 nanograms or more.
In a second aspect of the invention, the invention provides a method of constructing a sequencing library comprising: obtaining a target region enriched product of nucleic acid based on the method of any of the embodiments of the first aspect of the invention; enriching products based on the target region of the nucleic acid, and pooling to obtain a sequencing library.
According to an embodiment of the present invention, the above method for constructing a sequencing library may further include the following technical features:
In some embodiments of the invention, further comprising: after preparing microfluidic drops on a microfluidic chip, performing end repair and joint connection, and performing first PCR amplification on the obtained connection product so as to construct a pre-capture DNA library; performing hybridization capture and purification on the pre-capture DNA library using a specific probe to obtain a captured hybridization product;
and performing a second PCR amplification based on the captured hybridization product to construct a captured DNA library.
In some embodiments of the invention, the specific probe is a biotin-labeled specific probe and the purification is performed using streptavidin-labeled magnetic beads.
In some embodiments of the invention, further comprising: after preparing microfluidic droplets on a microfluidic chip, amplifying with a first amplification primer adapted to amplify different regions of the nucleic acid to obtain a first amplification product; amplifying the first amplification product with a universal primer, which is partially complementary to the first amplification primer, so as to obtain a second amplification product.
In some embodiments of the invention, further comprising: thermally denaturing the second amplification product to obtain single-stranded circular DNA; rolling circle amplification is performed based on the single-stranded circular DNA to obtain DNA nanospheres.
In some embodiments of the invention, the purification is performed using magnetic beads after amplification with the first amplification primer or after amplification of the first amplification product with the universal primer is performed, respectively.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
fig. 1 is a schematic cross-sectional view of a microfluidic chip provided according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of a part of a structure of a digital microfluidic platform according to an embodiment of the present invention.
Fig. 3 is a schematic structural diagram of a digital microfluidic platform according to an embodiment of the present invention.
Fig. 4 is a schematic diagram of a method provided by an embodiment of the present invention that is adapted to a microfluidic chip by off-chip conditions fumbling.
Fig. 5 is a flow chart of a biochemical reaction captured on a digital microfluidic platform by hybridization according to an embodiment of the present invention.
Fig. 6 is a flow chart of biochemical reactions of multiple PCR on a digital microfluidic platform according to an embodiment of the present invention.
FIG. 7 shows the detection results of real-time fluorescent quantitative PCR provided in example 1 according to the present invention.
FIG. 8 is a graph of agarose gel electrophoresis detection of reaction products at different surfactant concentrations provided in accordance with an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Reference herein to a "digital microfluidic platform" is generally understood in the art to be a platform that relies on the formation of droplets by the surface tension of a liquid, with the movement of the droplets being controlled by means of an electric and/or magnetic field. Such a digital microfluidic platform pass may include a microfluidic chip. In addition, other operation instruments matched with the microfluidic chip, such as a microfluidic device, can be also included, so long as the movement of the liquid drops can be controlled through an electric field and/or a magnetic field, and the digital operation can be realized. The microfluidic chip and the microfluidic device can be detachably connected, the microfluidic chip can be connected into the microfluidic device according to the requirement, and then the flow of microfluidic drops on the chip is controlled through the microfluidic device. The digital microfluidic platform can also contain other devices, such as a data processing device, according to the need, and the reaction result of the microfluidic chip is directly converted into an electric signal for output, so that the reaction process on the microfluidic chip can be conveniently controlled.
In at least some embodiments of the present invention, provided methods for enriching a target region of nucleic acid based on a digital microfluidic platform, based on hybrid capture techniques, accomplish enrichment of the target region, thereby achieving automated enrichment of low initial amounts of small volumes of nucleic acid. In at least some embodiments of the present invention, provided methods for enriching a target region of nucleic acid based on a digital microfluidic platform, based on multiplex PCR techniques, accomplish enrichment of the target region, thereby achieving automated enrichment of low initial amounts of small volumes of nucleic acid. Small volume droplets (also called microfluidic droplets) are prepared on a microfluidic chip, the microfluidic droplets contain a surfactant, a low viscosity silicone oil and nucleic acid, and then movement and mixing of the microfluidic droplets are achieved by an electrowetting method (as shown in fig. 1). As shown in fig. 1, the provided microfluidic chip includes a substrate on which an electrode matrix is disposed. The substrate used may be a PCB substrate. Meanwhile, an insulating layer can be arranged on the upper surface of the electrode matrix to prevent the liquid drops from conducting electricity so as to prevent nucleic acid, protein and the like in the liquid drops from being influenced by current. In addition, in order to facilitate the formation of microfluidic droplets, a hydrophobic coating may be provided on the surface of the microfluidic chip to increase the surface tension of the microfluidic droplet surface, thereby helping the formation of microfluidic droplets. The upper part of the microfluidic chip can be covered with a top layer.
Taking a hybrid capture technology as an example, enriching a nucleic acid target region or constructing a sequencing library based on a digital microfluidic platform, and adopting the principle that the fragmented nucleic acid is utilized to realize hybrid capture of the target region of the nucleic acid with a micro volume on a microfluidic chip, wherein the method comprises the steps of library construction, early amplification, denaturation, probe capture, washing and purification after hybridization, later amplification, concentration detection, single-strand cyclization, DNA nanosphere preparation and the like.
The principle of the method is that nucleic acid is utilized to realize amplification and enrichment of a micro-volume nucleic acid target region on a microfluidic chip, wherein the method comprises the steps of two rounds of multiplex PCR enrichment and amplification, magnetic bead purification, concentration detection, single-strand cyclization, DNA nanosphere preparation and the like.
The nucleic acid referred to may be genomic DNA. The provided methods are suitable for a plurality of sequencing platforms, such as a BGI platform, an illumina platform, and the like.
The provided digital microfluidic platform comprises: the microfluidic chip and the microfluidic device can be detachably connected, for example, the microfluidic chip can be connected with the microfluidic device in a jogged mode, the microfluidic chip comprises an operation area, the operation area is positioned on a substrate and comprises a sample adding area, a storage area, a reaction area and a collection area, and the sample adding area, the storage area, the reaction area and the collection area are sequentially connected. Accordingly, different modules, such as a magnetic control module, a temperature control module, a water cooling module and the like, can be arranged in the microfluidic device for assisting the reaction of each different stage completed on the microfluidic chip. According to the embodiment of the invention, the temperature control module is arranged opposite to the reaction zone, and is used for controlling the temperature of the reaction zone. According to the embodiment of the invention, the magnetic control module is arranged opposite to the collecting area, a magnet is arranged in the magnetic control module, and the magnetic control module controls the lifting of the magnet through a stepping motor. According to the embodiment of the invention, the water cooling module is arranged opposite to the storage area through the metal plate, and the water cooling module is used for controlling the temperature of the storage area. Thus, automated enrichment of the target region of nucleic acid or automated construction of sequencing libraries can be achieved. For ease of understanding, a partial structure of the digital microfluidic platform is shown in fig. 2, and a schematic structural diagram of the digital microfluidic platform is also shown in fig. 3. In addition, the microfluidic device can further comprise a fluorescence detection module according to requirements, wherein the fluorescence detection module is connected with the reaction zone and is used for carrying out fluorescence detection on the product of the reaction zone.
The method for enriching the target region of nucleic acid or the method for constructing a sequencing library based on the digital microfluidic platform will be described below by taking the hybridization capture technology as an example. By this method an automated enrichment of the target region of low starting amount of small volume nucleic acid can be achieved.
The automatic hybridization capture enrichment and library establishment of the nucleic acid target area are completed by utilizing the digital microfluidic platform, and the method can be used for clinical diagnosis of the target gene or the nucleic acid target area, whole exon capture sequencing and other products. The enrichment and purification process and the library building process can be realized in a system with the concentration lower than 16 mu L, and the system contains a surfactant with a certain concentration. The whole process can be automatically realized without manual work. In the research process, firstly, an off-chip small-volume manual hybridization capture technology is developed, the data performance of the technology is superior to that of a hybridization capture kit commonly used in the market in terms of the initial DNA input amount, the reagent use amount and the time, and the capture efficiency is equivalent (as shown in fig. 4). After the technology is applied to a digital microfluidic platform to realize automation, experiments prove that the capturing efficiency of the automatic target area hybridization capturing technology is equivalent to that of a conventional kit in the market, the capturing uniformity performance is good, the initial quantity of DNA required by the technology is 0.1-0.2 times of that of the conventional kit, and the required reagent dosage is about 0.1 time of that of the conventional kit. In addition, the automatic library construction process saves labor cost and can reduce pollution of the exogenous nucleic acid to the sample library in the library construction process. In at least some embodiments of the present invention, a capture banking process is provided as follows:
Firstly, low-viscosity silicone oil is filled into a microfluidic chip loaded on a microfluidic device, then a prepared small volume of reaction reagent containing a surfactant and a broken low initial quantity of DNA small liquid drop are added into a reagent storage area (the temperature of a storage area can be controlled by a water cooling module, for example, the temperature can be controlled at 4 ℃), then an operation button in a program is clicked, the reaction reagent can move and be mixed under the control of an electrode, and the bottom of the microfluidic chip, namely a temperature control module and a magnetic control module on the microfluidic device are utilized to realize reaction control and magnetic bead purification of a sample. The DNA in the liquid drop on the micro-fluidic chip is subjected to the steps of terminal repair, joint connection, magnetic bead purification and early-stage PCR amplification under the help of the reaction reagent and the temperature, and a proper amount of amplified products can be extracted by utilizing a fluorescent quantitative module in the digital micro-fluidic device to carry out the subsequent probe hybridization capturing step. After hybridization capture, the streptavidin magnetic beads are used for specifically combining with biotin groups on the probes, and secondary amplification is carried out after a plurality of rounds of automatic washing processes, so that the obtained product is a library after hybridization capture. And (3) continuing to carry out single-stranded cyclization and DNA nanosphere preparation on the library, and then taking out or directly connecting the library with a sequencer for sequencing. All of the above processes were automated on chip, with all of the reaction systems being less than 16 microliters and containing some amount of surfactant (as shown in fig. 5).
The application of hybrid capture technology to enrich a nucleic acid target region based on a digital microfluidic platform or to construct a sequencing library has many beneficial effects, for example, can be expressed as follows:
1. Compared with the hybridization capture products commonly used in the market, the hybridization capture technology with small volume and low initial quantity can reduce the reagent cost by about 10 times and save precious initial DNA samples.
2. The provided method is a full-automatic hybridization capture process, and no manual operation is needed in the reaction process except that manual operation is needed for loading reagent samples and taking out products.
3. All reaction processes are carried out on the microfluidic chip, so that the pollution of exogenous nucleic acid to the library establishment process can be reduced.
4. The used instruments and chips are portable equipment, and can be widely applied to clinical gene diagnosis and rapid detection in hospitals, whole-exon sequencing and the like.
The method simultaneously solves some technical difficulties which are difficult to solve, such as:
1. Incomplete and low initiation of DNA samples often results in risk or failure of library construction of the hybrid capture library, with the initial amount of genomic DNA typically greater than 50ng following disruption of the current conventional library construction method of hybrid capture. The method provided by the invention requires only 5ng of initial amount by optimizing a biochemical system.
2. The reaction volume of each step in the conventional hybridization capturing process is maximally 200uL (washing step) and minimally 20uL (hybridization step), the reaction volume of the washing step can be smaller than 16uL, the reaction volume of the hybridization step is smaller than 10uL, and the capturing efficiency cannot be influenced or even better through optimizing a biochemical system.
3. Compared with the traditional PCR instrument, the temperature control on the micro-fluidic chip has insufficient precision, so that the temperature tolerance of each step (including hybridization and library establishment) is tested and debugged, and finally the micro-fluidic chip is successfully combined.
The method for enriching the target region of nucleic acid or constructing sequencing library based on the digital microfluidic platform will be described below by taking the multiplex PCR technology as an example. By this method an automated enrichment of the target region of low starting amount of small volume nucleic acid can be achieved.
The automatic multiplex PCR target region enrichment is completed by utilizing a digital microfluidic platform, and the enrichment library building method can be applied to products such as clinical diagnosis of the nucleic acid target region. The enrichment and purification process and the warehouse building process can be realized in a system lower than 16uL, and the system contains a certain concentration of surfactant. The whole process can be automatically realized without manual work. In the research process, an off-chip small-volume multiplex PCR technology is developed first, and the data performance of the technology is superior to that of hybridization capture kits commonly used in the market in terms of DNA input initial quantity, reagent use quantity and time, and the enrichment efficiency is equivalent. After the technology is applied to a digital droplet microfluidic platform to realize automation, experiments prove that the multiplex PCR enrichment efficiency is equivalent to that of the conventional kit in the market, the gene loci to be detected in a positive sample (BRCA 1/2 mutation) can be successfully detected, the initial quantity of DNA required by the technology is 0.1-0.2 times that of the conventional kit, and the required reagent dosage is about 0.1 times that of the conventional kit. In addition, the automatic library construction process saves labor cost and can reduce the pollution of exogenous nucleic acid to a sample library in the enrichment and library construction process. In at least some embodiments of the invention, the amplification and pooling process of the method is as follows:
Firstly, filling low-viscosity silicone oil into a microfluidic chip loaded on a microfluidic device, then adding a prepared small-volume reaction reagent containing a surfactant and a small genomic DNA droplet with a low initial quantity into a storage area on the microfluidic chip, clicking an operation button in a program, moving and mixing the reaction reagent under the control of an electrode, and simultaneously realizing reaction temperature control and magnetic bead purification of a sample by using a temperature control module and a magnetic control module at the bottom of the microfluidic chip. The DNA in the liquid drop on the micro-fluidic chip is successively subjected to the steps of first round multiplex PCR (containing hundreds of pairs of primers) and magnetic bead purification under the help of reaction reagent and temperature, and the obtained product is the library enriched in the target area after multiplex PCR after the second round amplification PCR (a pair of universal primers) and magnetic bead purification. In addition, a proper amount of library can be extracted by utilizing a fluorescence quantitative module on the digital microfluidic platform, and the library can be taken out or directly connected with a sequencer for sequencing after subsequent single-chain cyclization and DNA nanosphere preparation. All of the above processes were automated on chip, with all of the reaction systems being less than 16 microliters and containing some amount of surfactant (as shown in figure 6).
The method has a plurality of advantages, and can be specifically expressed as follows:
1. compared with the enrichment products of multiple PCR target areas commonly used in the market, the small-volume low-initial-quantity multiple PCR technology can reduce the reagent cost by about 10 times, and can save precious initial DNA samples.
2. The full-automatic multiplex PCR process provided by the invention does not need any manual operation in the reaction process except manual operation for loading reagent samples and taking out products.
3. The reaction process provided by the invention is carried out on a chip, so that the pollution of exogenous nucleic acid to the library establishment process can be reduced.
4. The instrument and the chip used in the invention are portable equipment, and can be widely applied to clinical gene diagnosis, rapid detection and the like in hospitals.
The provided method simultaneously solves some technical difficulties which are difficult to solve, and the method is characterized in that:
1. the initial amount of genomic DNA required for conventional multiplex PCR enrichment methods is typically 10-20ng. The method provided above requires only 3ng of starting amount by optimizing the biochemical system.
2. The PCR amplification of conventional multiplex PCR enrichment methods is typically 50uL, and the PCR volumes can be reduced to 6uL by optimizing the biochemical system. Due to the excessive restriction of the primer pair number of multiplex PCR, a small volume may result in a decrease in PCR amplification specificity, but according to this example PCR system design, the specificity of primer amplification is not significantly changed and 7 mutation sites of positive BRCA1/2 can be effectively detected without changing the multiplex PCR primer pair design.
In addition, whether an automated multiplex PCR process is implemented based on a digital microfluidic platform or a hybrid capture process is implemented, it requires biochemical reactions in silicone oil and increases the surface tension of the droplets by surfactants, and the presence of weak currents cannot be ignored. Therefore, the influence of silicone oil, surfactant and current on multiple PCR steps or hybridization capturing process is solved through biochemical debugging and chip testing.
The test found that silicone oil generally did not affect biochemical reagents, but ethanol used for magnetic bead purification was soluble in silicone oil, so we used 20% polyethylene glycol 8000 and 2.5M sodium chloride solution as their alternatives. In addition, in multiplex PCR experiments, the PCR additive used was also changed to a silicone oil-insoluble reagent.
The non-ionic surfactant Tween 20 is selected as an additive, more than 1% of Tween 20 is found to seriously influence the biochemical reaction in experiments, the tests show that 0.01% -0.1% of Tween 20 has lower influence on the biochemistry and can reach the surface tension of digital microfluidics, and finally, 0.075% of Tween 20 is selected as the surfactant for increasing the surface tension of liquid drops.
In addition, digital microfluidics is believed to be free of current in the droplets due to a similar electrostatic shielding effect. However, in order to ensure that weak current during droplet movement does not affect movement of charged DNA or protein to the surface, the fragment size of sample DNA and the primer fragment size can be increased, and 1% polyethylene glycol 8000 is added during preparation of DNA nanospheres to protect the DNA nanospheres from directional movement so as to prevent the droplets from adhering to the surface of silicone oil.
Examples of the present invention will be explained below in connection with embodiments. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1 Low initial amount small volume automated hybridization Capture target region enrichment technique
Example 1 provides a method for hybrid capture of two target regions, the exon region of the RUNX1 gene and PRKG gene, respectively, and a non-target region, the non-exon region of the GAPDH gene, using a microfluidic platform.
The method comprises the following steps:
1. The preparation method of the genome DNA fragment comprises the following steps:
50ng of human genome DNA is taken as a raw material, an ultrasonic breaking instrument is used for breaking the genome, and magnetic beads are used for selecting the broken fragments, so that a DNA fragment with a main band of 280bp is obtained.
2. The construction method of the DNA library before capture comprises the following steps:
2.1 inserting microfluidic chips into microfluidic devices, filling the chips with silicone oil.
2.2 After quantifying the DNA selected from the fragment of step 1 or the extracted cell free DNA (cfDNA), 5 μl (5 ng) was added dropwise to the microfluidic chip and all reagents were loaded into a storage area maintained at 4 ℃ throughout the library construction.
All reagents used below were purchased from commercial kit MGIEasy exome capture V5 probe kit, manufacturer Hua Dazhi under the accession number 1000007746. Including end repair buffers, end repair enzyme mixtures, ligase buffers, ligases, polymerase chain reaction mixtures, RNA probes, RNase inhibitors, wash buffers, and the like. Streptavidin magnetic beads M280 were purchased from Invitrogen under the accession number 00456577. Tween 20 was added to all reagents at a final concentration of 0.075%.
The magnetic beads for magnetic bead purification, AMPure XP, were purchased from BECKMAN COULTER under the trade designation 17775900. Tween 20 was added to the beads at a final concentration of 0.075%.
The washing reagent used for magnetic bead purification comprises the following components: polyethylene glycol 8000,2.5M sodium chloride solution at final concentration of 20% and tween 20 at 0.075%.
2.3 The following steps are all program controlled electrode switch to control droplet movement:
from the storage area, 2. Mu.L of the end repair buffer and 1. Mu.L of the end repair enzyme mixture were separated, and after well mixing with the DNA (cfDNA) droplets, reacted at 37℃for 30 minutes, and then reacted at 65℃for 15 minutes.
2.4 From the storage area 3. Mu.L of ligation buffer containing a linker and 1. Mu.L of ligase were added to the product from step 2.3, and incubated at 25℃for 30 minutes after thoroughly mixing.
2.5 Separating the magnetic bead reagent from the storage area, purifying the product obtained in the step 2.4, and controlling the magnet to lift by utilizing a stepping motor to finish the adsorption, washing, release and elution of the magnetic beads. The product obtained after elution was added to 4uL of the polymerase chain reaction mixture from the reagent storage zone, and was subjected to Polymerase Chain Reaction (PCR) amplification in a total volume of 6. Mu.L: pre-denaturation at 98 ℃ for 3 min; 12 cycles: denaturation at 98℃for 20 sec, annealing at 60℃for 15 sec, extension at 72℃for 30 sec; final extension at 72℃for 10 min. The amplification reaction is also performed in the central reaction zone of the chip.
And purifying the amplified product again by using the separated magnetic bead reagent, eluting by separating 3 mu L of TE buffer solution, and obtaining the product after eluting, namely the DNA library before capturing.
3. The capture hybridization method comprises the following steps:
3.1 separating 3. Mu.L of COT-1DNA and 3. Mu.L of salmon sperm DNA from the reagent storage area, adding the product obtained in step 2, mixing thoroughly, separating out the magnetic bead reagent, concentrating it, and separating out 6. Mu.L of hybridization buffer containing DNA blocking nucleotide chain, eluting the magnetic beads.
3.2 The eluted product from step 3.1 was reacted in the central reaction zone at 95℃for 5 minutes followed by continuous incubation at 65 ℃. To the droplets incubated at 65℃3. Mu.L of a probe mixture containing a biotin-modified RNA probe and an RNase inhibitor was added and mixed well.
3.3 The mixed droplets obtained in step 3.2 were incubated at 65℃for 16-24 hours.
3.4 The product from step 3.3 was split into 8. Mu.L of streptavidin-modified M280 beads resuspended in binding buffer and thoroughly mixed. Incubate in the central reaction zone for 45 min at room temperature.
3.5, Controlling a stepping motor to lift the magnet by an automatic program, recycling the magnetic beads by using the magnet to remove the rest liquid from the product incubated in the step 3.4. From the reagent storage area of 10 u L washing buffer heavy suspension of magnetic beads (while the stepper motor lowered the magnet), move to the central reaction area room temperature incubation for 30 minutes.
And 3.6, controlling a stepping motor by an automatic program, recovering the magnetic beads by using a magnet from the product incubated in the step 3.5, and removing the rest liquid. From the reagent storage area 10. Mu.L of strong washing buffer to resuspend the beads, 65℃for 10 minutes incubation.
3.7 Repeat step 3.6 twice.
3.8, Controlling a stepping motor by an automatic program, recovering the magnetic beads by using a magnet from the product incubated in the step 3.7, removing the rest liquid, and distinguishing 4uL double-distilled water from a reagent storage area to resuspend the magnetic beads. Finally obtaining the captured hybridization product.
4. Post-capture library construction method:
4.1 the product obtained in step 3 was separated into 6. Mu.L of a polymerase chain reaction mixture, and the mixture was thoroughly mixed in a central reaction zone, followed by a PCR amplification reaction having a total volume of 10. Mu.L. The amplification procedure was: pre-denaturation at 95 ℃ for 45 seconds; 12 cycles: denaturation at 98℃for 15 sec, annealing at 60℃for 30 sec, extension at 72℃for 30 sec; final extension at 72℃for 1 min.
4.2, Purifying the product obtained in the step 4.1 by magnetic beads, and finally eluting by using TE buffer solution to obtain a DNA library after capturing hybridization, wherein the DNA library can be used for sequencing by a BGI sequencing platform. (depending on the kit and primer used, the library may also be suitable for use in other sequencing platforms such as illumine for sequencing, here exemplified by the BGI platform)
Meanwhile, enrichment is carried out without a microfluidic chip as a control experiment (enrichment by small-volume manual operation), namely all the reactions are completed in an analog system (centrifuge tube), and the temperature is controlled by a PCR instrument. All reagents remained identical and 30-75uL of silicone oil was added to the centrifuge tube to seal the droplets (see figure 4 simulation system).
The experimental results are as follows:
The enrichment results were quantified using real-time fluorescent PCR (containing two target regions and one non-target region) and the results are shown in FIG. 7. In FIG. 7, unentrained represents libraries not subjected to capture hybridization, and input quantification represents qPCR identification of three types of libraries at the same initial amount.
As can be seen from fig. 7, the small volume of the manual enrichment library and the chip automated library gave good enrichment in both target regions (the qPCR curve peaked earlier, about 3-4 cycles ahead) and no enrichment or even loss in the non-target regions (the qPCR curve peaked later) compared to the non-enriched library. This demonstrates that the biochemical method can successfully enrich the target region by hybridization capture, and that the chip automated library has more obvious advantages because it is automated without manual manipulation.
In addition, the normal reaction procedure was completed according to the instructions of the commercial kit MGIEasy exon group capture V5 probe kit, all reagents were not treated with tween 20 and no silicone oil (see standard system of fig. 4). The results are consistent with small volumes of manual manipulation (curve coincidence) and are therefore not shown in the experimental results.
Meanwhile, the influence of the addition amount of the surfactant Tween 20 on the result is studied, the addition amount of the used surfactant Tween 20 is adjusted to be respectively set to be 0.01%, 0.75%, 0.1%, 0.5% and 1% of the mass concentration of the Tween 20 in the microfluidic drop, and experimental results show that 0.01% -0.1% basically cannot influence the biochemical reaction and can meet the surface tension of the digital microfluidic drop, and when the concentration reaches 1%, the yield of the biochemical reaction is seriously reduced or even cannot be basically carried out.
The experimental results are shown in FIG. 7.
From this, it was found that tween 20 with a mass concentration of 1% or more severely affects the progress of biochemical reaction, while tween 20 with a mass concentration of 0.01% -0.1% has a low effect on biochemistry and can reach the surface tension of digital microfluidics, especially tween 20 with a mass concentration of 0.075% can be used as a surfactant for increasing the surface tension of liquid droplets.
Example 2 Low initial Low volume automated multiplex PCR target region enrichment technique
Embodiment 2 provides a method for multiplex PCR enrichment of a target region using a microfluidic platform, comprising the steps of:
The microfluidic chip is inserted into the microfluidic device, and filled with silicone oil. 1. Mu.L (3 ng) of human genomic DNA was used as a raw material, which was injected into a microfluidic chip, and all the reagents were loaded into a storage area maintained at 4℃throughout the whole library construction process.
All of the reagents used below were purchased from commercial kit MGICare BRCA, 1/2 sequencing library preparation kit, manufacturer Hua Dazhi. The PCR primer comprises a polymerase chain reaction mixed solution, a multiplex PCR primer mixed solution, a universal primer mixed solution, a PCR promoter, a single-stranded loop preparation buffer solution, ligase, a digestion mixed solution, a termination buffer solution, a DNB preparation primer, a DNB preparation buffer solution, a DNB preparation buffer enzyme and the like. Tween 20 was added to all reagents at a final concentration of 0.075%. Wherein, the DNB preparation buffer solution is additionally added with polyethylene glycol 8000 with the final concentration of 1 percent.
The magnetic beads for magnetic bead purification, AMPure XP, were purchased from BECKMAN COULTER under the trade designation 17775900. Tween 20 was added to the beads at a final concentration of 0.075%.
The washing reagent used for magnetic bead purification comprises the following components: polyethylene glycol 8000,2.5M sodium chloride solution at final concentration of 20% and tween 20 at 0.075%.
The following steps are all that the program controls the electrode switch so as to control the movement of the liquid drops:
1. first round PCR amplification and purification method after amplification
From the reagent storage area, 1. Mu.L (3 ng) of a human genomic DNA solution, 3. Mu.L of a polymerase chain reaction mixture, 1. Mu.L of a multiplex PCR primer mixture and 1. Mu.L of an LPCR promoter were transferred to the central reaction area and mixed well, followed by amplification.
The amplification procedure was: pre-denaturation at 95 ℃ for 10 min; 5 cycles: denaturation at 95℃for 1 min, annealing at 60℃for 15min, extension at 72℃for 1 min; final extension at 68℃for 10 min. The obtained product is purified by using magnetic bead reagents which are distinguished by a reagent storage area, a stepping motor is used for controlling the lifting of a magnet so as to control the adsorption of magnetic beads, and finally, a first round of amplification product is obtained after eluting by using 1 mu L of TE buffer solution.
2. Second round PCR amplification and purification method after amplification
From the reagent storage area of 3 u L polymerase chain reaction mixture, 1 u L universal primer mixture and 1 u LPCR promoter added to the step 1 product and fully mixed, then amplification.
The amplification procedure was: pre-denaturation at 95 ℃ for 10 min; 20 cycles: denaturation at 95℃for 1 min, annealing at 60℃for 1 min, extension at 72℃for 1 min; final extension at 68℃for 10 min. The obtained product is purified by using magnetic bead reagents which are distinguished by a reagent storage area, a stepping motor is used for controlling the lifting of a magnet so as to control the adsorption of magnetic beads, and finally, a second round of amplification product is obtained after eluting by using 1 mu L of TE buffer solution.
3. Preparation of DNA nanospheres
3.1 Controlling the product obtained in the step 2 by using an electrode, moving the product to a central reaction zone for denaturation at 95 ℃, and cooling the product to room temperature.
3.2 Separating 3. Mu.L of Single Strand preparation buffer and 1. Mu.L of ligase buffer from the storage area were added to the product obtained in step 3.1 and mixed well, followed by incubation at 37℃for 30 minutes.
3.3 3. Mu.L of digestion mixture was removed from the storage area and added to the product obtained in step 3.2 and thoroughly mixed and incubated at 37℃for 30 minutes.
3.4 1. Mu.L of stop buffer was removed from the storage area and added to the product obtained in step 3.3 and thoroughly mixed.
3.5 Separating the magnetic bead reagent from the storage area, purifying the product obtained in the step 3.4, controlling the magnet to lift by using a stepping motor so as to control the adsorption of the magnetic beads, and separating 3 mu L of TE buffer from the reagent storage area to elute the magnetic beads.
3.6 Preparation of primers from storage area 3. Mu. LDNB after addition to the product obtained in step 3.5 and thorough mixing, 95℃1min,65℃1min,40℃1min.
3.7 From the reagent reservoir, 6. Mu.L of DNB preparation mixture and 1. Mu.L of DNB preparation polymerase were added to the product obtained in step 3.6 and thoroughly mixed. Incubate at 30℃for 20 min.
3.8 From the reagent reservoir 3. Mu.L of stop buffer was added to the product obtained in step 3.7 and thoroughly mixed. The obtained product is the DNA nanospheres which can be used for sequencing by the BGI platform, and a sequencing library is obtained.
Meanwhile, a small-volume manual operation multiplex PCR method is used as a control, namely, all the reactions are completed in a simulation system (centrifuge tube), and the temperature is controlled by a PCR instrument. All reagents remained identical and 30-75uL of silicone oil was added to the centrifuge tube to seal the droplets (see figure 4 simulation system).
The normal reaction procedure was completed according to the instructions of the commercial kit MGICare BRCA/2 sequencing library preparation kit using the normal volume manual multiplex PCR method as a control, all reagents were not treated with Tween 20 and no silicone oil (see standard system of FIG. 4).
The enriched library was sequenced using BGISeq500,500 sequencer and the experimental results are shown in table 1 below:
TABLE 1 sequencing data analysis results
Wherein the genome matching ratio in Table 1 refers to the ratio of the base sequence obtained by sequencing to the human reference genome; the capture ratio refers to the ratio of the base sequence obtained by sequencing on two BRCA1/2 genes; the average sequencing depth relative duty ratio is 1 when the depth measured by manually operating multiplex PCR with normal volume, and the other two methods are relative to the duty ratio of 1 (the average sequencing depth refers to the average coverage times of a base sequence in each region of a genome, and the higher the depth is, the more data volume is indicated, the more data volume is in this case, the more data volume is required because of the enrichment library, so the higher the data volume is, the waste will be caused); coverage refers to the extent to which the base sequence obtained by sequencing can cover both BRCA1/2 gene regions; the uniformity refers to the uniformity of the base sequence obtained by sequencing covering different regions.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (14)
1. A method for enriching nucleic acid based on a digital microfluidic platform is characterized in that the digital microfluidic platform comprises a microfluidic chip and a microfluidic device, the microfluidic chip and the microfluidic device are detachably connected,
The method comprises the following steps:
Preparing microfluidic drops on a microfluidic chip, wherein the microfluidic drops contain a surfactant, silicone oil and nucleic acid;
Specifically capturing or amplifying a target region of the nucleic acid in the microfluidic droplet based on a specific probe or a specific primer to obtain an enriched nucleic acid product,
Wherein,
The surfactant is 0.075% tween 20,
The microfluidic chip includes:
a substrate on which an electrode matrix is disposed;
The operation area is positioned on the substrate and comprises a sample adding area, a storage area, a reaction area and a collection area, wherein the sample adding area, the storage area, the reaction area and the collection area are sequentially connected;
the microfluidic device includes:
the temperature control module is arranged opposite to the reaction zone and is used for controlling the temperature of the reaction zone;
The magnetic control module is arranged opposite to the collecting area, a magnet is arranged in the magnetic control module, and the magnetic control module controls the lifting of the magnet through a stepping motor;
The water cooling module is arranged opposite to the storage area through a metal plate and is used for controlling the temperature of the storage area.
2. The method of claim 1, wherein the silicone oil is a low viscosity silicone oil.
3. The method of claim 1, further comprising the step of purifying the enriched nucleic acid product using the microfluidic device.
4. The method of claim 1, wherein the microfluidic droplet has a volume of no more than 16 microliters.
5. The method of claim 1, wherein the metal plate is a parchment plate.
6. The method of claim 1, wherein the microfluidic device further comprises:
the fluorescence detection module is connected with the reaction zone and is used for carrying out fluorescence detection on the product of the reaction zone.
7. The method of claim 1, wherein the digital microfluidic platform further comprises:
The data processing device is used for converting the reaction result of the microfluidic chip into an electric signal and outputting the electric signal;
Optionally, a mechanical arm is further arranged on the data processing device, and the mechanical arm is used for realizing automatic operation of the microfluidic chip.
8. The method of claim 1, wherein the nucleic acid is genomic DNA and the genomic DNA is 5 nanograms or more.
9. A method of constructing a sequencing library, comprising:
Obtaining a target region enriched product of nucleic acid based on the method of any one of claims 1-8;
enriching products based on the target region of the nucleic acid, and pooling to obtain a sequencing library.
10. The method as recited in claim 9, further comprising:
After preparing microfluidic drops on a microfluidic chip, performing end repair and joint connection, and performing first PCR amplification on the obtained connection product so as to construct a pre-capture DNA library;
performing hybridization capture and purification on the pre-capture DNA library using a specific probe to obtain a captured hybridization product;
and performing a second PCR amplification based on the captured hybridization product to construct a captured DNA library.
11. The method of claim 10, wherein the specific probe is a biotin-labeled specific probe and the purification is performed using streptavidin-labeled magnetic beads.
12. The method as recited in claim 9, further comprising:
After preparing microfluidic droplets on a microfluidic chip, amplifying with a first amplification primer adapted to amplify different regions of the nucleic acid to obtain a first amplification product;
Amplifying the first amplification product with a universal primer, which is partially complementary to the first amplification primer, so as to obtain a second amplification product.
13. The method as recited in claim 12, further comprising:
denaturing and circularizing the second amplification product to obtain single-stranded circular DNA;
rolling circle amplification is performed based on the single-stranded circular DNA to obtain DNA nanospheres.
14. The method according to claim 12, wherein the purification is performed using magnetic beads after amplification using the first amplification primer or after amplification of the first amplification product using the universal primer, respectively.
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