CN114621307A - Oligonucleotide space coordinate coding method and microfluidic device thereof - Google Patents
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Abstract
The invention relates to an oligonucleotide space coordinate encoding method and a microfluidic device thereof, belonging to the technical field of molecular biology. The invention provides an oligonucleotide space coordinate coding method, presetting the oligonucleotide full sequence to be coded as being formed by connecting n + i sections of oligonucleotide sequence segments, and synthesizing n groups of different oligonucleotide sequence segments X and i groups of different oligonucleotide sequence segments Y according to the presetting; setting an oligonucleotide synthesis array formed by orthogonally setting an X flow channel and a Y flow channel, so that a coding region for connecting oligonucleotide sequence fragments is formed at the intersection of the X flow channel and the Y flow channel; and respectively applying the oligonucleotide sequence fragment X and the oligonucleotide sequence fragment Y to each X flow channel and each Y flow channel of the oligonucleotide synthesis array by wheel pair so as to gradually connect the oligonucleotide sequence fragment X and the oligonucleotide sequence fragment Y in the coding region to obtain the oligonucleotide complete sequence to be coded. The method greatly reduces the synthesis cost of the oligonucleotide.
Description
Technical Field
The invention relates to an oligonucleotide space coordinate encoding method and a microfluidic device thereof, belonging to the technical field of molecular biology.
Background
The analysis of the spatial biological information of cells in tissues is important for the subjects of oncology, immunology, neuroscience, genetic development, pathology and the like. For example, tumor heterogeneity in oncology, tumor microenvironment, tumor metastasis; embryonic development in genetic development, organ development maps, individual cell layers of the brain in neuroscience, and anatomical features of normal and abnormal parts of the brain. The spatial biological information analysis can be realized by means of spatial coding, namely, a unique code is constructed on each point of a two-dimensional space, a sample to be detected is marked by the code, and then the spatial biological information analysis is realized by decoding.
The oligonucleotide (Oligo) is formed by connecting nucleotides carrying four bases of ATCG, and up to 4 can be realized by the sequential combination of the basesnA combination of codes that can be used for spatial coding. Currently, thanks to advances in the art of artificial synthesis of DNA, high-throughput, low-cost in vitro synthesis of oligonucleotides has been achieved. However, there are technical difficulties in constructing oligonucleotide arrays of known sequence in two dimensions. At present, the main strategy for constructing oligonucleotide arrays with known sequences on two-dimensional space and the defects thereof are as follows:
1. firstly synthesizing and then fixing: firstly synthesizing a large number of oligonucleotide fragments with known sequences, then sequentially introducing the oligonucleotide fragments with the known sequences into a substrate target position through spotting according to required spatial arrangement, and then connecting and fixing to form an oligonucleotide array; this method is limited by the size of the drop of the printing medium, the spatial resolution is generally greater than 50 microns, and the cost increases significantly as the number of coding dots increases;
2. in-situ synthesis: namely, chemically synthesizing a required oligonucleotide array on a two-dimensional substrate in situ; the key technology of the method is selectively introducing ATCG raw materials, can be realized by technologies such as photoetching, ink-jet printing, type printing and the like, has the advantage of high spatial resolution (10-60 micrometers), but is limited by the chemical reaction efficiency of DNA synthesis, the coding length connected by the method is limited (generally not more than 20bp, the limit length is 60bp), and the oligonucleotide in the coding point has low density and low purity;
3. random fixation resequencing: firstly synthesizing all required coding sequences, mixing to form a primer pool, then adding the primers to be randomly connected to a two-dimensional substrate at one time, reading coding information in an in-situ sequencing mode, and determining coding sequence information connected to each spatial position; although the method can construct Oligo arrays with high spatial resolution and long coding sequences, the cost of the Oligo arrays is high and the Oligo arrays are difficult to prepare in batches due to early primer pool synthesis and subsequent sequencing.
Therefore, it is highly desirable to find oligonucleotide synthesis technology with high spatial resolution, long coding sequence and low cost to construct oligonucleotide arrays with known sequence in two-dimensional space, and further develop the spatial coding technology.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for encoding oligonucleotide spatial coordinates, the method comprising the steps of:
the method comprises the following steps: presetting the whole sequence of the oligonucleotide to be coded as n + i segments of oligonucleotide sequence segments, and synthesizing n groups of different oligonucleotide sequence segments X and i groups of different oligonucleotide sequence segments Y according to the presetting;
step two: setting an oligonucleotide synthesis array formed by orthogonally setting an X flow channel and a Y flow channel, so that a coding region for connecting oligonucleotide sequence fragments is formed at the intersection of the X flow channel and the Y flow channel;
step three: respectively applying oligonucleotide sequence segments X and oligonucleotide sequence segments Y to each X flow channel and each Y flow channel of the oligonucleotide synthesis array by wheel pairs so as to gradually connect the oligonucleotide sequence segments X and the oligonucleotide sequence segments Y in the coding region to obtain the whole sequence of the oligonucleotide to be coded;
wherein the number of the X runners is n; the number of the Y flow channels is i; when oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y are applied in rounds, the same oligonucleotide sequence fragment is applied to the coding region in the same flow channel, and different oligonucleotide sequence fragments are applied to the coding region in different flow channels, so that multiple rows can be coded in each round.
The invention also provides a microfluidic device for oligonucleotide synthesis, comprising a first microfluidic chip and a second microfluidic chip; a plurality of X flow channels are arranged on the first micro-fluidic chip; a plurality of Y flow channels are arranged on the second micro-fluidic chip; the X runner and the Y runner are arranged orthogonally; and the intersection of the X runner and the Y runner forms a coding region.
In one embodiment of the invention, the microfluidic device comprises a first microfluidic module and a second microfluidic module; the first microfluidic module is sequentially provided with a first substrate, a first coding chip, a first microfluidic chip, a first sample introduction adapter plate and a first upper fixing plate from bottom to top; the first microfluidic chip is provided with a plurality of first sample inlets and a plurality of first sample outlets; the first sample inlets are arranged in pairs, and the pair of first sample inlets are communicated through an X flow channel; the first sample outlets are arranged in pairs, and the pair of first sample outlets are communicated through an X runner; the first sample introduction adapter plate is provided with a plurality of first sample inlets and a plurality of first sample outlets; the first sample inlets correspond to the first sample inlets one to one; the first sample outlets correspond to the first sample outlets one to one; a plurality of first openings are formed in the first upper fixing plate; the first opening is communicated with at least one first sample inlet or first sample outlet; a first sealing cover plate is arranged on the first opening; the second microfluidic module is sequentially provided with a second substrate, a second coding chip, a second microfluidic chip, a second sample introduction adapter plate and a second upper fixing plate from bottom to top; the second microfluidic chip is provided with a plurality of second sample inlets and a plurality of second sample outlets; the second sample inlets are arranged in pairs, and the pair of second sample inlets are communicated through a Y flow channel; the second sample outlets are arranged in pairs, and the pair of second sample outlets are communicated through a Y flow channel; the second sample introduction adapter plate is provided with a plurality of second sample inlets and a plurality of second sample outlets; the second sample inlets correspond to the second sample inlets one to one; the second sample outlets correspond to the second sample outlets one to one; a plurality of second openings are formed in the second upper fixing plate; the second opening is communicated with at least one second sample inlet or second sample outlet; and a second sealing cover plate is arranged on the second opening.
In an embodiment of the present invention, the first coding chip and/or the second coding chip is provided with a coded photographing region.
In an embodiment of the present invention, the coded photographing region is disposed at a central position of the first coding chip and/or the second coding chip.
In one embodiment of the invention, a first sealing ring is arranged between the first sealing cover plate and the first opening.
In one embodiment of the invention, a second sealing ring is arranged between the second sealing cover plate and the second opening.
In one embodiment of the present invention, the first base and the first upper fixing plate are fixed by bolts.
In one embodiment of the present invention, the second substrate and the second upper fixing plate are fixed to each other by bolts.
In an embodiment of the present invention, the first microfluidic chip, the first sample injection adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample injection adapter plate, and the second upper fixing plate are made of a light-transmitting material.
In an embodiment of the present invention, the first microfluidic chip, the first sample introduction adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample introduction adapter plate, and the second upper fixing plate are made of a flexible light-transmitting material.
The present invention also provides a method for synthesizing oligonucleotides using the above method for encoding spatial coordinates of oligonucleotides on the above microfluidic device for oligonucleotide synthesis.
The invention also provides the application of the oligonucleotide space coordinate encoding method or the microfluidic device for oligonucleotide synthesis or the oligonucleotide synthesis method in oligonucleotide synthesis.
The technical scheme of the invention has the following advantages:
1. the invention provides an oligonucleotide space coordinate coding method, which comprises the following steps: firstly, presetting the oligonucleotide full sequence to be coded, which is formed by connecting n + i oligonucleotide sequence segments, synthesizing n groups of different oligonucleotide sequence segments X and i groups of different oligonucleotide sequence segments Y according to the presetting, then, arranging an oligonucleotide synthesis array formed by orthogonally arranging an X flow channel and a Y flow channel, enabling the intersection of the X flow channel and the Y flow channel to form a coding region for connecting the oligonucleotide sequence segments, finally, respectively applying the oligonucleotide sequence segments X and the oligonucleotide sequence segments Y to each X flow channel and each Y flow channel of the oligonucleotide synthesis array wheel by wheel, enabling the oligonucleotide sequence segments X and the oligonucleotide sequence segments Y to be gradually connected in the coding region, and obtaining the oligonucleotide full sequence to be coded, wherein the number of the X flow channels is n, the number of the Y flow channels is i, and when the oligonucleotide sequence segments X and the oligonucleotide sequence segments Y are applied wheel by wheel, the same oligonucleotide sequence segments are applied to the coding regions in the same flow path and different oligonucleotide sequence segments are applied to the coding regions in different flow paths so that multiple rows can be encoded per round. The method separates a section of oligonucleotide complete sequence to be coded into a plurality of sections for synthesis and connection, and ensures that each coding region has at least one section of coding sequence through a plurality of microfluidic selective region connection reactionsThe different columns ensure that the coding sequence of each site of the finally combined coding sequence is different. The method uses a parallel linear micro-channel array for positioning the coding region, the same channel applies the same oligonucleotide sequence segment to the flow-through region, different channels apply different oligonucleotide sequence segments, and each round can code a plurality of lines. In order to complete the dot matrix coding, the method can carry out multi-round coding application and connection through micro-channels, the direction or the angle of each round of micro-channels is different, and the applied oligonucleotide sequence fragments are ensured to be different at least in one round for different coding regions, therefore, if the number of coding rounds is q, the number of micro-channels in each round is p (namely the number of coding sequences applied in each round is p), only p multiplied by q oligonucleotide sequence fragments are needed to be synthesized, and then p can be completedq×4mCoding combinations that require a reduced number of oligonucleotide sequence fragments to be synthesized to achieve the same number of codes compared to conventional coding methodsq-1The/q times, greatly reduces the synthesis cost of the oligonucleotide.
2. The invention provides a micro-fluidic device for oligonucleotide synthesis, which comprises a first micro-fluidic chip and a second micro-fluidic chip, wherein a plurality of X flow channels are arranged on the first micro-fluidic chip, a plurality of X flow channels are arranged on the second micro-fluidic chip, the X flow channels and the Y flow channels are arranged in an orthogonal mode, and a coding area is formed at the intersection of the X flow channels and the Y flow channels. The microfluidic device adopts an independent parallel microfluidic channel design (an X runner and a Y runner) and is combined with a detachable pressure seal assembly module (a substrate, a coding chip, a microfluidic chip, a sample introduction adapter plate and an upper fixing plate), so that parallel oligonucleotide sequence fragment addition and multiple oligonucleotide sequence fragment addition, positioning and connection can be realized. The microfluidic device can regulate and control the density of the oligonucleotide coding array through the size design of a micro-channel. The upper fixing plate, the sample introduction adapter plate and the microfluidic chip of the microfluidic device are made of light-transmitting materials, so that optical photographing in a code photographing area of the code chip is facilitated, and optical information is acquired. The sample introduction adapter plate and the microfluidic chip of the microfluidic device are made of flexible light-transmitting materials such as PDMS, and the sealing effect of the detachable pressure sealing assembly module is improved.
3. The present invention provides a method for synthesizing oligonucleotides using the above oligonucleotide spatial coordinate encoding method on the above microfluidic device for oligonucleotide synthesis. The method adopts a step-by-step connection method, firstly prepares the required oligonucleotide sequence fragment and modification by a standard oligonucleotide synthesis method, and then sequentially connects the required oligonucleotide sequence fragment to a target position by combining a microfluidic device through connection methods such as chemical connection, enzyme connection and the like so as to obtain an oligonucleotide coding array with a determined sequence, wherein the prepared oligonucleotide sequence has long length and high purity.
Drawings
FIG. 1: schematic flow diagram of oligonucleotide synthesis.
FIG. 2: schematic view of a flow channel structure of a first micro-fluidic chip and a second micro-fluidic chip in the micro-fluidic device.
FIG. 3: cross-sectional views of a first microfluidic module and a second microfluidic module in a microfluidic device.
FIG. 4: schematic diagram of the overall structure of the first microfluidic module and the second microfluidic module in the microfluidic device.
FIG. 5 is a schematic view of: a top view of a first microfluidic chip in a microfluidic device.
FIG. 6: a top view of a second microfluidic chip in the microfluidic device.
FIG. 7: and a top view of the first encoding chip and the second encoding chip in the microfluidic device.
FIG. 8: and a top view of a first sample adapter plate and a second sample adapter plate in the microfluidic device.
FIG. 9: a top view of a first microfluidic module and a second microfluidic module in a microfluidic device.
FIG. 10: and the whole structure of the first upper fixing plate and the second upper fixing plate in the microfluidic device is schematically shown.
FIG. 11: the overall structure of the first sealing ring and the second sealing ring in the microfluidic device is shown schematically.
FIG. 12: the overall structure of the first sealing cover plate and the second sealing cover plate in the microfluidic device is schematically shown.
FIG. 13: the process of oligonucleotide synthesis.
FIG. 14: the result of oligonucleotide synthesis of the local coding region.
In fig. 2 to 12, a first microfluidic module 1, a second microfluidic module 2, a first substrate 3, a first encoding chip 4, a first microfluidic chip 5, a first sample adapter plate 6, a first upper fixing plate 7, a first sample inlet 8, a first sample outlet 9, an X-channel 10, a first sample inlet 11, a first sample outlet 12, a first opening 13, a first sealing cover plate 14, a second substrate 15, a second encoding chip 16, a second microfluidic chip 17, a second sample adapter plate 18, a second upper fixing plate 19, a second sample inlet 20, a second sample outlet 21, a Y-channel 22, an encoding region 23, a second sample inlet 24, a second sample outlet 25, a second opening 26, a second sealing cover plate 27, an encoding photographing region 28, a first sealing ring 29, and a second sealing ring 30.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples, where specific experimental procedures or conditions are not indicated, can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the art. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1: oligonucleotide space coordinate coding method
This example provides a method for encoding oligonucleotides in terms of their spatial coordinates (see FIG. 1 for a scheme for synthesizing oligonucleotides using this method), comprising the steps of:
the method comprises the following steps: presetting the whole sequence of the oligonucleotide to be coded as n + i segments of oligonucleotide sequence segments, and synthesizing n groups of different oligonucleotide sequence segments X and i groups of different oligonucleotide sequence segments Y according to the presetting;
step two: setting an oligonucleotide synthesis array formed by orthogonally setting an X flow channel and a Y flow channel, so that a coding region for connecting oligonucleotide sequence fragments is formed at the intersection of the X flow channel and the Y flow channel;
step three: respectively applying oligonucleotide sequence segments X and oligonucleotide sequence segments Y to each X flow channel and each Y flow channel of the oligonucleotide synthesis array by wheel pairs, so that the oligonucleotide sequence segments X and the oligonucleotide sequence segments Y are gradually connected in a coding region to obtain an oligonucleotide complete sequence to be coded;
wherein the number of the X runners is n; the number of the Y flow channels is i; when oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y are applied in rounds, the same oligonucleotide sequence fragment is applied to the coding region in the same flow channel, and different oligonucleotide sequence fragments are applied to the coding region in different flow channels, so that multiple rows can be coded in each round.
Example 2: micro-fluidic device
As shown in FIGS. 2 to 12, this example provides a microfluidic device for implementing the method for encoding spatial coordinates of oligonucleotides described in example 1 (the flow of oligonucleotide synthesis using this device is shown in FIG. 1), which comprises a first microfluidic module 1 and a second microfluidic module 2; the first microfluidic module 1 is sequentially provided with a first substrate 3, a first coding chip 4, a first microfluidic chip 5, a first sample introduction adapter plate 6 and a first upper fixing plate 7 from bottom to top; the first micro-fluidic chip 5 is provided with a plurality of first sample inlets 8, a plurality of first sample outlets 9 and a plurality of X flow channels 10; the first sample inlets 9 are arranged in pairs, and the pair of first sample inlets 9 are communicated through an X flow channel 10; the first sample outlets 9 are arranged in pairs, and the first sample outlets 9 are communicated with each other through an X flow channel 10; the first sample introduction adapter plate 6 is provided with a plurality of first sample inlets 11 and a plurality of first sample outlets 12; the first sample inlets 11 correspond to the first sample inlets 8 one by one; the first sample outlets 12 correspond to the first sample outlets 9 one by one; a plurality of first openings 13 are arranged on the first upper fixing plate 7; the first opening 13 is communicated with at least one first sample inlet 11 or first sample outlet 12; a first sealing cover plate 14 is arranged on the first opening 13; the second microfluidic module 2 is sequentially provided with a second substrate 15, a second coding chip 16, a second microfluidic chip 17, a second sample introduction adapter plate 18 and a second upper fixing plate 19 from bottom to top; the second microfluidic chip 17 is provided with a plurality of second sample inlets 20, a plurality of second sample outlets 21 and a plurality of Y flow channels 22; the second sample inlets 20 are arranged in pairs, and the pair of second sample inlets 20 are communicated through a Y flow channel 22; the second sample outlets 21 are arranged in pairs, and the pair of second sample outlets 21 are communicated through a Y flow passage 22; the X runner 10 and the Y runner 22 are orthogonally arranged; the intersection of the X runner 10 and the Y runner 22 forms a coding region 23; the second sample introduction adapter plate 18 is provided with a plurality of second sample inlets 24 and a plurality of second sample outlets 25; the second sample inlets 24 correspond to the second sample inlets 20 one by one; the second sample outlets 25 correspond to the second sample outlets 21 one by one; a plurality of second openings 26 are arranged on the second upper fixing plate 19; the second opening 26 is in communication with at least one second sample inlet 24 or second sample outlet 25; a second sealing cover plate 27 is arranged on the second opening 26; the first coding chip 4 and the second coding chip 16 are provided with a coding photographing region 28; the coded photographing region 28 is arranged at the central positions of the first coding chip 4 and the second coding chip 16; a first sealing ring 29 is arranged between the first sealing cover plate 14 and the first opening 13; a second sealing ring 30 is arranged between the second sealing cover plate 27 and the second opening 26; the first base 3 and the first upper fixing plate 7 are fixed by bolts (e.g., M3, M4, etc.); the second substrate 15 and the second upper fixing plate 19 are fixed by bolts; the first microfluidic chip 5, the first sample introduction adapter plate 6, the first upper fixing plate 7, the second microfluidic chip 17, the second sample introduction adapter plate 18 and the second upper fixing plate 19 are made of flexible light-transmitting materials (such as fluororubber, chloroprene rubber, PDMS, etc.).
Preferably, the width of the X flow channel and the Y flow channel is 2-200 μm; the size of the center distance between the X runner and the Y runner is 4-500 mu m; the height of the X runner and the height of the Y runner are 2-200 mu m; the number of the first sample inlet, the first sample outlet, the second sample inlet, the second sample outlet, the second sample inlet and the second sample outlet is 12-240, and the aperture is 0.5 mm-5.0 mm; the length of the bolt is 3 mm-12 mm.
Example 3: method for synthesizing oligonucleotide
This example provides a method for synthesizing oligonucleotides using the oligonucleotide spatial coordinates encoding method of example 1 on the microfluidic device for oligonucleotide synthesis of example 2, wherein the synthesis target is an oligonucleotide having a nucleotide sequence shown in SEQ ID No. 1(AAGCAGTGGTATCAACGCAGAGTACGTCTCTTTCCCTACACACGACGCTCTTCCGATCTNNNNNNNNNNGAGTGATTGCTTGTGACGCCTTNNNNNNNNNNNNNNNNNNNNNNT30VN), the oligonucleotide having a nucleotide sequence shown in SEQ ID No. 1 is first prepared by ligating oligo1, oligo2 and oligo3 according to the oligonucleotide spatial coordinates encoding method of example 1, wherein oligo1 is a universal head sequence having a nucleotide sequence shown in SEQ ID No. 2(TTAAGCAGTGGTATCAACGCAGAGTACGTCTCTTTCCCTACAC), oligo2 and oligo3 are coding sequences including 4 sets (i.e., n ═ 4) of different oligonucleotide sequence segments X and 4 sets (i ═ 4) of different oligonucleotide sequence segments Y, respectively, and the 4 sets of different oligonucleotide segments X1 to X4 (nucleotide sequences shown in SEQ ID nos. 3 to 6: 6) are synthesized according to the preparation ACGACGCTCTTCCGATCTGCTTACGCAGGAGTGATTGC, ACGACGCTCTTCCGATCTCTACAGAGCGGAGTGATTGC, ACGACGCTCTTCCGATCTGATCTGGTCCGAGTGATTGC, ACGACGCTCTTCCGATCTTACAGCGTTAGAGTGATTGC) and 4 different oligonucleotide sequence fragments Y1-Y4 (the nucleotide sequences are shown as SEQ ID NO: 7-SEQ ID NO:10, TTGTGACGCCTTCCGATTGGATNNNNNNNNNNNNT30VN, TTGTGACGCCTTAGGCCTCATANNNNNNNNNNNNT30VN, TTGTGACGCCTTTTGTACGGCCNNNNNNNNNNNNT30VN, TTGTGACGCCTTTACTATCGGANNNNNNNNNNNNT30VN), and then the oligonucleotide ligation is performed using the microfluidic device for oligonucleotide synthesis of example 2 (the ligation process is shown in FIG. 13), and the specific steps are as follows:
1. preparing a substrate, carrying out surface treatment on the substrate, and then connecting a universal head sequence Oligo 1;
2. constructing an X-direction micro-channel on a substrate;
3. applying Oligo2 coding sequence to a micro flow channel in the X direction by positive pressure pump suction, and connecting the oligonucleotide sequence fragment X1 to the head sequence by a connection reaction;
4. disassembling the X-direction micro-channel and reconstructing a new Y-direction micro-channel, wherein the fluid directions of the new micro-channel and the pre-positioned micro-channel are arranged in an orthogonal direction (the first micro-fluidic module and the second micro-fluidic module share one substrate, and the disassembly and reconstruction of the X-direction micro-channel and the Y-direction micro-channel on the same substrate are realized through bolts);
5. applying Oligo3 coding sequence through micro flow channel, and connecting the oligonucleotide sequence fragment Y1 to the oligonucleotide sequence fragment X1 through connection reaction;
6. repeating the steps 4 and 5 until the requirement of the number of the encoding rounds is met, and disassembling the micro flow channel to obtain the Oligo encoding array on the substrate;
wherein the Oligo1, Oligo2 and Oligo3 are synthesized by the company of Toyobo Biotechnology, Inc. (Shanghai), the reagent for oligonucleotide ligation is T4 DNAligase (Novosa, cat # N103-01), and the oligonucleotide ligation is performed at room temperature (25 ℃);
the specific parameters of the microfluidic device for oligonucleotide synthesis of example 2 were: the width of the X runner and the Y runner is 50 μm; the size of the center distance between the X runner and the Y runner is 100 mu m; the height of the X runner and the height of the Y runner are 50 mu m; the number of the first sample inlet, the first sample outlet, the second sample inlet, the second sample outlet, the second sample inlet and the second sample outlet is 48, and the aperture is 1.0 mm; the first microfluidic chip, the first sample introduction adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample introduction adapter plate and the second upper fixing plate are made of PDMS materials; the bolt was M4 and had a length dimension of 8 mm.
In the process of oligonucleotide synthesis, 4 oligonucleotide sequence segments X and 4 oligonucleotide sequence segments Y are respectively and continuously communicated in the X flow channel and the Y flow channel, and theoretically, 16 cross-over points with clear boundaries exist, so that after a fluorescent hybridization probe is introduced in the Y flow channel, a local coding photographing region (namely, a half coding photographing region) after oligonucleotide synthesis is carried out is photographed by a confocal microscope, and the observation result is shown in fig. 14. As can be seen from FIG. 14, the confocal microscope photographed 8 clearly-defined intersections in the imaged region of the local code after oligonucleotide synthesis, and the results were as expected.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Sequence listing
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<120> oligonucleotide space coordinate coding method and microfluidic device thereof
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Claims (10)
1. A method for encoding oligonucleotides in spatial coordinates, said method comprising the steps of:
the method comprises the following steps: presetting the whole sequence of the oligonucleotide to be coded as n + i segments of oligonucleotide sequence segments, and synthesizing n groups of different oligonucleotide sequence segments X and i groups of different oligonucleotide sequence segments Y according to the presetting;
step two: setting an oligonucleotide synthesis array formed by orthogonally setting an X flow channel and a Y flow channel, so that a coding region for connecting oligonucleotide sequence fragments is formed at the intersection of the X flow channel and the Y flow channel;
step three: respectively applying oligonucleotide sequence segments X and oligonucleotide sequence segments Y to each X flow channel and each Y flow channel of the oligonucleotide synthesis array by wheel pairs, so that the oligonucleotide sequence segments X and the oligonucleotide sequence segments Y are gradually connected in a coding region to obtain an oligonucleotide complete sequence to be coded;
wherein the number of the X runners is n; the number of the Y flow channels is i; when oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y are applied in rounds, the same oligonucleotide sequence fragment is applied to the coding region in the same flow channel, and different oligonucleotide sequence fragments are applied to the coding region in different flow channels, so that multiple rows can be coded in each round.
2. A microfluidic device for oligonucleotide synthesis, comprising a first microfluidic chip and a second microfluidic chip; a plurality of X flow channels are arranged on the first micro-fluidic chip; a plurality of Y flow channels are arranged on the second micro-fluidic chip; the X runner and the Y runner are arranged orthogonally; and the intersection of the X runner and the Y runner forms a coding region.
3. The microfluidic device for oligonucleotide synthesis according to claim 2, wherein the microfluidic device comprises a first microfluidic module and a second microfluidic module; the first microfluidic module is sequentially provided with a first substrate, a first coding chip, a first microfluidic chip, a first sample introduction adapter plate and a first upper fixing plate from bottom to top; the first microfluidic chip is provided with a plurality of first sample inlets and a plurality of first sample outlets; the first sample inlets are arranged in pairs, and the pair of first sample inlets are communicated through an X flow channel; the first sample outlets are arranged in pairs, and the pair of first sample outlets are communicated through an X runner; the first sample introduction adapter plate is provided with a plurality of first sample inlets and a plurality of first sample outlets; the first sample inlets correspond to the first sample inlets one to one; the first sample outlets correspond to the first sample outlets one to one; a plurality of first openings are formed in the first upper fixing plate; the first opening is communicated with at least one first sample inlet or first sample outlet; a first sealing cover plate is arranged on the first opening; the second microfluidic module is sequentially provided with a second substrate, a second coding chip, a second microfluidic chip, a second sample introduction adapter plate and a second upper fixing plate from bottom to top; the second microfluidic chip is provided with a plurality of second sample inlets and a plurality of second sample outlets; the second sample inlets are arranged in pairs, and the pair of second sample inlets are communicated through a Y flow channel; the second sample outlets are arranged in pairs, and the pair of second sample outlets are communicated through a Y flow channel; the second sample introduction adapter plate is provided with a plurality of second sample inlets and a plurality of second sample outlets; the second sample inlets correspond to the second sample inlets one by one; the second sample outlets correspond to the second sample outlets one to one; a plurality of second openings are formed in the second upper fixing plate; the second opening is communicated with at least one second sample inlet or second sample outlet; and a second sealing cover plate is arranged on the second opening.
4. The microfluidic device according to claim 3, wherein the first coding chip and/or the second coding chip has a coded camera area.
5. The microfluidic device according to claim 4, wherein the encoded imaging region is located at the center of the first encoding chip and/or the second encoding chip.
6. The microfluidic device according to any of claims 3 to 5, wherein a first sealing ring is disposed between the first sealing cover plate and the first opening; and a second sealing ring is arranged between the second sealing cover plate and the second opening.
7. The microfluidic device according to any of claims 3 to 6, wherein the first substrate and the first upper fixing plate are fixed by bolts; the second base and the second upper fixing plate are fixed through bolts.
8. The microfluidic device according to any of claims 3 to 6, wherein the first microfluidic chip, the first sample adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample adapter plate and the second upper fixing plate are made of a light-transmitting material.
9. A method for synthesizing oligonucleotides, wherein the method comprises performing oligonucleotide synthesis on the microfluidic device for oligonucleotide synthesis according to any one of claims 2 to 8 using the oligonucleotide spatial coordinate encoding method according to claim 1.
10. Use of the method for spatial coordinate encoding of oligonucleotides according to claim 1 or the microfluidic device for oligonucleotide synthesis according to any one of claims 1 to 8 or the method for oligonucleotide synthesis according to claim 9 for oligonucleotide synthesis.
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