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DNA encoding microsphere and synthetic method thereof

A technology for coding microspheres and synthesis methods, which is applied in the fields of DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of high cost and complex synthesis of DNA-coded microspheres, and achieves a small number, simple and reliable design, and low cost. Effect

Active Publication Date: 2016-09-07
苏州德运康瑞生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the complex and expensive synthesis of existing DNA-encoded microspheres, the present invention proposes a simple, cheap and efficient method for synthesizing DNA-encoded microspheres

Method used

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  • DNA encoding microsphere and synthetic method thereof
  • DNA encoding microsphere and synthetic method thereof
  • DNA encoding microsphere and synthetic method thereof

Examples

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Embodiment 1

[0065] Synthesis and purification of embodiment 1 nucleic acid molecule

[0066] Using common controllable microporous glass beads (CPG) as a solid phase carrier and DNA monomer bases as raw materials, the DNA sequences shown in Table 1 were synthesized from the 3' end to the 5' end on a DNA synthesizer. After the synthesis, the above CPG was transferred to a 2 mL clean and sterilized Eppendorf tube, and 0.5 mL of methylamine:ammonia = 1:1 solution was added, and the DNA was cleaved from the CPG by ammonolysis at 65°C for 30 min. After ammonium hydrolysis, extract the supernatant, wash the CPG with a small amount of ultrapure water, and combine the supernatant. Add 2.5 times the volume of frozen absolute ethanol and 0.1 times the volume of 3mol / L NaCl to the system, and carry out alcohol precipitation in a -20°C refrigerator for 30 minutes. After the alcohol precipitation is complete, centrifuge at 14,000rpm for 10min and discard the supernatant. The obtained crude product w...

Embodiment 2 10

[0067] Example 2 Fabrication of a cross-shaped glass microfluidic chip

[0068] Use AutoCAD software to draw the two-dimensional structure of the chip and make a mask. Use soft lithography technology to transfer the pattern to the chrome plate glass, develop the hard film and remove the chrome layer, then place the chip in the etching solution (HF:HNO 3 :H 2 O=1:2:17) and etch for 100min, the chip structure can be obtained, and the remaining photoresist and chromium layer can be removed. Drill holes with a 1.8mm drill bit. Then put the chip into piranha washing solution (concentrated sulfuric acid / H 2 o 2 , v:v=3:1) to heat for 40min to ensure the cleanliness of the chip. Ultrapure water, acetone, ultrapure water, sulfuric acid, and ultrapure water were used to ultrasonically clean the chip in sequence to ensure the cleanliness of the chip. The etched glass chip and the flat glass of the same material are fixed together with iron clips, and placed in a muffle furnace wit...

Embodiment 3

[0070] The generation of embodiment 3 polyacrylamide microspheres

[0071] Prepare the oil phase and dispersed phase in advance, wherein the proportioning of each component in the oil phase is: 40% (w / w) Dow Corning 5225C, 30% (w / w) Dow Corning 749, 30% (w / w) silicone oil Ar20, The dispersed phase is 20% (w / v) acrylamide, 0.1% N,N'-methylene acrylamide, 0.14% (w / v) ammonium persulfate, and 16 μM acrydite-modified primer aqueous solution. The process of generating uniformly sized acrylamide droplets on the cross-flow focusing chip prepared in Example 2: the acrylamide solution as the dispersed phase and the mixed silicone oil as the continuous phase in the syringe were respectively transferred from the chip by using the Harvard ultra-high pressure precision syringe pump Horizontal channel and vertical channel injection on both sides. The flow rate of the oil phase is 1mL / h, and the flow rate of the water phase is 0.1mL / h. The generated acrylamide droplets are collected into th...

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Abstract

The invention discloses a DNA encoding microsphere and a synthetic method thereof. The encoding microsphere consists of five parts: microspheres, a universal primer, a cell encoder, a molecule encoder and a capture probe. The synthetic method comprises the following steps: (1) coupling the universal primer with the microspheres; (2) uniformly mixing the microspheres coupled with the universal primer and a PCR solution, wrapping each single microsphere into a water-in-oil droplet and then amplifying cell encoding DNA onto the microsphere; (3) sorting out microspheres with fluorescence, removing microspheres without fluorescence, processing the obtained microspheres with a denaturing reagent, and changing double-stranded DNA products on the microspheres into single-stranded DNA products; (4) mixing the microspheres amplified with the cell encoding DNA and a reaction solution mixed with a molecule encoding DNA library, and reacting molecule encoding DNA onto the microspheres; (5) processing the microspheres washed by PBS by using the denaturing reagent and changing the double-stranded DNA products into the single-stranded DNA products to finish the synthesis of the DNA encoding microsphere. The method has the advantages of being simple in design, low in lost, convenient to operate and the like.

Description

technical field [0001] The invention relates to a method for synthesizing DNA-encoded microspheres, which belongs to the technical field of single-cell analysis methods. Background technique [0002] Cells are the most basic unit of the composition of living organisms and life activities. Traditional methods of analyzing and processing the average signal of a large number of cells make the averaging of the signal obscure people's understanding of the heterogeneity among the brain, blood system, immune system, and the cells that make up these systems. With the development of high-throughput sequencing technology, single-cell sequencing technology has become the most important means of single-cell analysis, which greatly improves the efficiency and accuracy of single-cell analysis. The analysis of the single-cell gene level can reveal the mutations and structural variations in the genome of cells (especially cancer cells), and to understand the differences in the response of ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10
CPCC12N15/11C12Q1/686C12Q1/6862C12Q2563/159C12Q2563/149C12Q2521/501
Inventor 杨朝勇邹远许醒宋彦龄张明霞朱志
Owner 苏州德运康瑞生物科技有限公司
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