A kind of solid phase carrier for in situ synthesis of nucleic acid and its preparation method
An in-situ synthesis, solid-phase carrier technology, applied in the field of gene chips, can solve the problems of reduced oligonucleotide synthesis capacity, CPG preparation technology defects, and few types of oligonucleotide sequences, and achieves reduction of steric hindrance effect, The effect of reducing the effect of electronic interference and optimizing the load capacity
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[0039] Therefore, according to the first aspect of the present invention, there is provided a method for preparing a solid phase carrier for nucleic acid in situ synthesis, wherein the method comprises:
[0040] (1) performing a first amination treatment on the solid phase substrate with a first amination modification reagent to obtain a first aminated solid phase substrate;
[0041] (2) modifying the first aminated solid phase substrate with a linker modification reagent to obtain a grafted solid phase substrate;
[0042] (3) performing a second amination treatment on the grafted solid phase substrate with a second amination modification reagent to obtain a second aminated solid phase substrate; and
[0043] (4) Linking a cleavable linker with a protecting group to the second aminated solid phase substrate to obtain a solid phase carrier.
[0044] In a specific embodiment, the method for preparing a solid phase carrier for nucleic acid in situ synthesis comprises:
[0045] (1...
Embodiment 1
[0079] In this embodiment, a nucleic acid chip is prepared by using a quartz glass slide as a solid-phase substrate, wherein the specification of the quartz glass slide is 25 mm·75 mm for a standard slide glass.
[0080] The first amination treatment of quartz glass slides: take two quartz glass slides and place them in the processed reaction vessel, add 20ml (concentrated sulfuric acid: hydrogen peroxide = 7:3) reaction solution to it, and react under ultrasonic conditions at 70°C for 3h , washed with deionized water, blown dry with nitrogen, and then added 20ml of ethanol solution of 5% 3-aminopropyltriethoxysilane to it, and reacted for 24h under ultrasonic conditions at 50°C. After the reaction was completed, use Washed with deionized water and placed in an oven at 110°C for overnight curing to obtain the first aminated glass slide.
[0081] Long-chain grafting of quartz glass slides: Place active aminated quartz glass slides in a reaction vessel, add 600mg 11-bromoundecan...
Embodiment 2
[0085] In this example, a nucleic acid chip was prepared using porous glass beads as a solid phase substrate.
[0086] The first amination treatment of porous glass beads: take 2g of porous glass beads and place them in a 50ml centrifuge tube, wherein the diameter of the porous glass beads is about 90 μm-110 μm, and the pore size is Add 40ml of reaction solution (concentrated sulfuric acid:hydrogen peroxide=7:3) to it, react under ultrasonic conditions at 70°C for 3h, centrifugally wash with deionized water, blow dry with nitrogen, and then add 3-aminopropane with a concentration of 5% 40ml of ethanol solution of triethoxysilane was reacted at 50°C for 24h under ultrasonic conditions. After the reaction was completed, it was centrifuged and washed with deionized water and placed in an oven at 110°C for overnight curing to obtain the first aminated porous glass beads .
[0087] Long-chain branching of porous glass beads: Weigh 100 mg of aminated porous glass beads and place t...
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