Nucleotides modified by aggregation-induced fluorescent molecules and their applications in DNA sequencing and SNPs detection
An aggregation-induced fluorescence and nucleotide technology, applied in the field of gene sequencing
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Embodiment 1
[0064] Example 1 Synthesis of aggregation-induced fluorescent molecule HCAP
[0065] 1. Synthesis of 4-Dimethylamino-2'-hydroxychalcone (4-Dimethylamino-2'-hydroxychalcone, HCA)
[0066] Weigh 4-dimethylaminobenzaldehyde (12mmol, 1.79g) and 2-hydroxyacetophenone (13.2mmol, 1.80g) in a 100mL round bottom flask, add 30mL of ethanol and stir to dissolve. Weigh KOH (2.8 g, 50 mmol) and dissolve it in 2 mL of water, then add it into a round bottom flask, and stir at room temperature for 24 hours. After the reaction, adjust the pH of the reaction system to 7.0 with dilute hydrochloric acid, filter with suction and wash the precipitate with ethanol three times, and dry the filter cake. The dried filter cake was dissolved in dichloromethane, suction filtered and washed 3 times with dichloromethane, the filtrate was collected, and the product obtained by rotary evaporation under reduced pressure was analyzed by proton nuclear magnetic resonance ( 1 H NMR), proton NMR ( 13 C NMR), hi...
Embodiment 2
[0075] Example 2: Synthesis of aggregation-induced fluorescent molecularly modified nucleotides (dNTPs-HCAP)
[0076] The nucleotides (dNTPs-HCAP) modified by aggregation-induced fluorescent molecules in this example include the modified nucleotides dATP-HCAP (A-H), dTTP-HCAP (T-H), dCTP-HCAP (C-H) and dGTP- HCAP(G-H), the synthesis steps are the same, take dATP-HCAP(A-H) as an example to describe in detail:
[0077] dATP (12.5 μmol, 125 μL) was acidified with a BioRad AG-50w-XB ion-exchange column, and then tri-n-butylamine (12.5 μmol, 3.0 μL) was added to the solution and stirred at room temperature for 5 minutes. The water was removed by rotary evaporation under reduced pressure, then spin-dried twice with 2 mL of ultra-dry N,N-dimethylformamide (DMF), and then dissolved in 0.5 mL of DMF. Under nitrogen protection, 1,1'-carboxydiimidazole (63 μmol, 10.1 mg) dissolved in 0.5 mL DMF was added to the DMF solution, and stirred at room temperature for 12 hours. After the react...
Embodiment 3
[0086] Example 3 dNTPs-HCAP is used for rolling circle amplification reaction to detect SNPs
[0087] The principle of dNTPs-HCAP for the detection of SNPs by rolling circle amplification reaction is as follows Figure 16 As shown in A, when the wild-type target molecule is perfectly complementary to the lock probe Lock, Lock can be circularized into circular DNA by T4 ligase. Then primer primer2 starts the rolling circle amplification reaction. In the amplification reaction, the HCAP molecule on the dNTPs-HCAP is shed under the action of DNA polymerase, and the phosphate radical is hydrolyzed under the action of shrimp alkaline phosphatase to form HCA, which emits red fluorescence at 640nm. The mutant target molecule cannot be completely complementary to Lock, so it cannot be circularized under the action of T4 ligase, so the subsequent rolling circle amplification reaction cannot be performed, so there is no 640nm red fluorescence. Therefore, according to the change of the...
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