Three-color high-throughput sequencing reagent and sequencing method
A high-throughput, sequencing technology, applied in the field of high-throughput sequencing, can solve the problems of reducing signal-to-noise ratio, affecting sequencing read length, sequencing efficiency and sequencing throughput, etc.
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Embodiment 1
[0045] Example 1 Synthesis of 3'-O-Azuromethyl DATP
[0046] like figure 2 As shown, 410.4 mg (FW 410.4, 1 Hao Moore) is labeled by the base and proton sponge (254 mg, 1.17 Hao Moore) Dry overnight after drying overnight in a vacuum dryer with phosphorus phosphorus. Ester. After adding 134 microlituted fresh distilled trichlorophos (1.17 Hao Mole) at 0 ° C, stirred at 0 ° C for 2 hours. Subsequently 1.817 grams of triaxadium tract (FW 363.33, 5 Hao Moore) and 1.84 ml of tributylamine (185.35, D = 0.778, 7.7 Hao Mocetra) were mixed in 8 ml of no water DMF (dimethylformamide) After mixing, stir after the next time, stirred for 30 minutes. After adding 50 ml of 0.1 M Teab (carbonate) (pH 8.0) at room temperature for 1 hour. After adding 50 ml of concentrated concentrated ammonia at room temperature, stirring overnight, extols off the water and ammonia after dissolving in 15 ml of water. The 3'-O-azide methyl DATP was purified by HPLC (high performance liquid chromatography) using 0.1...
Embodiment 2
[0047] Example 2 Synthesis 3'-O-azide methyl DCTP
[0048] like image 3 As shown, the reactant of 386.4 mg (FW 386.4, 1 Hao Moore) and the proton sponge (254 mg, 1.17 Hao Moore) in P 2 O 5 Drying overnight after drying overnight in a vacuum dryer was dissolved in trimethyl phosphate. Add 134 microlituted fresh distilled POCl at 0 ° C 3 (1.17 Hao Moore) was stirred at 0 ° C for 2 hours. Subsequently 1.817 grams of triaxamate (FW 363.33, 5 Hao Moore) and 1.84 ml of tributylamine (185.35, D = 0.778, 7.7 Hao Mole) were mixed in 8 ml of no water DMF after mixing at room temperature. After stirring for 30 minutes. After stirring 50 ml of TEAb (pH 8.0) at room temperature for 1 hour. After adding 50 ml of concentrated concentrated ammonia at room temperature, stirring overnight, extols off the water and ammonia after dissolving in 15 ml of water. The 3'-O-azide methyl DCTP was purified with EtOAc (EtOAc) EtOAc.
Embodiment 3
[0049] Example 3 Synthesis of 3'-O-Azuromethyl DGTP
[0050] like Figure 4 As shown, 587.6 mg (FW 587.6, 1 Hao Moore) reactants and proton sponges (254 mg, 1.17 Hao Moore) in P 2 O 5 Drying overnight after drying overnight in a vacuum dryer was dissolved in trimethyl phosphate. Add 134 microlituted fresh distilled POCl at 0 ° C 3 (1.17 Hao Moore) was stirred at 0 ° C for 2 hours. Subsequently 1.817 grams of triazine-jam (FW363.33, 5 Hao Moore) and 1.84 ml of tributylamine (185.35, D = 0.778, 7.7 Hao Mole) were mixed in 8 ml of no water DMF after mixing at room temperature. After stirring, stir it for 30 minutes. After stirring 50 ml of TEAb (pH 8.0) at room temperature for 1 hour. After adding 50 ml of concentrated concentrated ammonia at room temperature, stirring overnight, extols off the water and ammonia after dissolving in 15 ml of water. The 3'-O-azide methyl DGTP was purified with EtOAc (EtOAc) / TEAB.
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