초록 ▼

Provided are improved processes and apparatuses for solid phase oligonucleotide synthesis, the improvements comprising carrying out detritylation of the nascent oligonucleotide using a composition of an organic solvent, a protic solvent and a selected concentration, or selected concentration range,

Provided are improved processes and apparatuses for solid phase oligonucleotide synthesis, the improvements comprising carrying out detritylation of the nascent oligonucleotide using a composition of an organic solvent, a protic solvent and a selected concentration, or selected concentration range, of water. In some embodiments, an oligonucleotide synthesis apparatus includes means for adding water to a detritylation reagent. In some embodiments, an apparatus can include a water detector for analyzing the water concentration of a detritylation reagent to be reacted with a nascent oligonucleotide. An apparatus can comprise a feedback loop to control the concentration of water at the point of the detritylation reaction and/or to control the detritylation reaction time. The apparatuses and methods reduce batch-to-batch variations in the manufacture of oligonucleotides immobilized on the surface of various substrates.

대표청구항 ▼

1. An apparatus for synthesizing an oligonucleotide, the apparatus comprising: a manifold,a source of detritylating reagent upstream of said manifold,a conduit that operatively connects said manifold to said source of detritylating reagent,means for measuring the water concentration of detritylating

1. An apparatus for synthesizing an oligonucleotide, the apparatus comprising: a manifold,a source of detritylating reagent upstream of said manifold,a conduit that operatively connects said manifold to said source of detritylating reagent,means for measuring the water concentration of detritylating reagent in said conduit between said manifold and said source of detritylating reagent,a source of a compatible aqueous reagent that is: a) operatively connected to said conduit by a controllable valve, andb) upstream of said means for measuring water concentration, anda valve controller that is operatively connected to said controllable valve and said means for measuring water concentration,wherein said valve controller maintains said water concentration of said detritylating reagent above a selected minimal concentration. 2. The apparatus of claim 1, wherein said detritylating reagent comprises a composition suitable for detritylating a nascent oligonucleotide having one or more trityl group. 3. The apparatus of claim 2, wherein the water concentration is in the range from 100 ppm to 3000 ppm. 4. The apparatus of claim 3, wherein the water concentration is in the range from 100 ppm to 500 ppm. 5. The apparatus of claim 1, wherein said detritylating reagent comprises: an organic solvent,a protic acid in the range from 0.0125% mole fraction to 12.5% mole fraction, andwater at a concentration greater than 100 ppm. 6. The apparatus of claim 1, wherein said means for measuring water concentration comprises an inline detector. 7. The apparatus of claim 6, wherein said means for measuring water concentration comprises an infrared detector. 8. The apparatus of claim 7, wherein said detector comprises fiber optics. 9. The apparatus of claim 7, comprising a computer for controlling said manifold and for receiving data from said detector. 10. The apparatus of claim 7, wherein said means for measuring water concentration comprises a computer and wherein said computer is configured for calculating the water concentration in said detritylation reagent in response to electrical signals received from said detector. 11. The apparatus of claim 10, wherein said computer comprises a feedback loop algorithm for calculating a detritylation reaction time dependent upon the concentration of water in said detritylating reagent. 12. The apparatus of claim 11, wherein said algorithm comprises a PID (proportional, integral, derivative). 13. The apparatus of claim 6, wherein said apparatus further comprises a computer for controlling said manifold and for receiving data from said detector. 14. The apparatus of claim 13, wherein said computer is interfaced with said detector and with said valve controller, wherein said means for measuring water concentration comprises said computer and wherein said computer is configured for calculating the water concentration in said detritylation reagent in response to electrical signals received from said detector. 15. The apparatus of claim 14, wherein said computer comprises a feedback loop algorithm for regulating said valve dependent upon the concentration of water in said mixture in order to maintain a selected concentration of water in said mixture. 16. The apparatus of claim 15, wherein said algorithm comprises a PID (proportional, integral, derivative). 17. The apparatus of claim 15, wherein said water concentration is in the range of between 100 ppm and 500 ppm. 18. The apparatus of claim 1, wherein said source of a compatible aqueous reagent is capable of mixing with said detritylating reagent to form a mixture whereby the water concentration in the mixture is higher than said detritylating reagent. 19. The apparatus of claim 18, wherein said aqueous reagent comprises water. 20. The apparatus of claim 19, wherein said aqueous reagent comprises water mixed with an organic solvent. 21. The apparatus of claim 19, wherein said aqueous reagent comprises water mixed with a detritylating reagent. 22. The apparatus of claim 18, comprising means for mixing said compatible aqueous reagent with detritylating reagent in said conduit to form a mixture. 23. The apparatus of claim 22, comprising, upstream of said detector and in fluidic communication with said conduit, means for mixing said compatible aqueous reagent with said detritylating reagent in said conduit. 24. The apparatus of claim 23, wherein said means for mixing comprises a mixing tee. 25. The apparatus of claim 24, comprising a conduit connecting said source of aqueous reagent with said mixing tee. 26. The apparatus of claim 18, wherein said source of compatible aqueous reagent comprises a syringe.