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US 2024/0207805 A1Published application
METHODS FOR PERFORMING IN VITRO TRANSCRIPTION USING dUTP
OwnerHung Pin Kaoinventor
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Abstract
A method includes incubation of an amplification mixture containing uracil-DNA glycosy lase enzyme (UNG enzyme), a deoxyribonucleotide triphosphate (dNTP) mixture, a DNA polymerase, a template including a sequence of interest, and at least one primer pair, inactivation of UNG enzyme in the amplification mixture, and amplification of a sequence of interest to form an in vitro transcription template. The in vitro transcription template may be used to produce a therapeutic polynucleotide.
Claims21
1-89. (canceled)
A method comprising:
- incubating an amplification mixture comprising a uracil-DNA glycosylase enzyme (UNG enzyme), a deoxyribonucleotide triphosphate (dNTP) mixture, a DNA polymerase, a template comprising a sequence of interest, and at least one primer pair;
- inactivating said UNG enzyme in said amplification mixture;
- amplifying said sequence of interest from said template to form an in vitro transcription (IVT) template, said IVT template comprising a uracil-containing polynucleotide sequence corresponding to said sequence of interest;
- and
- performing in vitro transcription (IVT) from said IVT template to produce a therapeutic polynucleotide;
- said deoxyribonucleotide triphosphate (dNTP) mixture comprising a dUTP, a modified dUTP, or combinations thereof.
3 more elements
An apparatus comprising:
- a first chamber to:
- incubate a UNG enzyme with an amplification mixture comprising a deoxyribonucleotide triphosphate (dNTP) mixture, a DNA polymerase, a template, and at least one primer pair;
- inactivate said UNG enzyme in said amplification mixture;
- amplify a polynucleotide sequence of interest from said template to form a uracil-containing polynucleotide;
- and
- a second chamber to:
- perform in vitro transcription (IVT) from said uracil-containing polynucleotide to produce a therapeutic polynucleotide.
4 more elements
A system comprising:
- one apparatus comprising one or more chambers, said apparatus removably inserted into the system;
- and
- a controller to drive communication of fluid through the one or more chambers to thereby:
- incubate uracil-DNA glycosylase (UNG) enzyme with an amplification mixture comprising a deoxyribonucleotide triphosphate (dNTP) mixture, a DNA polymerase, a template material, and at least one primer pair;
- inactivate said UNG enzyme in said amplification mixture;
- amplify a polynucleotide sequence of interest from said template precursor material to form a uracil-containing polynucleotide;
- and
- perform in vitro transcription (IVT) from said uracil-containing polynucleotide to produce a therapeutic polynucleotide.
5 more elements
Dependencies are inferred from the claim text.