Thermally responsive conjugates for DNA polymerase inhibition
Inventors
Guerreiro, Antonio • Canfarotta, Francesco • Kinkaid, Adrian • Groves, Alistair
Assignees
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Publication Number
US-12428677-B2
Publication Date
2025-09-30
Expiration Date
Abstract
A method of amplifying a target nucleic acid in a DNA sample comprising (a) contacting the DNA sample containing the target nucleic acid with a DNA polymerase, at least two oligonucleotide primers designed to flank the target nucleic acid, a mixture of dATP, dGTP, dCTP, and dTTP, and a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer, (b) heating the output of step (a) to a temperature at which the conjugate precipitates and thus the DNA polymerase is no longer inhibited and (c) amplifying the target nucleic acid, e.g. by performing PCR steps of denaturing the target nucleic acid, annealing the primers to the target nucleic acid, and extending the primers, wherein step (c) is repeated at least two times. Also provided is a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer and kits and aqueous compositions comprising the same.
Core Innovation
The invention relates to thermally responsive conjugates for controlling nucleic acid amplification. A DNA sample containing a target nucleic acid is contacted with a DNA polymerase, at least two oligonucleotide primers flanking the target nucleic acid, and a mixture of dATP, dGTP, dCTP, and dTTP, together with a conjugate in which a DNA polymerase inhibitor is covalently attached to a negative temperature sensitive polymer.
The conjugate is configured to precipitate on heating at a temperature at which the DNA polymerase is no longer inhibited. The precipitation-based removal of inhibition enables PCR steps including denaturing the target nucleic acid, annealing the primers to the target nucleic acid, and extending the primers, with repetition at least two times.
The invention also provides thermally responsive conjugates for reverse transcription of RNA. An RNA sample is contacted with a reverse transcriptase, at least one primer, and a mixture of dATP, dGTP, dCTP, and dTTP, together with a conjugate comprising a reverse transcriptase inhibitor covalently attached to a negative temperature sensitive polymer.
Claims Coverage
The partial content provides three independent claims. The main inventive features are the thermally triggered precipitation of an inhibitor-conjugate and the resulting relief of enzyme inhibition for PCR, a kit, or reverse transcription.
Thermally responsive DNA polymerase inhibitor conjugate for PCR amplification
Contacting a DNA sample containing a target nucleic acid with a DNA polymerase, at least two oligonucleotide primers designed to flank the target nucleic acid, a mixture of dATP, dGTP, dCTP, and dTTP, and a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer; heating the output to a temperature at which the conjugate precipitates and thus the DNA polymerase is no longer inhibited; and amplifying the target nucleic acid by performing PCR steps of denaturing, annealing, and extending wherein the steps are repeated at least two times.
PCR kit with thermally responsive DNA polymerase inhibitor conjugate
A kit comprising a DNA polymerase, and a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer.
Thermally responsive reverse transcriptase inhibitor conjugate for reverse transcription
Contacting an RNA sample with a reverse transcriptase, at least one primer, a mixture of dATP, dGTP, dCTP and dTTP, and a conjugate comprising a reverse transcriptase inhibitor covalently attached to a negative temperature sensitive polymer; heating the output to a temperature at which the conjugate precipitates and thus the reverse transcriptase is no longer inhibited; and incubating the output for a sufficient time to allow reverse transcription to occur.
Across the independent claims, the shared inventive concept is the use of a conjugate with a covalently attached enzyme inhibitor to a negative temperature sensitive polymer, where heating causes conjugate precipitation and relief of inhibition to enable PCR amplification or reverse transcription.
Stated Advantages
Enables PCR amplification by making the DNA polymerase no longer inhibited after heating to a temperature where the conjugate precipitates.
Reduces inhibition of reverse transcriptase after heating to a temperature where the conjugate precipitates, enabling reverse transcription to occur.
Documented Applications
Amplifying a target nucleic acid in a DNA sample by PCR using thermally responsive precipitation of a covalently attached DNA polymerase inhibitor conjugate.
Reverse transcribing RNA by contacting an RNA sample with a reverse transcriptase and a thermally responsive precipitation-based inhibitor conjugate, followed by incubating to allow reverse transcription.
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