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US 12,173,351 B2Granted
DNA-tagged methanol responsive polymer for single-stranded nucleic acid production
AssigneesDana-Farber Cancer Institute, Inc., President and Fellows of Harvard College
InventorsElisha Krieg, William M. Shih, Dionis Minev, Richard Guerra
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Abstract
Provided herein, in some embodiments, are methods and compositions for the production of long single-stranded DNA.
Claims21
A method for producing single-stranded deoxyribonucleic acid (DNA), the method comprising:
- (a) amplifying, in solution, a DNA template in a polymerase chain reaction mixture that comprises the DNA template, a pair of primers, one of which is tagged with a methanol-responsive polymer (MeRPy) comprising acrylic monomers (a MeRPy-tagged primer) and comprises a cleavage site, a polymerase, and deoxynucleoside triphosphates to produce a double-stranded MeRPy-tagged amplicon;
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- (b) dehybridizing, in solution, the double-stranded MeRPy-tagged amplicon into an untagged DNA strand and a MeRPy-tagged DNA strand comprising the cleavage site, and optionally isolating the untagged DNA strand.
A method for producing single-stranded deoxyribonucleic acid (DNA), the method comprising:
- (a) copolymerizing, in solution, a 5′-acrylamide-tagged single-stranded DNA (ssDNA) forward primer with acrylamide monomers to produce a methanol-responsive polymer (MeRPy)-tagged forward primer, wherein the MeRPy comprises acrylamide monomers, wherein the ssDNA forward primer comprises a cleavage site;
- (b) optionally purifying the MeRPy-tagged forward primer;
- (c) amplifying, in solution, a DNA template in a polymerase chain reaction mixture that comprises the DNA template, a pair of primers comprising the MeRPy-tagged forward primer and a reverse primer, a polymerase, and deoxynucleoside triphosphates to produce a double-stranded MeRPy-tagged amplicon;
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- (d) dehybridizing, in solution, the double-stranded MeRPy-tagged amplicon into an untagged DNA strand and a MeRPy-tagged DNA strand comprising the cleavage site, and optionally isolating the untagged DNA strand.
2 more elements
Dependencies are inferred from the claim text.