Methods for making nucleic acids
AssigneeThe Regents of the University of California
InventorsTito Serafini, Percy Luu, John Ngai, David Lin
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Abstract
Nucleic acids are made by converting a primed single-stranded DNA to a double-stranded DNA by a method comprising the step contacting the single-stranded DNA with a DNA polymerase having 5′ exonuclease activity under conditions whereby the DNA polymerase converts the-single stranded DNA to the double-stranded DNA, wherein the single-stranded DNA is primed with oligonucleotide primer comprising a sequence complementary to the 3′ end of the single-stranded DNA, and at least one of the 5′ end of the primer and the single-stranded DNA comprises an RNA polymerase promoter joined to an upstream (5′) flanking moiety which protects the promoter from the 5′ exonuclease activity of the DNA polymerase.
Claims28
A method for making a nucleic acid comprising the step of:
- converting a primed single-stranded DNA to a double-stranded DNA by a method comprising the step of contacting the single-stranded DNA with a DNA polymerase having 5′ exonuclease activity under conditions whereby the DNA polymerase converts the single-stranded DNA to the double-stranded DNA,
- wherein the 5′ end of the single-stranded DNA comprises an RNA polymerase promoter joined to an upstream (5′) flaking moiety which protects the promoter from the 5′ exonuclease activity of the DNA polymerase, wherein the upstream flanking moiety is selected from the group consisting of a dye, nucleotides −50 to −16 of the T7 promoter, modified nucleotide, a sterol, and an affinity tag.
A method for making a nucleic acid comprising the step of:
- converting a primed single-stranded DNA to a double-stranded DNA by a method comprising the step of contacting the single-stranded DNA with a DNA polymerase having 5′ exonuclease activity under conditions whereby the DNA polymerase converts the single-stranded DNA to the double-stranded DNA,
- wherein the single-stranded DNA is primed with an oligonucleotide primer comprising a sequence complementary to the 3′ end of the single-stranded DNA and the 5′ end of the primer comprises an RNA polymerase promoter joined to an upstream (5′) flanking moiety which protects the promoter from the 5′ exonuclease activity of the DNA polymerase, wherein the upstream flanking moiety is selected from the group consisting of a dye, nucleotides −50 to −16 of the T7 promoter a modified nucleotide, a sterol, and an affinity tag.
Dependencies are inferred from the claim text.