US6582936B1
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Methods for making nucleic acids
Assignee: University of California, San Diego
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Filing: 2000-05-08
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Granted: 2003-06-24
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Expires: 2020-05-08
Abstract
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.
Inventors
- 👤 Tito Serafini
- 👤 Percy Luu
- 👤 John Ngai
- 👤 David Lin
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 04:20
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6853; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1096; C12Q1/6809
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
116 words
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.
Claim 2
Independent Claim
116 words
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.
Claim 3
Dependent Claim
29 words
A method according to claim 1 , wherein the RNA polymerase promoter is selected from the group consisting of a T7, a T3 and an SP-6 RNA polymerase promoter.
Claim 4
Dependent Claim
16 words
A method according to claim 1 , wherein the RNA polymerase promoter comprises SEQ ID NO:1.
Claim 5
Dependent Claim
30 words
A method according to claim 1 , wherein the upstream flanking moiety is selected from the group consisting of a dye, a modified nucleotide, a sterol, and an affinity tag.
Claim 6
Dependent Claim
25 words
A method according to claim 1 , wherein the upstream flanking moiety is selected from the group consisting of fluorescein, rhodamine, digoxigenin, cholesterol and biotin.
Claim 7
Dependent Claim
14 words
A method according to claim 1 , wherein the upstream flanking moiety is biotin.
Claim 8
Dependent Claim
30 words
A method according to claim 1 , wherein the RNA polymerase promoter comprises SEQ ID NO:1 and the upstream flanking moiety is nucleotides −50 to −16 of the T7 promoter.
Claim 9
Dependent Claim
18 words
A method according to claim 1 , further comprising the step of repeatedly transcribing the double stranded DNA.
Claim 10
Dependent Claim
64 words
A method according to claim 1 , wherein the primed single-stranded DNA is made by the steps of: reverse transcribing an RNA to form a heterohybrid of the RNA and the single-stranded DNA; and nicking the RNA with an RNA endonuclease under conditions whereby the single-stranded DNA retains a 5′ most fragment of the RNA hybridized at the 3′ end of the single-stranded DNA.
Claim 11
Dependent Claim
37 words
A method according to claim 9 , wherein the RNA is mRNA and the reverse transcribing step comprises hybridizing to a 3′ poly(A) tail of the mRNA a poly(T) oligonucleotide joined to the RNA polymerase promoter sequence.
Claim 12
Dependent Claim
49 words
A method according to claim 9 , wherein the RNA is aRNA made by amplifying an mRNA and the reverse transcribing step comprises adding a known sequence onto the 3′ end of the aRNA, hybridizing to the known sequence a complementary oligonucleotide joined to the RNA polymerase promoter sequence.
Claim 13
Dependent Claim
52 words
A method according to claim 9 , wherein the RNA is aRNA made by amplifying an mRNA and the reverse transcribing step comprises using polyadenyltransferase to add a known sequence onto the 3′ end of the aRNA, hybridizing to the known sequence a complementary oligonucleotide joined to the RNA polymerase promoter sequence.
Claim 14
Dependent Claim
54 words
A method according to claim 9 , wherein the RNA is aRNA made by amplifying an mRNA and the reverse transcribing step comprises using polyadenyltransferase to add a poly(A) tail onto the 3′ end of the aRNA, hybridizing to the poly(A) tail sequence a complementary poly(T) oligonucleotide joined to the RNA polymerase promoter sequence.
Claim 15
Dependent Claim
29 words
A method according to claim 9 , wherein the RNA polymerase promoter is selected from the group consisting of a T7, a T3 and an SP-6 RNA polymerase promoter.
Claim 16
Dependent Claim
28 words
A method according to claim 9 , wherein the upstream flanking moiety is selected from the group consisting of a dye, modified nucleotide, a sterol, and affinity tag.
Claim 17
Dependent Claim
25 words
A method according to claim 9 , wherein the upstream flanking moiety is selected from the group consisting of fluorescein, rhodamine, digoxigenin, cholesterol and biotin.
Claim 18
Dependent Claim
14 words
A method according to claim 9 , wherein the upstream flanking moiety is biotin.
Claim 19
Dependent Claim
16 words
A method according to claim 9 , wherein the RNA polymerase promoter comprises SEQ ID NO:1.
Claim 20
Dependent Claim
30 words
A method according to claim 9 , wherein the RNA polymerase promoter comprises SEQ ID NO:1 and the upstream flanking moiety is nucleotides −50 to −16 of the T7 promoter.
Claim 21
Dependent Claim
18 words
A method according to claim 9 , further comprising the step of repeatedly transcribing the double stranded DNA.
Claim 22
Independent Claim
138 words
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.
Claim 23
Independent Claim
138 words
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.
Claim 24
Dependent Claim
29 words
A method according to claim 21 , wherein the RNA polymerase promoter is selected from the group consisting of a T7, a T3 and an SP-6 RNA polymerase promoter.
Claim 25
Dependent Claim
30 words
A method according to claim 21 , wherein the upstream flanking moiety is selected from the group consisting of a dye, a modified nucleotide, a sterol, and an affinity tag.
Claim 26
Dependent Claim
25 words
A method according to claim 21 , wherein the upstream flanking moiety is selected from the group consisting of fluorescein, rhodamine, digoxigenin, cholesterol and biotin.
Claim 27
Dependent Claim
14 words
A method according to claim 21 , wherein the upstream flanking moiety is biotin.
Claim 28
Dependent Claim
16 words
A method according to claim 21 , wherein the RNA polymerase promoter comprises SEQ ID NO:1.
Claim 29
Dependent Claim
30 words
A method according to claim 21 , wherein the RNA polymerase promoter comprises SEQ ID NO:1 and the upstream flanking moiety is nucleotides −50 to −16 of the T7 promoter.
Claim 30
Dependent Claim
18 words
A method according to claim 21 , further comprising the step of repeatedly transcribing the double stranded DNA.
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