Methods for making nucleic acids
First Claim
Patent Images
1. 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.
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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.
34 Citations
28 Claims
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1. A method for making a nucleic acid comprising the step of:
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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.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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.
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10. 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.
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11. 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.
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12. 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.
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13. 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.
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14. 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.
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15. 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.
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16. A method according to claim 9, wherein the upstream flanking moiety is selected from the group consisting of fluorescein, rhodamine, digoxigenin, cholesterol and biotin.
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17. A method according to claim 9, wherein the upstream flanking moiety is biotin.
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18. A method according to claim 9, wherein the RNA polymerase promoter comprises SEQ ID NO:
- 1.
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19. 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.
- 1 and the upstream flanking moiety is nucleotides −
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20. A method according to claim 9, further comprising the step of repeatedly transcribing the double stranded DNA.
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21. A method for making a nucleic acid comprising the step of:
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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.- View Dependent Claims (22, 23, 24, 25, 26, 27, 28)
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Specification
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Current AssigneeRegents of the University of California (University of California)
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Original AssigneeRegents of the University of California (University of California)
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InventorsSerafini, Tito, Lin, David, Ngai, John, Luu, Percy
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Primary Examiner(s)LeGuyader, John L.
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Assistant Examiner(s)SCHMIDT, MARY M
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Application NumberUS09/566,570Time in Patent Office1,142 DaysField of Search435/91.2, 435/91.3, 435/91.1, 435/91.5, 435/91.51, 435/91.52, 536/23.1, 536/24.3, 536/24.1US Class Current435/91.2CPC Class CodesC12N 15/1096 cDNA Synthesis; Subtracted ...C12Q 1/6809 Methods for determination o...C12Q 1/6853 using modified primers or t...C12Q 2525/107 incorporating a peptide nuc...C12Q 2525/125 incorporating agents result...C12Q 2525/173 incorporating a polynucleot...C12Q 2525/191 incorporating an adaptorC12Q 2531/143 Promoter based amplificatio...
