US6114152A
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Methods for making nucleic acids
Assignee: University of California
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Filing: 1998-03-27
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Granted: 2000-09-05
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Expires: 2018-03-27
Abstract
Abstract Nucleic acids are made by adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. In one embodiment, the first RNA is an amplified mRNA, the known sequence at the 5' end comprises a poly(T) sequence, the adding step comprises using a polyadenyltransferase to add a poly(A) sequence to the 3' end, the reverse transcribing step is initiated at a duplex region comprising the poly(T) sequence hybridized to the poly(A) sequence, the cDNA is converted to double-stranded cDNA by a polymerase initiating from a noncovalently joined duplex region, and the double-stranded cDNA is transcribed to form one or more third RNAs.
Inventors
- 👤 Tito Serafini
- 👤 Percy Luu
- 👤 John Ngai
- 👤 David Lin
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 14:41
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6809; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1096; C12Q1/6844; C12Q1/6853
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
47 words
A method for making a nucleic acid comprising the steps of adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA.
Claim 2
Independent Claim
47 words
A method for making a nucleic acid comprising the steps of adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA.
Claim 3
Dependent Claim
55 words
A method according to claim 1, wherein the adding step comprises contacting the first RNA with at least one of (a) a nucleotide and polyadenyltransferase and (b) an oligonucleotide and a ligase, whereby said polyadenyltransferase and/or said ligase adds said known nucleotide to the 3' end of said first RNA to form said second RNA.
Claim 4
Dependent Claim
18 words
A method according to claim 1, wherein the known sequence at the 3' end comprises, a poly(A) sequence.
Claim 5
Dependent Claim
37 words
A method according to claim 1, wherein the known sequence at the 5' end comprises at least one of (a) a poly(T) or poly(A) sequence and (b) an internal sequence of an mRNA or the complement thereof.
Claim 6
Dependent Claim
24 words
A method according to claim 1, wherein the known sequence at the 5' end comprise a poly(T) sequence and an RNA polymerase promoter sequence.
Claim 7
Dependent Claim
40 words
A method according to claim 1, wherein the known sequence at the 3' end comprises a poly(A) sequence and the reverse transcribing step is initiated at a noncovalently joined duplex region comprising a poly(T) sequence hybridized to the poly(A) sequence.
Claim 8
Dependent Claim
61 words
A method according to claim 1, wherein the known sequence at the 3' end comprises a poly(A) sequence and the reverse transcribing step is initiated at a noncovalently joined duplex region comprising a poly(T) sequence hybridized to the poly(A) sequence, wherein the poly(T) sequence is covalently joined to at least one of a RNA polymerase promoter sequence and a primer sequence.
Claim 9
Dependent Claim
17 words
A method according to claim 1, wherein the cDNA is single-stranded and isolated from the second RNA.
Claim 10
Dependent Claim
40 words
A method according to claim 1, wherein the cDNA is single-stranded and isolated from the second RNA by a method comprising the step of contacting the RNA with at least one of an RNase H, a denaturant, and an alkali.
Claim 11
Dependent Claim
17 words
A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA.
Claim 12
Dependent Claim
28 words
A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA and the conversion is initiated at a noncovalently joined duplex region.
Claim 13
Dependent Claim
53 words
A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with as RNase H and contacting the single-stranded cDNA with a DNA polymerase whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region.
Claim 14
Dependent Claim
80 words
A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3' end of the single-stranded cDNA, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
Claim 15
Dependent Claim
85 words
A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3' end of the single-stranded cDNA and an RNA polymerase promoter, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
Claim 16
Dependent Claim
95 words
A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an oligonucleotide primer comprising a sequence complementary to the 3' end of the single-stranded cDNA and an RNA polymerase promoter comprising a natural class III T7 RNA polymerase promoter sequence, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
Claim 17
Dependent Claim
102 words
A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA by a method comprising the steps of contacting the RNA with a denaturant and contacting the single-stranded cDNA with a DNA polymerase and an at oligonucleotide primer comprising a sequence complementary to the 3' end of the single-stranded cDNA and an RNA polymerase promoter comprising SEQ ID NO:1 joined to an upstream flanking sequence of about 3 to 100 nucleotides, whereby the DNA polymerase initiates the conversion at a noncovalently joined duplex region of the 3' end of the single-stranded cDNA and the oligonucleotide primer.
Claim 18
Dependent Claim
22 words
A method according to claim 1, further comprising the step of repeatedly transcribing the cDNA to form a plurality of third RNAs.
Claim 19
Dependent Claim
37 words
A method according to claim 1, wherein the cDNA is single-stranded and converted to a double-stranded cDNA, and the method further comprises the step of repeatedly transcribing the double-stranded cDNA to form a plurality of third RNAs.
Claim 20
Dependent Claim
16 words
A method according to claim 1, wherein the first RNA is made by amplifying a mRNA.
Claim 21
Dependent Claim
64 words
A method according to claim 1, wherein the first RNA is made by amplifying a mRNA by the steps of hybridizing to the poly(A) tail of the mRNA a poly(T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.
Claim 22
Dependent Claim
20 words
A method according to claim 1, wherein the adding step comprises contacting the first RNA with a nucleotide and polyadenyltransferase.
References Cited (Backward Citations)
US4952496A
Cited
US5514545A
Cited
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2003-09-05 | 2004-03-06 | 2004-09-05 | $2000.00 | Window Closed / Lapsed |
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