Method for linear mRNA amplification
First Claim
1. A method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:
- (a) converting mRNA to double-stranded cDNA, wherein one terminus of said double-stranded cDNA comprises an RNA polymerase promoter region; and
(b) transcribing said double-stranded cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of a reverse transcriptase that is incapable of RNA-dependent DNA polymerase activity during said transcribing step.
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Abstract
Methods for linearly amplifying mRNA to produce antisense RNA are provided. In the subject methods, mRNA is converted to double-stranded cDNA using a promoter-primer having a poly-dT primer site linked to a promoter sequence so that the resulting double-stranded cDNA is recognized by an RNA polymerase. The resultant double-stranded cDNA is then transcribed into antisense RNA in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods find use a variety of different applications in which the preparation of linearly amplified amounts of antisense RNA is desired. Also provided are kits for practicing the subject methods.
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Citations
31 Claims
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1. A method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:
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(a) converting mRNA to double-stranded cDNA, wherein one terminus of said double-stranded cDNA comprises an RNA polymerase promoter region; and (b) transcribing said double-stranded cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of a reverse transcriptase that is incapable of RNA-dependent DNA polymerase activity during said transcribing step. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:
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(a) converting mRNA to cDNA with a promoter-primer comprising an mRNA binding site linked to a promoter sequence, wherein one terminus of said cDNA comprises an RNA polymerase promoter region; and (b) transcribing said cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of a reverse transcriptase that has been rendered ineffective for RNA-dependent DNA polymerase activity prior to said transcribing step. - View Dependent Claims (11, 12, 13, 14, 15)
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16. A method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:
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(a) converting mRNA to double-stranded cDNA, wherein one terminus of said double-stranded cDNA comprises an RNA polymerase promoter region by; (i) contacting mRNA with a promoter-primer under conditions wherein said mRNA forms a complex with said promoter-primer, wherein said promoter-primer comprises an mRNA binding site linked to a promoter sequence; and (ii) converting said complex to double-stranded cDNA using a combination of RNA-dependent DNA polymerase activity, RNaseH activity and DNA-dependent DNA polymerase activity; and (b) transcribing said double-stranded cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of a reverse transcriptase that is incapable of RNA-dependent DNA polymerase activity during said transcribing step;
whereby said mRNA is linearly amplified into antisense RNA. - View Dependent Claims (17, 18, 19)
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20. A method for producing linearly amplified amounts of antisense RNA from mRNA, said method comprising:
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(a) contacting mRNA with a promoter-primer in the presence of a first polymerase having RNA-dependent DNA polymerase activity and lacking RNaseH activity under conditions sufficient for first strand cDNA synthesis to occur to produce a hybrid of said mRNA and a first strand cDNA, wherein said promoter-primer comprises an mRNA binding site linked to a RNA polymerase promoter sequence; (b) contacting said hybrid with an enzyme catalyzing RNaseH activity under conditions sufficient to convert said complex to a double-stranded cDNA molecule; and (c) transcribing said double-stranded cDNA with an RNA polymerase and ribonucleotides into antisense RNA in the presence of ddNTPs;
whereby said mRNA is linearly amplified into antisense RNA. - View Dependent Claims (21, 22, 23, 24, 25)
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26. A kit for use in linearly amplifying mRNA into antisense RNA, said kit comprising:
- an oligonucleotide promoter-primer comprising an RNA polymerase promoter sequence; and
ddNTPs. - View Dependent Claims (27, 28, 29, 30, 31)
- an oligonucleotide promoter-primer comprising an RNA polymerase promoter sequence; and
Specification