Rolling circle amplification of RNA

Abstract

Disclosed are compositions and methods for amplification of RNA molecules. The disclosed method involves synthesizing first strand cDNA molecules from RNA molecules, circularizing the first strand cDNA molecules and replicating the circularized first strand cDNA molecules using rolling circle replication. The method can be aided by the use of specialized primers for cDNA synthesis and specialized probes for circularizing the first strand cDNA molecules. The method can be used to replicate and amplify multiple RNA molecules, such as all RNA molecules in a sample or all mRNA molecules in a sample, or be used to replicate and amplify specific RNA molecules. Rolling circle replication of the circularized first strand cDNA molecules results in long DNA strands containing tandem repeats of the cDNA sequence. The tandem sequence DNA can be used directly (for detection of sequences, for example), further amplified, or used any other purpose. Double-stranded tandem sequence DNA can be used to produce unit lengths of the cDNA sequence. Tandem sequence DNA can also be transcribed to produce transcripts having sequence complementary to or matching the sequence of RNA molecules.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of copending application Ser. No. 10 / 335,573, filed Dec. 31, 2002. Application Ser. No. 10 / 335,573, filed Dec. 31, 2002, is hereby incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention is in the field of nucleic acid amplification, and specifically in the area of rolling circle amplification of RNA molecules. BACKGROUND OF THE INVENTION

[0003] Numerous nucleic acid amplification techniques have been devised, including strand displacement cascade amplification (SDCA) (referred to herein as exponential rolling circle amplification (ERCA)) and rolling circle amplification (RCA) (U.S. Pat. No. 5,854,033; PCT Application No. WO 97 / 19193; Lizardi et al., Nature Genetics 19(3):225-232 (1998)); multiple displacement amplification (MDA) (PCT Application WO 99 / 18241); strand displacement amplification (SDA) (Walker et al., Nucleic Acids Research 20:1691-1696 (1992),...

Examples

example 1

A. Example 1

Circularization-dependent Amplification of Human RNA

[0363] This example demonstrates a basic form of the disclosed method by amplifying polyadenylated RNA from a human adenorectal carcinoma. Control reactions indicate that amplification was dependent on circularization of first strand cDNA molecules.

[0364] First strand cDNA was synthesized from 1 μg poly A+ mRNA (human adenorectal carcinoma, SV-480) using a 5′-P-Not-1-(dT)20 cDNA primer (SEQ ID NO:1) and Superscript II reverse transcriptase. RNAse H+ reverse transcriptase was added to digest RNA templates. 0.1 M NaOH was then added and the mixture was incubated for 10 minutes. The reaction was then neutralized by adding 0.1 M HCl. The first strand cDNA molecules were purified using a Qiagen column to remove dNTPs and the primer. The first strand cDNA molecules were then circularized using T4 DNA ligase and a generic circularization probe (5′-AAAAGCGGCCGCACNNNNNN-3′; SEQ ID NO:2). The NNN segment of this circularization...

example 2

B. Example 2

Analysis of Amplification Products

[0366] Following the design concept (FIG. 1), first strand cDNA molecules were synthesized from 1 μg poly A+ mRNA from Adenocarcinoma cells (Clontech) using a 5′-phosphorylated cDNA primer as shown in FIG. 1 (oligo˜dT at the 3′-end and Not I endonuclease sequence on its 5′-phosphorylated end) using Superscript II reverse transcriptase (Invitrogen) at 42° C. for 1 hr followed by AMV reverse transcriptase (Invitrogen) at 37° C. for 1 hr. The first strand cDNA was isolated from the reaction mixture using Qiagen PCR product purification kit. First strand cDNA representing 100 ng mRNA ( 1 / 10th reaction product) was annealed to an excess of bridge oligonucleotide (circularization probe) (1 μg) in 20 μl×T4 DNA ligase buffer (heating to 80° C. followed by slow cooling to 30° C.) followed by the addition of 40U T4 DNA ligase for 12 hrs at 30° C. A fraction of the ligated cDNA reaction mixture, representing 5 ng mRNA, was then used for 100 μl amp...

example 3

C. Example 3

Analysis of Amplification Bias of Amplification Products

[0369] In order to determine the bias generated by amplification of a specific transcript from two different RNA samples, first strand cDNA synthesis was carried out from poly-A+ mRNA derived from human adult brain and fetal brain (Clontech). The first strand cDNA was ligated at 4 ng / μl concentration in the presence of a circularization probe. 10 ng of ligated first strand cDNA molecules were used for transcript amplification in a 30 μl reaction volume using Φ29 DNA polymerse and exonuclease-resistant p(dN)6 primers. To determine the relative abundance of DNA damage-inducible transcript (Dle3) targets per ng of the sample, Taqman assays were carried out using amplified and unamplified cDNA molecules representing adult and fetal brain mRNA. Transcript abundance in these samples was calculated by utilizing Taqman assay standard curve using varying amounts of genomic DNA (333 copies per ng genomic DNA). This procedure...

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of copending application Ser. No. 10 / 335,573, filed Dec. 31, 2002. Application Ser. No. 10 / 335,573, filed Dec. 31, 2002, is hereby incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention is in the field of nucleic acid amplification, and specifically in the area of rolling circle amplification of RNA molecules. BACKGROUND OF THE INVENTION

[0003] Numerous nucleic acid amplification techniques have been devised, including strand displacement cascade amplification (SDCA) (referred to herein as exponential rolling circle amplification (ERCA)) and rolling circle amplification (RCA) (U.S. Pat. No. 5,854,033; PCT Application No. WO 97 / 19193; Lizardi et al., Nature Genetics 19(3):225-232 (1998)); multiple displacement amplification (MDA) (PCT Application WO 99 / 18241); strand displacement amplification (SDA) (Walker et al., Nucleic Acids Research 20:1691-1696 (1992),...

Examples

example 1

A. Example 1

Circularization-dependent Amplification of Human RNA

[0363] This example demonstrates a basic form of the disclosed method by amplifying polyadenylated RNA from a human adenorectal carcinoma. Control reactions indicate that amplification was dependent on circularization of first strand cDNA molecules.

[0364] First strand cDNA was synthesized from 1 μg poly A+ mRNA (human adenorectal carcinoma, SV-480) using a 5′-P-Not-1-(dT)20 cDNA primer (SEQ ID NO:1) and Superscript II reverse transcriptase. RNAse H+ reverse transcriptase was added to digest RNA templates. 0.1 M NaOH was then added and the mixture was incubated for 10 minutes. The reaction was then neutralized by adding 0.1 M HCl. The first strand cDNA molecules were purified using a Qiagen column to remove dNTPs and the primer. The first strand cDNA molecules were then circularized using T4 DNA ligase and a generic circularization probe (5′-AAAAGCGGCCGCACNNNNNN-3′; SEQ ID NO:2). The NNN segment of this circularization...

example 2

B. Example 2

Analysis of Amplification Products

[0366] Following the design concept (FIG. 1), first strand cDNA molecules were synthesized from 1 μg poly A+ mRNA from Adenocarcinoma cells (Clontech) using a 5′-phosphorylated cDNA primer as shown in FIG. 1 (oligo˜dT at the 3′-end and Not I endonuclease sequence on its 5′-phosphorylated end) using Superscript II reverse transcriptase (Invitrogen) at 42° C. for 1 hr followed by AMV reverse transcriptase (Invitrogen) at 37° C. for 1 hr. The first strand cDNA was isolated from the reaction mixture using Qiagen PCR product purification kit. First strand cDNA representing 100 ng mRNA ( 1 / 10th reaction product) was annealed to an excess of bridge oligonucleotide (circularization probe) (1 μg) in 20 μl×T4 DNA ligase buffer (heating to 80° C. followed by slow cooling to 30° C.) followed by the addition of 40U T4 DNA ligase for 12 hrs at 30° C. A fraction of the ligated cDNA reaction mixture, representing 5 ng mRNA, was then used for 100 μl amp...

example 3

C. Example 3

Analysis of Amplification Bias of Amplification Products

[0369] In order to determine the bias generated by amplification of a specific transcript from two different RNA samples, first strand cDNA synthesis was carried out from poly-A+ mRNA derived from human adult brain and fetal brain (Clontech). The first strand cDNA was ligated at 4 ng / μl concentration in the presence of a circularization probe. 10 ng of ligated first strand cDNA molecules were used for transcript amplification in a 30 μl reaction volume using Φ29 DNA polymerse and exonuclease-resistant p(dN)6 primers. To determine the relative abundance of DNA damage-inducible transcript (Dle3) targets per ng of the sample, Taqman assays were carried out using amplified and unamplified cDNA molecules representing adult and fetal brain mRNA. Transcript abundance in these samples was calculated by utilizing Taqman assay standard curve using varying amounts of genomic DNA (333 copies per ng genomic DNA). This procedure...

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