Method for Generating Transcripts

• US 20070260053A1
• Filed: 09/23/2005
• Published: 11/08/2007
• Est. Priority Date: 10/01/2004
• Status: Abandoned Application

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Mallet, Francois, Pichon, Jean-Philippe, Oriol, Guy
• Application Number:
  US11/660,952
• Publication Number:
  US 20070260053A1
• Time in Patent Office: Days
• US Class Current: 536/25.300
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/301 Endonuclease

Abstract

The present invention relates to a method for generating transcripts from: at least one RNA sequence to be amplified comprising a “primer” region and a region of interest, and an amplification primer comprising a promoter region, and a region capable of hybridizing to said “primer” region of the RNA sequence to be amplified, said method being carried out at constant temperature, and comprising the following steps: a) said primer is hybridized with the RNA to be amplified, b) the primer is extended by means of a reverse transcriptase enzymatic activity in order to generate a complementary deoxyribonucleic acid (cDNA) sequence of the RNA to be amplified, c) the RNA to be amplified, hybridized to said cDNA, is cleaved by means of an enzyme that has a ribonuclease H activity, so as to obtain fragments of RNA to be amplified, hybridized to said cDNA, d) the ends of said fragments of RNA to be amplified are extended by means of a reverse transcriptase and strand displacement enzyme, so as to obtain RNA-DNA/DNA hybrids, e) RNA transcripts are obtained from the RNA-DNA/DNA hybrids formed in step d), by means of an enzyme that has an RNA polymerase activity.

Claims

1. A method for generating transcripts from:

at least one RNA sequence to be amplified comprising a “

primer”

region and a region of interest, and

an amplification primer comprising a promoter region, and a region capable of hybridizing to said “

primer”

region of the RNA sequence to be amplified,

said method being carried out at constant temperature, and comprising the following steps;

a) said primer is hybridized with the RNA to be amplified,

b) the primer is extended by means of a reverse transcriptase enzymatic activity in order to generate a complementary deoxyribonucleic acid (cDNA) sequence of the RNA to be amplified,

c) the RNA to be amplified, hybridized to said cDNA, is cleaved by means of an enzyme that has a ribonuclease H activity, so as to obtain fragments of RNA to be amplified, hybridized to said cDNA,

d) the ends of said fragments of RNA to be amplified are extended by means of a reverse transcriptase and strand displacement enzyme, so as to obtain RNA-DNA/DNA hybrids,

e) RNA transcripts are obtained from the RNA-DNA/DNA hybrids formed in step d), by means of an enzyme that has an RNA polymerase activity.

2. The method for generating transcripts as claimed in claim 1, also comprising the following step:

f. a hybridization probe specific for said region of interest is used for detecting the RNA transcripts generated.

3. The method for generating transcripts as claimed in claim 1, according to which steps a) to e) are carried our for a period of time sufficient to obtain a sufficient number of transcripts.

4. The method for generating transcripts as claimed in claim 1, in which the enzyme that has an RNA polymerase activity is a bacteriophage RNA polymerase, preferably the RNA polymerase of the T7 bacteriophage.

9. The method for generating transcripts as claimed in claim 2, according to which steps a) to e) are carried our for a period of time sufficient to obtain a sufficient number of transcripts.

10. The method for generating transcripts as claimed in claim 2, in which the enzyme that has an RNA polymerase activity is a bacteriophage RNA polymerase, preferably the RNA polymerase of the T7 bacteriophage.

11. The method for generating transcripts as claimed in claim 3, in which the enzyme that has an RNA polymerase activity is a bacteriophage RNA polymerase, preferably the RNA polymerase of the T7 bacteriophage.

5. A method for generating transcripts from:

at least a first RNA sequence to be amplified comprising a “

primer”

region and a region of interest, and a first amplification primer comprising a promoter region and a region capable of hybridizing to said “

primer”

region of the first RNA sequence to be amplified,

at least a second RNA sequence to be amplified, different than said first RNA sequence to be amplified, and comprising a “

primer”

region and a region of interest, and a second amplification primer comprising a promoter region and a region capable of hybridizing to said “

primer”

region of the second RNA sequence to be amplified,

said method being carried out at constant temperature, and comprising the following steps;

a). said first and second primers are hybridized with said first and second RNAs to be amplified,

b). said first and second primers are extended by means of a reverse transcriptase enzymatic activity in order to generate a first complementary deoxyribonucleic acid (cDNA) sequence of the first RNA to be amplified and a second complementary deoxyribonucleic acid (cDNA) sequence of the second RNA to be amplified,

c). the first and second RNAs to be amplified, hybridized respectively to the first and second cDNAs, are cleaved by means of an enzyme that has a ribonuclease H activity, so as to obtain first fragments of RNA to be amplified, hybridized to the first cDNA, and second fragments of RNA to be amplified, hybridized to the second cDNA,

d). the ends of said first and second fragments of RNA to be amplified are extended by means of a reverse transcriptase and strand displacement enzyme, so as to obtain RNA-DNA/DNA hybrids,

e). transcripts of said first and second RNAs to be amplified are obtained from the RNA-DNA/DNA hybrids formed in step d), by means of an enzyme that has an RNA polymerase activity.

6. The method for generating transcripts as claimed in claim 5, also comprising the following step:

f. a first hybridization probe is used for detecting the transcripts of the first RNA to be amplified and a second hybridization probe is used for detecting the transcripts of the second RNA to be amplified.

7. The method for generating transcripts as claimed in claim 5, according to which steps a) to e) are carried out for a period of time sufficient to obtain a sufficient number of transcripts.

8. The method for generating transcripts as claimed in claim 5, in which the enzyme that has an RNA polymerase activity is a bacteriophage RNA polymerase, preferably the RNA polymerase of the T7 bacteriophage.

12. The method for generating transcripts as claimed in claim 6, according to which steps a) to e) are carried out for a period of time sufficient to obtain a sufficient number of transcripts.

13. The method for generating transcripts as claimed in claim 6, in which the enzyme that has an RNA polymerase activity is a bacteriophage RNA polymerase, preferably the RNA polymerase of the T7 bacteriophage.

14. The method for generating transcripts as claimed in claim 7, in which the enzyme that has an RNA polymerase activity is a bacteriophage RNA polymerase, preferably the RNA polymerase of the T7 bacteriophage.