Method for the amplificaton and detection of dna using a transcription based amplification

• US 20040152090A1
• Filed: 03/23/2004
• Published: 08/05/2004
• Est. Priority Date: 03/07/2001
• Status: Abandoned Application

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Strijp, Arnoldina Margaretha Wilhelmina, Deiman, Birgit Alberta Louisa Maria, Frantzen, Inge Marjolein
• Application Number:
  US10/471,136
• Publication Number:
  US 20040152090A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/301 Endonuclease

Abstract

The present invention is directed to a transcription based amplification method for the amplification of DNA targets starting from ds or ssDNA optionally present in a sample, comprising the steps of:—incubating the sample in an amplification buffer with one or more restriction enzymes capable of cleaving DNA at a selected restriction site, said restriction enzyme creating a defined 3′ end on the said DNA strand(s), and a promoter-primer, said promoter-primer having a 5′ region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′ region complementary to the defined 3′ end of the DNA strand, a second primer, having the opposite polarity of the promoter-primer and comprising the 5′ end of the target sequence, and in case of ssDNA as the target DNA, a restriction primer;—maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place;—subjecting the sample to a heat treatment at a temperature and time sufficient to inactivate the restricting enzyme and/or to render a double strand single stranded;—adding the following reagents to the sample: an enzyme having RNA dependent DNA polymerase activity, an enzyme having DNA dependent DNA polymerase activity, an enzyme having Rnase H activity, an enzyme having RNA polymerase activity; and—maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place.

Claims

1. Method for the transcription based amplification of a target nucleic acid sequence starting from DNA optionally present in a sample, comprising the steps of,

incubating the sample in an amplification buffer with one or more restriction enzymes capable of cleaving DNA at a selected restriction site, said restriction enzyme creating a defined 3′

end on the said DNA strand(s), and

a promoter-primer, said promoter-primer having a 5′

region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′

region complementary to the defined 3′

end of the DNA strand,

a second primer, having the opposite polarity of the promoter-primer and comprising the 5′

end of the target sequence, and

in case of ssDNA as the target nucleic acid sequence, a restriction primer,

maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for a digestion by the restriction enzyme to take place,

subjecting the sample to a heat treatment at a temperature and time sufficient to inactivate the restriction enzyme and/or to render a double strand single stranded,

adding the following reagents to the sample;

an enzyme having RNA dependent DNA polymerase activity

an enzyme having DNA dependent DNA polymerase activity

an enzyme having RNase H activity

an enzyme having RNA polymerase activity, and

maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place.

2. Method according to claim 1, wherein the DNA is single stranded and the function of the promoter primer and the function of the restriction primer are combined in using a combined promoter and restriction primer comprising a sequence complementary to the region including the restriction site of the target ssDNA and the sequence of a promoter recognized by a DNA-dependent RNA polymerase.

3. Method according to claim 1 or 2 in which the appropriate nucleosidetriphosphates are added to the initial incubation mixture prior to the heat treatment.

4. Method according to claim 1 or 2 in which a reverse transcriptase is used combining the

activities of the enzyme having RNA dependent DNA polymerase activity and the enzyme having DNA dependent DNA activity.

5. Method according to claim 4 in which a reverse transcriptase is used having inherent

RNase H activity replacing 3 enzymes, namely the enzyme having RNA dependent DNA polymerase activity, the enzyme having DNA dependent DNA activity as well as the enzyme having RNase H activity.

6. Method according to claim 1, in which the incubation temperature is from 35°

C. to about 45°

C. and preferably about 37-41°

C.

7. Method according to claim 1 in which to heating step is carried out at a temperature between 92°

C. and 98°

C. and preferably at about 95°

C.