Method for one step isothermal non-transcription based amplification of nucleic acids

• US 6,033,881 A
• Filed: 04/06/1998
• Issued: 03/07/2000
• Est. Priority Date: 06/13/1995
• Status: Expired due to Term

• Current Assignee: Lumigen, Inc. (Danaher Corporation)
• Original Assignee: Lumigen, Inc. (Danaher Corporation)
• Inventors: Himmler, Gottfried, Schlederer, Thomas
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US08/973,965
• Time in Patent Office: 701 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.21, 435/91.5, 435/91.51, 435/91.52, 536/23.1, 536/24.3, 536/24.31, 536/24.32
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]

Abstract

The present invention relates to a process for the transcriptionless amplification of nucleic acids or nucleic acid sequences by means of enzymes in which the nucleic acids or sequences are divided at least partially into their individual strands before amplification and/or transcribed and the reaction mixture contains oligonucleotides with a base sequence essentially complementary to those of the ends of the nucleic acids or sequences to be amplified, where said oligonucleotides can, in certain conditions, form starting points and/or chemical components for the synthesis of nucleic acids and are built into the amplification product to be formed. The product of the components itself again corresponds to the nucleic acid or sequence to be amplified. The reaction product formed from the oligonucleotide components, possibly from the templates and other chemical components, reacts again in one step with the oligonucleotide components, the reaction being conducted substantially isothermally.

Claims

1. A process for the exponential amplification of a nucleic acid in a reaction mix by means of at least one enzyme, wherein the reaction mix comprises a nucleic acid to be amplified and oligonucleotides having base sequences substantially complementary to base sequences of the nucleic acid to be amplified, which oligonucleotides may, under appropriate conditions, form starting points and/or chemical oligonucleotide building blocks for nucleic acid synthesis, said process comprising the steps of

forming a reaction product comprising said oligonucleotides which is substantially complementary to the nucleic acid to be amplified,

reacting the reaction product with oligonucleotides such that additional reaction product is formed and the nucleic acid to be amplified is amplified,

whereby the steps are performed at a temperature which destabilizes the double strand form of the reaction product, and whereby said temperature is held essentially isothermal throughout amplification.

2. A process according to claim 1, wherein the at least one enzyme used for amplification is a polymerase.

3. A process according to claim 2, wherein the at least one polymerase used for amplification shows reverse transcriptase activity.

4. A process according to claim 2, wherein in addition to polymerase also a ligase is used as an enzyme for amplification.

5. A process according to claim 1, wherein at least one enzyme used for amplification is a ligase.

6. A process according to claim 1, further comprising the steps of

reacting the reaction product with oligonucleotides or oligonucleotide building blocks and polymerase,

at a temperature which destabilizes the double strand form of the reaction product, the temperature being held substantially isothermal,

wherein the polymerase demonstrates strand displacement activity.

7. A process according to claim 6, characterized wherein at least one of the oligonucleotides used is immobilized on a carrier.

8. A process according to claim 1, further comprising carrying out the amplification at a temperature above the melting temperature of the oligonucleotides used.

9. A process according to claim 6, wherein the oligonucleotides are substantially complementary to adjacent base sequences on the nucleic acid to be amplified.

10. A process according to claim 6, further comprising using helper oligonucleotides for the improvement of amplification.

11. A process according to claim 10, wherein the oligonucleotides are exactly complementary to at least a part of the template sequence.

12. A method for diagnosis by detection of a target sequence outside a human or animal body, comprising amplifying a nucleic acid according to the process of claim 5, whereby the target sequence is detected.

13. A process according to claim 1, further comprising at least partially separating the nucleic acid to be amplified into single strands and/or transcribing the nucleic acid prior to amplification.

14. A process according to claim 7, wherein said carrier is a plastic surface.

15. A process according to claim 6, further comprising at least partially separating the nucleic acid to be amplified into single strands and/or transcribing the nucleic acid prior to amplification.

16. A method for diagnosis by detection of a target sequence outside of a human or animal body, comprising amplifying a nucleic acid according to the process of claim 6, whereby the target sequence is detected.

17. A process for the exponential amplification of a nucleic acid in a reaction mix by means of enzymes, wherein the reaction mix comprises a nucleic acid to be amplified and oligonucleotides having base sequences substantially complementary to base sequences of the nucleic acid to be amplified, which oligonucleotides may, under appropriate conditions, form starting points and/or chemical oligonucleotide building blocks for nucleic acid synthesis, said process comprising the steps of

forming a reaction product comprising said oligonucleotides which is substantially complementary to the nucleic acid to be amplified,

reacting the reaction product with oligonucleotides such that additional reaction product is formed and the nucleic acid to be amplified is amplified,

whereby the steps are performed at a temperature which is above the melting temperature of the oligonucleotides used, whereby said temperature destabilizes the double strand form of the reaction product, and whereby said temperature is held essentially isothermal throughout amplification.