Process for amplifying nucleic acids using DNA/PNA primers

• US 6,063,571 A
• Filed: 09/11/1997
• Issued: 05/16/2000
• Est. Priority Date: 09/13/1996
• Status: Expired due to Fees

• Current Assignee: Steven A. Benner
• Original Assignee: Hoechst Aktiengesellschaft (Sanofi )
• Inventors: Breipohl, Gerhard, Benner, Steven A., Lutz, Michael, Uhlmann, Eugen
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/927,274
• Time in Patent Office: 978 Days
• Field of Search: 432/6, 432/91.1, 432/91.2, 435/91.1, 435/91.2
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention relates to a novel advantageous process for amplifying nucleic acids using DNA/PNA primers and a temperature-stable polymerase enzyme.

Claims

1. A process for amplifying nucleic acids comprising:

obtaining a nucleic acid template;

hybridizing at least one PNA/DNA primer, each having at least one nucleoside unit with a 3'"'"'-hydroxyl group on the 3'"'"' end, to said nucleic acid template, to produce PNA/DNA primer-template hybrids; and

amplifying linearly said PNA/DNA primer-template hybrid using at least one temperature-stable polymerase enzyme, wherein said temperature-stable polymerase enzyme is VENT™

polymerase, Pwo polymerase, TTH™

polymerase, or 9°

N DNA POLYMERASE™

, to produce linear amplification products.

4. A process according to claim 1, wherein said temperature-stable polymerase enzyme comprises a combination of temperature-stable polymerase enzymes comprising VENT™

polymerase and TTH™

polymerase.

2. A process for amplifying nucleic acids comprising:

obtaining a nucleic acid template;

hybridizing at least one PNA/DNA primer, each having one to three nucleoside units at the 3'"'"' end and at least one nucleoside unit with a 3'"'"'-hydroxyl group on the 3'"'"' end, to said nucleic acid template, to produce PNA/DNA primer-template hybrids; and

amplifying linearly said PNA/DNA primer-template hybrid using at least one temperature-stable polymerase enzyme, to produce linear amplification products.

3. A process for amplifying nucleic acids comprising:

obtaining a nucleic acid template;

hybridizing at least one PNA/DNA primer, each having one nucleoside unit at the 3'"'"' end and at least one nucleoside unit with a 3'"'"'-hydroxyl group on the 3'"'"' end, to said nucleic acid template, to produce PNA/DNA primer-template hybrids; and

amplifying linearly said PNA/DNA primer-template hybrid using at least one temperature-stable polymerase enzyme, to produce linear amplification products.

5. A method of diagnosing diseases comprising:

obtaining a nucleic acid template from a biological sample;

mixing at least one PNA/DNA primer, specific for diseases associated with the expression of one or more genes, and having at least one nucleoside unit with a 3'"'"'-hydroxyl group on the 3'"'"' end, with said nucleic acid template under conditons wherein specific hybridization would occur to produce PNA/DNA primer-template hybrids;

amplifying linearly said PNA/DNA primer-template hybrid using at least one temperature-stable polymerase enzyme, wherein said temperature-stable polymerase enzyme is VENT™

polymerase, Pwo polymerase, TTH™

polymerase, or 9°

N DNA POLYMERASE™

, to produce linearly amplified nucleic acid sequences; and

diagnosing said diseases by detecting the presence, absence or the amount of linearly amplified nucleic acid sequences present in said biological sample.

6. The method of claim 5, wherein said temperature-stable polymerase enzyme comprises a combination of temperature-stable polymerase enzymes comprising VENT™

polymerase and TTH™

polymerase.

7. A method of diagnosing diseases comprising:

obtaining a nucleic acid template from a biological sample;

mixing at least one PNA/DNA primer, specific for diseases associated with the expression of one or more genes, and having one to three nucleoside units at the 3'"'"' end and at least one nucleoside unit with a 3'"'"'-hydroxyl group on the 3'"'"' end, with said nucleic acid template under conditions wherein specific hybridization would occur to produce PNA/DNA primer-template hybrids;

amplifying linearly said PNA/DNA primer-template hybrid using at least one temperature-stable polymerase enzyme to produce linearly amplified nucleic acid sequences; and

diagnosing said diseases by detecting the presence, absence or the amount of linearly amplified nucleic acid sequences present in said biological sample.

8. A method of diagnosing diseases comprising:

obtaining a nucleic acid template from a biological sample;

mixing at least one PNA/DNA primer, specific for diseases associated with the expression of one or more genes, and having one nucleoside unit at the 3'"'"' end and at least one nucleoside unit with a 3'"'"'-hydroxyl group on the 3'"'"' end, with said nucleic acid template under conditions wherein specific hybridization would occur to produce PNA/DNA primer-template hybrids;

amplifying linearly said PNA/DNA primer-template hybrid using at least one temperature-stable polymerase enzyme to produce linearly amplified nucleic acid sequences; and

diagnosing said diseases by detecting the presence, absence or the amount of linearly amplified nucleic acid sequences present in said biological sample.