3 blocked nucleic acid amplification primers

• US 6,509,157 B1
• Filed: 10/13/2000
• Issued: 01/21/2003
• Est. Priority Date: 11/05/1999
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Martinez, Tomás Ramon
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): MAUPIN, CHRISTINE L
• Application Number:
  US09/687,910
• Time in Patent Office: 830 Days
• Field of Search: 435/6, 435/91.2, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The present invention provides reversibly blocked primers for use in the amplification of a nucleic acid sequence. Amplifications carried out using the blocked primers result in less non-specific amplification product, in particular, primer dimer, and a concomitant greater yield of the intended amplification product compared to amplifications carried out using unblocked primers.

Claims

1. A kit for carrying out a nucleic acid amplification reaction, wherein said kit comprises a pair of primers, wherein at least one primer of said pair is reversibly blocked by the covalent attachment of a triarylmethyl group to the 3′

terminal OH.

2. A kit of claim 1, wherein said triarylmethyl group has the formula:

wherein R₁, R₂, and R₃ represent independently an aryl group;

a nitrogen, sulfur, or oxygen-containing heterocyclic ring;

or an aryl group with a monosubstituent selected from the group consisting of halide, nitro, lower alkyl, lower alkoxy, and aryl, aralkyl, and cycloalkyl containing up to 10 carbon atoms;

wherein R₂ and R₃ each also may be alkyl, aralkyl, or cycloalkyl containing up to 10 carbon atoms.

3. A kit of claim 1, wherein said triarylmethyl group is dimethoxytrityl.

4. A kit of claim 3, wherein both primers of said pair of primers are reversibly blocked.

5. A method for amplifying a nucleic acid target sequence, wherein said method comprises carrying out an amplification reaction using a pair of primers, wherein at least one primer of said pair is reversibly blocked by the covalent attachment of a triarylmethyl group to the 3′

terminal OH.

6. A method of claim 5, wherein said triarylmethyl group has the formula:

wherein R₁, R₂, and R₃ represent independently an aryl group;

a nitrogen, sulfur, or oxygen-containing heterocyclic ring;

or an aryl group with a monosubstituent selected from the group consisting of halide, nitro, lower alkyl, lower alkoxy, and aryl, aralkyl, and cycloalkyl containing up to 10 carbon atoms;

wherein R₂ and R₃ each also may be alkyl, aralkyl, or cycloalkyl containing up to 10 carbon atoms.

7. A method of claim 5, wherein said triarylmethyl group is dimethoxytrityl.

8. A method of claim 7, wherein both primers of said pair of primers are reversibly blocked.