Method for using unequal primer concentrations for generating nucleic acid amplification products

• US 6,391,544 B1
• Filed: 05/15/1998
• Issued: 05/21/2002
• Est. Priority Date: 05/15/1998
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories
• Original Assignee: Abbott Laboratories
• Inventors: Salituro, John A., Carrino, John J.
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/079,675
• Publication Number:
  US 20020045163A1
• Time in Patent Office: 1,467 Days
• Field of Search: 435/6, 435/91.2, 935/76, 935/77, 935/78, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe

Abstract

The method provided herein is a method for detecting a target sequence in a test sample. Generally, the method comprises forming a reaction mixture comprising a test sample, amplification reagents, a first primer, and a second primer wherein the concentration of the first primer in the reaction mixture is 15% to 250% percent greater than the concentration of the second primer. The target sequence is amplified according to any amplification protocol that employs the primer sequences to generate copies of the target sequence that include a product from the first and second primers. A probe is hybridized to the amplification product from the first primer to form a hybrid complex; and the hybrid complex is detected as an indication of the presence of the target sequence in the test sample.

Claims

1. In a method for amplifying and detecting a target nucleic acid sequence in a test sample comprising the steps of:

(a) forming an amplification mixture comprising a test sample, a first and a second primer sequence, and amplification reagents,

(b) amplifying the target sequence to generate copies of the target sequence comprising an amplification product from the first and second primers; and

(c) detecting the copies of the target sequence as an indication of the presence of the nucleic sequence in the test sample;

wherein the improvement comprises providing the first primer sequence in 15% to 250% excess over the second primer and wherein a probe is hybridized to the amplification product from the first primer to form a hybrid complex and the hybrid complex is detected as an indication of the presence of the nucleic acid sequence in the test sample.

2. The method of claim 1 wherein the first primer is labeled.

3. The method of claim 1 wherein the probe is labeled.

4. The method of claim 1 wherein the first primer is in 20% to 150% excess over the second primer.

5. The method of claim 1 wherein the probe is labeled with a specific binding member and the first primer is labeled with a specific binding member and said hybrid complex is detected with a capture reagent and a conjugate.

6. A method of detecting a target sequence in a test sample comprising the steps:

(a) forming a reaction mixture comprising a test sample, amplification reagents, a first primer, and a second primer wherein the concentration of the first primer in the reaction mixture is 15% to 250% percent greater than the concentration of the second primer;

(b) amplifying the target sequence to generate copies of the target sequence comprising an amplification product from the first and second primers;

(c) hybridizing a probe to the amplification product from the first primer to form a hybrid complex; and

(d) detecting the hybrid complex as an indication of the presence of the target sequence in the test sample.

7. The method of claim 6 wherein the first primer is labeled.

8. The method of claim 6 wherein the concentration of the first primer in the reaction mixture is 20% to 150% greater than the concentration of the second primer.

9. The method of claim 8 wherein said probe is labeled.

10. A kit comprising a first and second primer wherein the first primer is contained in the kit such that its concentration is 15% to 250% greater than the second primer.