Method for using unequal primer concentrations for generating nucleic acid amplification products
First Claim
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.
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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.
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Citations
10 Claims
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1. In a method for amplifying and detecting a target nucleic acid sequence in a test sample comprising the steps of:
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(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. - View Dependent Claims (2, 3, 4, 5)
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6. A method of detecting a target sequence in a test sample comprising the steps:
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(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. - View Dependent Claims (7, 8, 9)
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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.
Specification