Method for detecting specific nucleic acid sequences

Abstract

The present invention relates to a method and test kit for detecting specific nucleic acid sequences, comprising the steps of: 1. matrix-dependent new synthesis of the target nucleic acid; 2. target-specific probe hybridization; and 3. detection of the hybridization event. The invention is characterized in that, in the first step, an oligonucleotide 1, which is marked by a marker 1 and is entirely or partially complementary to the target sequence, acts as a primer in the matrix-dependent new synthesis of the target nucleic acid and, in the second step, an oligonucleotide 2, which is marked by a marker 2 and, owing to its melting temperature being lower than that of the oligonucleotide 1, is not involved in the first step, partially or completely hybridizes with the DNA new synthesis product of oligonucleotide 1.The detection of the hybridization reaction can take place both fluorometrically in the form of a homogeneous assay and, for verification of the result, subsequently immunologically. The detection reaction always takes place in time after the matrix-dependent new synthesis has been carried out.

1 . A method for detecting a specific nucleic acid sequence, the method comprising:
 a) synthesizing a DNA target nucleic acid in a matrix-dependent de novo manner,   b) hybridizing the target nucleic acid with a probe, and   c) detecting the hybridizing,   wherein:   a primer in a) comprises an oligonucleotide 1 labeled with a label 1, which is completely or partly complementary to the target nucleic acid, and   the probe in b) comprises an oligonucleotide 2 labeled with a label 2, which has a lower melting temperature than oligonucleotide 1 and does not participate in the synthesizing, but which hybridizes partly or completely with the target nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein the detecting is performed in a reaction cavity in which the synthesizing and hybridizing are also performed. 
     
     
         3 . The method of  claim 1 , wherein the detecting is performed on a solid phase, either inside or outside of a reaction cavity in which the synthesizing and hybridizing are performed. 
     
     
         4 . The method of  claim 2 , wherein the two labels 1 and 2 as are a FRET pair, and the detecting comprises measuring a decrease in fluorescence. 
     
     
         5 . The method to of  claim 4 , wherein the detecting comprises end-point detecting. 
     
     
         6 . The method of  claim 1 , further comprising, after a), heating a reaction batch heated to a temperature of greater than 90° C., and thereafter cooling the reaction batch is to a hybridization temperature of oligonucleotide 2. 
     
     
         7 . The method of  claim 1 , wherein the hybridized oligonucleotide 2 is not destroyed by a Taq polymerase during the synthesizing or hybridizing, but instead remains in a hybridized state even after the synthesizing and hybridizing. 
     
     
         8 . The method of  claim 1 , wherein the detecting is performed only after the synthesis and hybridization are finished. 
     
     
         9 . The method of  claim 3 , wherein the solid phase comprises: a site that binds to at least one selected from the group consisting of label 1, label 2, an antibody against label 1, an antibody against label 2, a binding molecule against label 1, and a binding molecule against label 2; and wherein the solid phase comprises a detection molecule that visualizes or measures the hybridizing. 
     
     
         10 . The method of  claim 1 , wherein oligonucleotide 2 is protected against a 5′Δ3′-polymerase activity. 
     
     
         11 . A test kit that performs the method of  claim 1 , comprising:
 the oligonucleotide 1, labeled with the label 1,   the oligonucleotide 2 labeled with the label 2, and   a mixture of chemicals, enzymes, or both, optionally with a further unlabeled oligonucleotide.   
     
     
         12 . The test kit of  claim 11 , wherein the melting temperature of the oligonucleotide 2 is 5° C. to 15° C. lower than that of the oligonucleotide 1. 
     
     
         13 . The method of  claim 4 , wherein the FRET pair is selected from the group consisting of FITC/TAMRA, FAM/TAMRA, and FAM/BHQ1. 
     
     
         14 . The method of  claim 1 , wherein the oligonucleotide 2 is protected against a 5′→3′-polymerase activity by the label 2. 
     
     
         15 . The method of  claim 1 , wherein the oligonucleotide 2 is protected against a 5′→3′-polymerase activity by phosphorylation. 
     
     
         16 . The method of  claim 1 , wherein the oligonucleotide 1 is at least 50% complementary to the target nucleic acid. 
     
     
         17 . The method of  claim 1 , wherein the oligonucleotide 1 is at least 70% complementary to the target nucleic acid. 
     
     
         18 . The method of  claim 1 , wherein the oligonucleotide 1 is completely complementary to the target nucleic acid. 
     
     
         19 . The method of  claim 1 , wherein the melting temperature of the oligonucleotide 2 is 5° C. to 15° C. lower than that of the oligonucleotide 1. 
     
     
         20 . The method of  claim 1 , wherein after the hybridizing, the labels 1 and 2 are located 1 to 50 base pairs apart from each other.