Kit for detecting a mutated target base sequence

Abstract

Kits for detecting a mutated target base sequence are provided. For example, the kits can be for detecting a target base sequence (A) containing a nucleotide with a mutated base from a nucleic acid sample. The kits contain a fluorescent-labeled detection probe and a competitive probe combination that is to improve detection by reducing noise. The probe combination is produced using a process that includes, for example, determining the base length and the base sequence of each of the fluorescent-labeled detection probe and the competitive probe. The determining can include experimentally determining the amount to be added to the nucleic acid sample of each of the fluorescent-labeled detection probe and the competitive probe. The kits provide a functional result of a first order derivative curve for the control target reaction sample having a substantial peak (maximum value), but a first order derivative curve for the control non-target reaction sample not having a substantial peak, the functional result improving detection by reducing noise.

We claim: 
     
         1 . A kit for detecting a target base sequence (A) containing a nucleotide with a mutated base from a nucleic acid sample, the kit comprising
 a combination of a fluorescent-labeled detection probe and a competitive probe, the combination designed by a process including
 determining the base length and the base sequence of each of the fluorescent-labeled detection probe and the competitive probe, the determining including
 experimentally determining the amount to be added to the nucleic acid sample of each of the fluorescent-labeled detection probe and the competitive probe, the experimentally determining including: 
 
 (a) adding the fluorescent-labeled detection probe and the competitive probe to each of
 a control target nucleic acid sample that contains the target nucleic acid, but does not substantially contain a non-target nucleic acid having the non-target base sequence (B); and, 
 a control non-target nucleic acid sample that does not substantially contain the target nucleic acid, but contains the non-target nucleic acid; 
 
 (b) measuring a fluorescence intensity while changing the temperature of each of the control reaction samples; and, 
 (c) performing first order differentiation of each of the temperature-fluorescence intensity curves obtained from the measurement results,
 wherein the adding, measuring, and performing are done so that a functional result of a first order derivative curve for the control target reaction sample has a substantial peak (maximum value), but a first order derivative curve for the control non-target reaction sample does not have a substantial peak. 
 
   
     
     
         2 . The kit of  claim 1 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 5° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe is lower than the Tm value of the fluorescent-labeled detection probe.   
     
     
         3 . The kit of  claim 1 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 10° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe is lower than the Tm value of the fluorescent-labeled detection probe.   
     
     
         4 . The kit of  claim 1 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 10° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe does not exceed the Tm value of the fluorescent-labeled detection probe+5° C.   
     
     
         5 . The kit of  claim 1 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 15° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe does not exceed the Tm value of the fluorescent-labeled detection probe+5° C.   
     
     
         6 . The kit of  claim 1 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 15° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe is lower than the Tm value of the fluorescent-labeled detection probe.   
     
     
         7 . The kit of  claim 1 , wherein
 (I) a region that hybridizes with the fluorescent-labeled detection probe in the target nucleic acid contains a region that hybridizes with the competitive probe;   (II) a region that hybridizes with the competitive probe in the target nucleic acid contains a region that hybridizes with the fluorescent-labeled detection probe; or   (III) a region that hybridizes with the competitive probe in the target nucleic acid coincides with a region that hybridizes with the fluorescent-labeled detection probe.   
     
     
         8 . The kit of  claim 1 , wherein the amount to be added of the competitive probe to the nucleic acid sample is at least 10 times (molar ratio) the amount to be added of the fluorescent-labeled detection probe. 
     
     
         9 . The kit of  claim 1 , wherein the amount to be added of the competitive probe to the nucleic acid sample is at least 20 times (molar ratio) the amount to be added of the fluorescent-labeled detection probe. 
     
     
         10 . The kit of  claim 1 , wherein the fluorescent dye is at least one type of fluorescent dye selected from the group consisting of tetramethyl rhodamine; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-propionic acid; 3-carboxy-6,8-difluoro-7-hydroxycoumarin; and carboxy rhodamine 6G. 
     
     
         11 . The kit of  claim 1 , wherein the combination is further produced by a process including
 synthesizing the detection probe and the competitive probe having the base length and the base sequence of each of the fluorescent-labeled detection probe and the competitive probe; and,   labeling the oligonucleotide of the fluorescent-labeled detection probe with a fluorescent dye.   
     
     
         12 . The kit of  claim 11 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 5° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe is lower than the Tm value of the fluorescent-labeled detection probe.   
     
     
         13 . The kit of  claim 11 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 10° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe is lower than the Tm value of the fluorescent-labeled detection probe.   
     
     
         14 . The kit of  claim 11 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 10° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe does not exceed the Tm value of the fluorescent-labeled detection probe+5° C.   
     
     
         15 . The kit of  claim 11 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 15° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe does not exceed the Tm value of the fluorescent-labeled detection probe+5° C.   
     
     
         16 . The kit of  claim 11 , wherein
 with respect to the non-target nucleic acid, the Tm value of the competitive probe is higher than the Tm value of the fluorescent-labeled detection probe by at least 15° C.; and,   with respect to the target nucleic acid, the Tm value of the competitive probe is lower than the Tm value of the fluorescent-labeled detection probe.   
     
     
         17 . The kit of  claim 11 , wherein
 (I) a region that hybridizes with the fluorescent-labeled detection probe in the target nucleic acid contains a region that hybridizes with the competitive probe;   (II) a region that hybridizes with the competitive probe in the target nucleic acid contains a region that hybridizes with the fluorescent-labeled detection probe; or   (III) a region that hybridizes with the competitive probe in the target nucleic acid coincides with a region that hybridizes with the fluorescent-labeled detection probe.   
     
     
         18 . The kit of  claim 11 , wherein the amount to be added of the competitive probe to the nucleic acid sample is at least 10 times (molar ratio) the amount to be added of the fluorescent-labeled detection probe. 
     
     
         19 . The kit of  claim 11 , wherein wherein the amount to be added of the competitive probe to the nucleic acid sample is at least 20 times (molar ratio) the amount to be added of the fluorescent-labeled detection probe. 
     
     
         20 . The kit of  claim 11 , wherein wherein the fluorescent dye is at least one type of fluorescent dye selected from the group consisting of tetramethyl rhodamine; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-propionic acid; 3-carboxy-6,8-difluoro-7-hydroxycoumarin; and carboxy rhodamine 6G.