Method for designing a probe combination

Abstract

Methods for designing and producing a fluorescent-labeled detection probe and a competitive probe combination are provided to improve detection by reducing noise. A method for designing the fluorescent-labeled detection probe and a competitive probe combination 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 methods 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 method for designing a fluorescent-labeled detection probe and a competitive probe combination, the method comprising:
 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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  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 . A method for producing a fluorescent-labeled detection probe and a competitive probe combination, the method comprising:
 the method of  claim 1 , further 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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  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 method according to  claim 11 , 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 method according to  claim 11 , 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.