Cooperative primers, probes, and applications thereof

Abstract

Disclosed are compositions and a method relating to amplifying and detecting nucleic acids.

We claim: 
     
         1 . A cooperative nucleic acid molecule comprising:
 a. a first nucleic acid sequence, wherein the first nucleic acid sequence is complementary to a first region of a target nucleic acid, and wherein the first nucleic acid is extendable on the 3′ end;   b. a second nucleic acid sequence, wherein the second nucleic acid sequence is complementary to a second region of the target nucleic acid, such that in the presence of the target nucleic acid it hybridizes to the target nucleic acid downstream from the 3′ end of the first nucleic acid sequence;   c. a linker connecting said first and second nucleic acid sequences in a manner that allows both the said first and second nucleic acid sequences to hybridize to the target at the same time.   
     
     
         2 . The cooperative nucleic acid molecule of  claim 1  wherein the first nucleic acid molecule will not hybridize to the target without the second nucleic acid molecule hybridizing to the target. 
     
     
         3 . The cooperative nucleic acid molecule of  claim 1  wherein the second nucleic acid molecule will not hybridize to the target without the first nucleic acid molecule hybridizing to the target. 
     
     
         4 . The cooperative nucleic acid molecule of  claim 1  wherein neither the first nor the second nucleic acid molecule will hybridize to the target without the other hybridizing to the target. 
     
     
         5 . The cooperative nucleic acid molecule of  claim 1  wherein the effective melting temperature (Tm) of the first nucleic acid molecule is increased by at least 1° C. as compared to the isolated Tm of the first nucleic acid sequence without the second nucleic acid sequence attached to it. 
     
     
         6 . The cooperative nucleic acid molecule of  claim 1  wherein the cooperative nucleic acid molecule comprises a label. 
     
     
         7 . The nucleic acid of  claim 6 , wherein the second nucleic acid sequence comprises a label. 
     
     
         8 . A method for synthesizing a nucleic acid, the method comprising:
 a. contacting a target nucleic acid with   b. a cooperative nucleic acid molecule comprising:
 i. a first nucleic acid sequence, wherein the first nucleic acid sequence is complementary to a first region of a target nucleic acid, and wherein the first nucleic acid is extendable on the 3′ end; 
 ii. a second nucleic acid sequence, wherein the second nucleic acid sequence is complementary to a second region of the target nucleic acid, such that it hybridizes to the target nucleic acid downstream from the 3′ end of the first nucleic acid sequence; 
 iii. a linker connecting said first and second nucleic acid sequences in a manner that allows both the said first and second nucleic acid sequences to hybridize to the target at the same time; 
   c. and providing conditions appropriate for nucleic acid synthesis, thereby synthesizing a nucleic acid.   
     
     
         9 . The method of  claim 8  wherein the cooperative nucleic acid molecule comprises a label. 
     
     
         10 . The method of  claim 8 , wherein more than one cooperative nucleic acid molecule with different sequences are provided. 
     
     
         11 . A method for detecting a target nucleic acid, the method comprising:
 a. contacting a sample containing the target nucleic acid with   b. a cooperative nucleic acid molecule comprising:
 i. a first nucleic acid sequence, wherein the first nucleic acid sequence is complementary to a first region of a target nucleic acid, and wherein the first nucleic acid is extendable on the 3′ end; 
 ii. a second nucleic acid sequence, wherein the second nucleic acid sequence is complementary to a second region of the target nucleic acid, such that it hybridizes to the target nucleic acid downstream from the 3′ end of the first nucleic acid sequence; 
 iii. a linker connecting said first and second nucleic acid sequences in a manner that allows both the said first and second nucleic acid sequences to hybridize to the target at the same time; 
   c. and detecting the target analyte.   
     
     
         12 . The method of  claim 11  wherein the label is attached to said second nucleic acid sequence. 
     
     
         13 . The method of  claim 12  wherein the change in signal is derived from the change in signal due to nuclease cleavage of the second nucleic acid sequence. 
     
     
         14 . A method for amplifying a target nucleic acid, the method comprising:
 a) providing the cooperative nucleic acid molecule of  claim 1 ;   b) providing a target nucleic acid; and   c) amplifying the target nucleic acid under appropriate conditions for amplification   The method of  claim 14 , wherein the first nucleic acid sequence is a primer.   
     
     
         15 . The method of  claim 14 , wherein the second nucleic acid sequence is a probe. 
     
     
         16 . The method of  claim 14 , wherein more than one cooperative nucleic acid molecule with different sequences are provided. 
     
     
         17 . The method of  claim 14 , wherein the first nucleic acid sequence without the second nucleic acid sequence attached to it is a normal primer. 
     
     
         18 . A method for detecting a nucleic acid, the method comprising:
 a) providing the cooperative nucleic acid molecule of  claim 1 , wherein the cooperative nucleic acid comprises a detectable label;   b) providing a target nucleic acid; and   c) detecting the target nucleic acid.   
     
     
         19 . The method of  claim 18 , wherein the detectable label is attached to the first nucleic acid sequence. 
     
     
         20 . The method of  claim 18 , wherein the detectable label is attached to the second nucleic acid sequence.