Methods For Amplification of Nucleic Acids Using Spanning Primers

Abstract

The teachings relate to methods and kits for detecting whether target nucleic acid sequences are present and/or quantitating target nucleic acid sequences.

1 . A method for amplifying at least one target nucleic acid sequence comprising:
 forming an amplification reaction composition comprising:
 a target nucleic acid sequence; 
 a polymerase; 
 a first primer comprising (i) a sequence complementary to the 5′ end of the target nucleic acid sequence and (ii) a sequence complementary to the 3′ end of the target nucleic acid sequence; and 
   subjecting the amplification reaction composition to at least one amplification reaction to form at least one amplification product.   
     
     
         2 . The method of  claim 1 , wherein the amplification reaction composition further comprises a second primer comprising (i) a sequence complementary to the 3′ end of a complement of the target nucleic acid sequence and (ii) a sequence complementary to any portion of the first primer. 
     
     
         3 . The method of  claim 1 , wherein the amplification reaction composition further comprises a second primer comprising (i) a sequence complementary to the 3′ end of a complement of the target nucleic acid sequence and (ii) a sequence complementary to the 5′ end of the first primer. 
     
     
         4 . The method of  claim 2 , wherein the second primer comprises a thymidine between (i) the sequence complementary to the 3′ end of a complement of the target nucleic acid sequence and (ii) the sequence complementary to any portion of the first primer. 
     
     
         5 . The method of  claim 3 , wherein the second primer comprises a thymidine between (i) the sequence complementary to the 3′ end of a complement of the target nucleic acid sequence and (ii) the sequence complementary to the 5′ end of the first primer. 
     
     
         6 . The method of  claim 1 , wherein the 3′ end of the target nucleic acid sequence is blocked. 
     
     
         7 . The method of  claim 1 , wherein the amplification reaction composition further comprises at least one probe. 
     
     
         8 . The method of  claim 7 , further comprising detecting the at least one amplification product. 
     
     
         9 . The method of  claim 1 , wherein the amplification reaction comprises an annealing step that takes place at a predetermined annealing temperature, and wherein the annealing temperature of two first cycles of amplification is 65° C., and is increased to at least 70° C. for subsequent cycles of amplification. 
     
     
         10 . The method of  claim 1 , wherein the amplification reaction comprises an annealing step that takes place at a predetermined annealing temperature, and wherein the annealing temperature is 70° C. or greater.