Methods For Amplification of Nucleic Acids Using Spanning Primers
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.