Compositions, methods and kits for enhancing PCR specificity
The present disclosure provides novel primers and method for the detection of specific nucleic acid sequences. The primers and methods provided herein are useful in a wide variety of molecular biology applications and are particularly useful in allele-specific PCR.
1. A composition comprising
a first oligonucleotide primer comprising a 5′ Sequence-Targeted Amplification Restrictive (STAR) tag sequence portion and a target nucleic acid hybridization sequence portion; and
a second oligonucleotide primer comprising a target nucleic acid hybridization sequence portion, wherein the STAR tag sequence is the same as all or a portion of the target nucleic acid hybridization sequence of the second oligonucleotide primer and the STAR tag sequence is the same as a sequence in the target nucleic acid 5′ of the first oligonucleotide target nucleic acid hybridization sequence hybridization site.
2. The composition of claim 1 , wherein the sequence shared between the first and the second primers is between about 3 and about 15 nucleotides in length.
3. The composition of claim 1 , wherein the first oligonucleotide primer and the second oligonucleotide primer are a pair of forward and reverse amplification primers.
4. The composition of claim 1 further comprising a polynucleotide extension product comprising the STAR tag sequence at the 5′ end and a sequence complementary to the STAR tag sequence to the 3′ side of the STAR tag sequence, wherein the STAR tag sequence and the complementary sequence render the extension product capable of forming a stem-loop structure.
5. A method of detecting a nucleic acid comprising:
contacting the target nucleic acid with a composition of claim 1 ;
hybridizing the target nucleic acid with the first oligonucleotide primer;
extending the oligonucleotide to form an extension product;
amplifying the extension product to form an amplification product; and
detecting the presence or absence of the amplification product, thereby detecting the target nucleic acid.
6. A method of inhibiting or substantially blocking undesired amplification of a target nucleic acid comprising:
contacting the target nucleic acid with a composition of claim 1 ;
hybridizing the target nucleic acid with the first oligonucleotide primer;
extending the oligonucleotide to form an extension product;
incubating the extension product with an amplification reaction mixture; and
detecting the presence or absence of the extension product.
7. A method of inhibiting or substantially blocking undesired amplification of a target nucleic acid comprising:
contacting the target nucleic acid with a composition of claim 1 ;
hybridizing the target nucleic acid with the first oligonucleotide primer; and
extending the oligonucleotide to form an extension product, wherein the extension product inhibits or substantially reduces undesired amplification of the target nucleic acid.
8. The composition of claim 1 , wherein the STAR tag sequence further comprises a sequence the same as a portion of an internal amplicon sequence of the target nucleic acid.
9. The composition of claim 1 , wherein the STAR tag sequence is about 10 to about 40 nucleotides in length.
10. The composition of claim 2 , wherein the sequence shared between the first and the second primers is 11, 12, or 13 nucleotides in length.
11. A composition comprising
a first oligonucleotide primer comprising a 5′ Sequence-Targeted Amplification Restrictive (STAR) tag sequence portion and a target nucleic acid hybridization sequence portion; and
a second oligonucleotide primer comprising a target nucleic acid hybridization sequence portion, wherein the STAR tag sequence is complementary to all or a portion of the target nucleic acid hybridization sequence of the second oligonucleotide primer and the STAR tag sequence is the same as a sequence in the target nucleic acid 5′ of the first oligonucleotide target nucleic acid hybridization sequence hybridization site.
12. The composition of claim 11 , wherein the STAR tag sequence further comprises a sequence the same as an internal amplicon sequence of the target nucleic acid.
13. The composition of claim 11 , wherein the STAR tag sequence is about 10 to about 40 nucleotides in length.
14. The composition of claim 11 , wherein the complementary sequence between the first and the second primers is between about 3 and about 15 nucleotides in length.
15. The composition of claim 11 further comprising a polynucleotide extension product comprising the STAR tag sequence at the 5′ end and a sequence complementary to the STAR tag sequence to the 3′ side of the STAR tag sequence, wherein the STAR tag sequence and the complementary sequence render the extension product capable of forming a stem-loop structure.