Methods for detecting nucleic acid sequence variants
View Patent ↗The present invention provides methods for detecting the presence or absence of a nucleic acid variant in a target region. These methods include amplifying the target region with a forward primer and a reverse primer in the presence of a selector blocker. The selector blocker includes a sequence complementary to the target region in the absence of the nucleic acid variant. The methods further include detecting amplification of the target region where amplification of the target region indicates the presence of the nucleic acid variant in the target region. The nucleic acid variant can include deletions, mutations or insertions.
1. A primer-switch comprising a forward primer linked or conjugated covalently or non-covalently to a selector blocker, wherein the forward primer and selector blocker are not linked or conjugated at the 3′ end of the forward primer wherein the forward primer and selector blocker each comprise a sequence complementary to a sequence in a target region of a nucleic acid, wherein the target region comprises a region to which both the forward primer and the selector blocker are complementary, wherein the selector blocker is complementary to a sequence 5′ of the sequence to which the forward primer is complementary or that overlaps with the sequence to which the forward primer is complementary, wherein the selector blocker is non-extendable, wherein the primer-switch is not attached to a solid support, and wherein (i) the selector blocker portion of the primer-switch functions as a steric blocker when bound to its target sequence, preventing extension of the forward primer, or (ii) the selector blocker portion of the primer-switch is not capable of binding tightly to the target region such that the forward primer can extend.
2. The primer-switch of claim 1 , wherein the forward primer and selector blocker are linked non-covalently or conjugated via a crosslinker or S18 spacer.
3. The primer-switch of claim 1 , wherein the selector blocker comprises a sequence complementary to a sequence in the target region in the absence of a nucleic acid variant.
4. The primer-switch of claim 1 , wherein the forward primer and selector blocker target sequences that overlap by 5, 10, 15, or more nucleotides.
5. The primer-switch of claim 1 , wherein the target region comprises one or more positions at which a nucleic acid variant may be present or absent, and wherein the forward primer and the selector blocker each have a sequence complementary to the same target region except that they differ from each other at one or more locations where the nucleic acid variant occurs.
6. The primer-switch of claim 5 , wherein the forward primer has a sequence fully complementary to the target region when the nucleic acid variant is present and the selector blocker has a sequence fully complementary to the target region when the nucleic acid variant is absent.
7. The primer-switch of claim 5 , wherein the forward primer has a sequence fully complementary to the target region when the nucleic acid variant is absent and the selector blocker has a sequence fully complementary to the target region when the nucleic acid variant is present.
8. The primer-switch of claim 1 , wherein the primer-switch is generated by synthesizing the forward primer with 3′ to 5′ chemistry, inserting at least one S18 spacer, and reversing strand direction to synthesize the selector blocker with 5′ to 3′ chemistry.
9. The primer-switch of claim 1 , wherein the primer-switch further comprises a detectable entity.
10. The primer-switch of claim 9 , wherein the detectable entity is selected from the group consisting of fluorescent labels, chemiluminescent labels, and FRET pairs.
11. The primer-switch of claim 1 , wherein the selector blocker has a higher Tm than the forward primer.
12. The primer-switch of claim 1 , wherein the selector blocker has a lower Tm than the forward primer.
13. A method for detecting the presence or absence of a nucleic acid variant in a target region comprising:
amplifying the target region in the presence of the primer-switch of claim 3 and a reverse primer, wherein amplification of the target region is indicative of the presence of the nucleic acid variant in the target region, and wherein the absence of amplification is indicative of the absence of the nucleic acid variant in the target region.
14. The method of claim 13 , wherein the nucleic acid variant includes a deletion, mutation, or insertion.
15. The method of claim 13 , wherein the amplification is performed in the presence of an enzyme possessing 3′ exonuclease repair activity.
16. A composition comprising: (i) a reverse primer, and (ii) a primer-switch comprising a forward primer linked or conjugated covalently or non-covalently to a selector blocker, wherein the forward primer and selector blocker are not linked or conjugated at the 3′ end of the forward primer, wherein the forward primer and selector blocker each comprise a sequence complementary to a sequence in a target region of a nucleic acid, wherein the target region comprises a region to which both the forward primer and the selector blocker are complementary, wherein the selector blocker is complementary to a sequence 5′ of the sequence to which the forward primer is complementary or that overlaps with the sequence to which the forward primer is complementary, wherein the selector blocker is non-extendable, and wherein (i) the selector blocker portion of the primer-switch functions as a steric blocker when bound to its target sequence, preventing extension of the forward primer, or (ii) the selector blocker portion of the primer-switch is not capable of binding tightly to the target region such that the forward primer can extend.