Polynucleotide and use thereof
Provided is a polynucleotide including, from the 3′ terminus of the polynucleotide to the 5′ terminus of the polynucleotide, a first region including a nucleotide sequence complementary to a nucleotide sequence of a portion of a target nucleic acid; a second region including a nucleotide sequence identical to a nucleotide sequence of a portion of the target nucleic acid; and a third region including a nucleotide sequence that self-hybridizes to form a stem-loop structure, and compositions, kits, and methods related thereto.
1. A method of amplifying a target nucleic acid, comprising:
incubating a primer polynucleotide and a sample comprising a target nucleic acid to form a hybridized product, wherein the primer polynucleotide comprises
a first region consisting of a sequence of at least two consecutive nucleotides from the 3′ terminus thereof that are complementary to at least two consecutive nucleotides of a first region of a target nucleic acid,
a second region consisting of a sequence of at least two consecutive nucleotides from the 5′ terminus of the first region of the primer polynucleotide, wherein the second region is identical to at least two consecutive nucleotides of a second region of the target nucleic acid,
and a third region beginning from the 5′ terminus of the second region of the primer polynucleotide and comprising a stem loop structure,
wherein the first region of the primer polynucleotide hybridizes to the first region of target nucleic acid, which is located at the 3′ terminus from the second region of the target nucleic acid;
incubating the hybridized product in the presence of a first nucleic acid polymerase to form a extended product having a sequence complementary to the target nucleic acid sequence, wherein the extended product comprises a nucleotide sequence complementary to the second region of the polynucleotide;
incubating the extended product in the presence of a ligase to form a circular extended product; and
incubating the circular extended product in the presence of a second nucleic acid polymerase to amplify the target nucleic acid.
2. The method according to claim 1 , wherein the first nucleic acid polymerase is a DNA polymerase.
3. The method according to claim 2 , wherein the DNA polymerase is DNA polymerase I, Klenow fragment, T4 DNA polymerase, T7 DNA polymerase, or a combination thereof.
4. The method according to claim 1 , further comprising cooling off the extended product followed by heating to form a self-hybridized extended product, after forming the extended product.
5. The method according to claim 1 , wherein the ligase is DNA ligase.
6. The method according to claim 5 , wherein the DNA ligase is T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNA ligase, temperature-stable DNA ligase, single-stranded DNA ligase or a combination thereof.
7. The method according to claim 1 , further comprising incubating the extended product in the presence of an exonuclease to remove single-stranded DNA, after forming the circular extended product.
8. The method according to claim 1 , wherein the second nucleic acid polymerase is DNA polymerase.
9. The method according to claim 8 , wherein the DNA polymerase is Bst DNA polymerase, HIV reverse transcriptase, pyrophage 3173 DNA polymerase, Tth polymerase, BcaBEST DNA polymerase, or a combination thereof.
10. The method according to claim 1 , wherein the amplifying is performed via isothermal amplification.
11. The method according to claim 10 , wherein the isothermal amplification is rolling circle amplification (RCA), strand-displacement amplification (SDA), multiple-displacement amplification (MDA), or a combination thereof.
12. The method according to claim 1 , wherein the amplifying is performed in the presence of a primer complementary to the third region of the polynucleotide or a primer complementary to the extended product.
13. The method according to claim 1 , further comprising incubating the sample containing a target nucleic acid with a blocking polynucleotide before forming the extended product, wherein the blocking polynucleotide lacks a hydroxyl group at the 3′-terminus thereof and is complementary to at least two consecutive nucleotides from the 5′-terminus of the target nucleic acid.
14. The method according to claim 1 , wherein the at least two consecutive nucleotides of the first region of the target nucleic acid includes the 3′ terminus of the target nucleic acid.
15. The method according to claim 1 , wherein the at least two consecutive nucleotides of the second region of the target nucleic acid includes the 5′ terminus of the target nucleic acid.
16. The method according to claim 1 , wherein the third region of the primer polynucleotide is phosphorylated at its 5′ terminus.
17. The method according to claim 1 , wherein the third region of the primer polynucleotide comprises a primer-binding site, a restriction enzyme recognition site, a probe-binding site, or a combination thereof.