Methods and polynucleotides for amplifying a target polynucleotide
A method of amplifying a target polynucleotide, comprising: providing a template polynucleotide comprising a 5′ hairpin, a target polynucleotide and a 3′ hairpin, wherein the 5′ hairpin comprises one or more non-canonical nucleotides; and contacting the template polynucleotide with a polymerase and canonical nucleotides, wherein the polymerase extends, using the canonical nucleotides, the target polynucleotide from its 3′ end to form a first extended polynucleotide comprising the 5′ hairpin at its 5′ end and the complement of the 5′ hairpin at its 3′ end, wherein the complement of the 5′ hairpin forms a 3′ hairpin; and the polymerase extends the first extended polynucleotide from its 3′ end to form a second extended polynucleotide comprising the 5′ hairpin at its 5′ end and the complement of the 5′ hairpin at its 3′ end, wherein the complement of the 5′ hairpin forms a 3′ hairpin.
1. A method of amplifying a target polynucleotide, comprising:
a) providing a template polynucleotide comprising a 5′ hairpin, a target polynucleotide and a 3′ hairpin, wherein the 5′ hairpin comprises one or more non-canonical nucleotides;
b) contacting the template polynucleotide with a polymerase and canonical nucleotides,
c) extending
the template polynucleotide, using the polymerase and canonical nucleotides, from the 3′ hairpin to form a first extended polynucleotide comprising the 5′ hairpin at its 5′ end and the complement of the 5′ hairpin at its 3′ end, wherein the complement of the 5′ hairpin forms a 3′ hairpin; and
d) extending
the first extended polynucleotide, using the polymerase, from its 3′ hairpin to form a second extended polynucleotide comprising the 5′ hairpin at its 5′ end and the complement of the 5′ hairpin at its 3′ end, wherein the complement of the 5′ hairpin forms a 3′ hairpin wherein the 5′ hairpin comprises a selectively cleavable group and the method is terminated using an enzyme that cleaves the polynucleotide at the selectively cleavable group.
2. A method according to claim 1 , wherein the polymerase extends the second extended polynucleotide from its 3′ hairpin to form a third extended polynucleotide comprising the 5′ hairpin at its 5′ end and the complement of the 5′ hairpin at its 3′ end, wherein the complement of the 5′ hairpin forms a 3′ hairpin.
3. A method according to claim 2 , wherein the polymerase extends the third extended polynucleotide from its 3′ hairpin, and optionally any further extended polynucleotides produced by extending the third extended polynucleotide and/or by subsequent extensions, to produce further extended polynucleotides comprising the 5′ hairpin at their 5′ ends and the complement of the 5′ hairpin at their 3′ ends, wherein the complement of the 5′ hairpin forms a 3′ hairpin.
4. A method according to claim 1 , wherein the polymerase is a strand displacing polymerase.
5. A method according to claim 1 , wherein the method is carried out at a constant temperature.
6. A method according to claim 1 , further comprising the initial step of preparing the template polynucleotide by primer hybridization and extension.
7. A method according to claim 6 , wherein a target polynucleotide is amplified using: (i) a first primer comprising a 5′ hairpin and a sequence at its 3′ end that is complementary to a sequence in the 3′ end of the first strand of the target polynucleotide; and (ii) a second primer comprising a 5′ hairpin and a sequence at its 3′ end that is complementary to a sequence in the 3′ end of the second strand of the target polynucleotide.
8. A method according to claim 6 , wherein the template polynucleotide is produced by PCR.
9. A method according to claim 6 , wherein the template polynucleotide is produced using an isothermal amplification method using a strand displacing polymerase.
10. A method according to claim 1 , further comprising adding sequencing adaptors to one or both ends of the extended polynucleotide.