Methods, compositions, systems, apparatuses and kits for nucleic acid amplification
In some embodiments, the present teachings provide methods for paired end sequencing. In some embodiment, a polynucleotide template to be subjected to paired end sequencing comprises at least one cross linking moiety and at least one scissile moiety. In some embodiments, a paired end sequencing reaction comprises (a) a forward sequencing step, (b) a cleavage step, and (c) a reverse sequencing step. In some embodiments, a paired end sequencing reaction comprises (a) a forward sequencing step, (b) a cross-linking step, (c) a cleavage step, and (d) a reverse sequencing step.
1. A method for paired end sequencing comprising:
(a) conducting a forward sequencing reaction on a template polynucleotide attached to a surface and having at least one cleavable scissile moiety by (i) hybridizing a first primer to the template polynucleotide, (ii) extending the first primer by conducting primer extension reactions by polymerase-catalyzed successive nucleotide incorporations and forming a first extension strand hybridized to the template polynucleotide, and (iii) identifying the incorporated nucleotides in the first extension strand;
(b) inducing cross-linking between a nucleoside of the template polynucleotide and a nucleoside of the first extension strand;
(c) cleaving the template polynucleotide by reacting the scissile moiety with heat, irradiation, at least one chemical, or at least one enzyme, to form a truncated template polynucleotide and forming a terminal 3′ phosphate group, converting the terminal 3′ phosphate group to an —OH group, and removing a portion of the cleaved template polynucleotide; and
(d) conducting a reverse sequencing reaction on the first extension strand by extending the terminal 3′ OH group on the truncated template polynucleotide with primer extension reactions by polymerase-catalyzed successive nucleotide incorporations and forming a second extension strand, and (iii) identifying the incorporated nucleotides in the second extension strand.
2. The method of claim 1 , wherein the 5′ end of the template polynucleotide is attached to a surface.
3. The method of claim 1 , wherein the template polynucleotide further comprises a cross linking moiety.
4. The method of claim 3 , wherein the cross linking moiety comprises a 3-cyanovinylcarbazole nucleoside.
5. The method of claim 1 , wherein the cleavable scissile moiety comprise a uracil base.
6. The method of claim 1 , wherein an appropriate wavelength of irradiation comprises 366 nm.
7. The method of claim 1 , wherein the at least one enzyme that cleaves the scissile moiety comprise a glycosylase.
8. The method of claim 1 , wherein the at least one enzyme that cleaves the scissile moiety comprises a lyase.
9. The method of claim 1 , wherein the terminal 3′ phosphate group is converted to an —OH group by a kinase enzyme.
10. The method of claim 1 , wherein the portion of the cleaved template polynucleotide is removed by denaturation.
11. A method for paired end sequencing comprising:
(a) conducting a forward sequencing reaction on a template polynucleotide attached to a surface and having at least one cross linking moiety and at least one cleavable scissile moiety by (i) hybridizing a first primer to the template polynucleotide, (ii) extending the first primer by conducting primer extension reactions by polymerase-catalyzed successive nucleotide incorporations and forming a first extension strand hybridized to the template polynucleotide, and (iii) identifying the incorporated nucleotides in the first extension strand;
(b) inducing cross linking between a nucleoside of the template polynucleotide and a nucleoside of the first extension strand with a heat, chemical or photochemical condition;
(c) cleaving the template polynucleotide by reacting the scissile moiety with heat, irradiation, at least one chemical, or at least one enzyme, to form a truncated template polynucleotide and forming a terminal 3′ phosphate group, converting the terminal 3′ phosphate group to an —OH group, and removing a portion of the cleaved template polynucleotide; and
(d) conducting a reverse sequencing reaction on the first extension strand by extending the terminal 3′ OH group on the truncated template polynucleotide with primer extension reactions by polymerase-catalyzed successive nucleotide incorporations and forming a second extension strand, and (iii) identifying the incorporated nucleotides in the second extension strand.
12. The method of claim 11 , wherein the 5′ end of the template polynucleotide is attached to a surface.
13. The method of claim 11 , wherein the cross linking moiety comprises a 3-cyanovinylcarbazole nucleoside.
14. The method of claim 11 , wherein the cleavable moiety comprise a uracil base.
15. The method of claim 11 , wherein an appropriate wavelength of irradiation comprises 366 nm.
16. The method of claim 11 , wherein the at least one enzyme that cleaves the scissile moiety comprise a glycosylase.
17. The method of claim 11 , wherein the at least one enzyme that cleaves the scissile moiety comprises a lyase.
18. The method of claim 11 , wherein the terminal 3′ phosphate group is converted to an —OH group by a kinase enzyme.
19. The method of claim 11 , wherein the portion of the cleaved template polynucleotide is removed by denaturation.