METHODS, COMPOSITIONS, SYSTEMS, APPARATUSES AND KITS FOR NUCLEIC ACID AMPLIFICATION

• US 20130203607A1
• Filed: 03/14/2013
• Published: 08/08/2013
• Est. Priority Date: 12/17/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: LAO, Kai Qin, MA, Zhaochun, LI, Bin, O'NEIL, Jennifer, KUNKEL, Jennifer, HALEY, Kellie, KASINSKAS, Rachel, BRZOSKA, Pius
• Granted Patent: US 9,309,566 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2535/119 Double strand sequencing

Abstract

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.

Claims

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) 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 optionally forming a terminal 3′

phosphate group, and optionally converting the terminal 3′

phosphate group to an —

OH group, and optionally removing a portion of the cleaved template polynucleotide; and

(c) 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 the 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 the template polynucleotide and 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 optionally forming a terminal 3′

phosphate group, and optionally converting the terminal 3′

phosphate group to an —

OH group, and optionally 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 the 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.