Methods, compositions, systems, apparatuses and kits for nucleic acid amplification
First Claim
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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.
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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.
85 Citations
19 Claims
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1. A method for paired end sequencing comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for paired end sequencing comprising:
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(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. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19)
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Specification