Adaptor-based sequence analysis
First Claim
1. In a method of adaptor-based sequencing of one or more nucteotides in a target polynucleotide, which method comprises forming a target polynucleotide-adaptor adduct, the improvement comprising the steps of;
- (a) providing (i) a double-stranded target polynucleotide comprising a first strand and a second strand, wherein, at one end of the polynucleotide, the first strand terminates with a 5'"'"'-hydroxyl group, and, at the same said polynucleotide end, the second strand terminates with a 3'"'"'-phosphate group, and (ii) a double-stranded polynucleotide adaptor comprising a first strand and a second strand, wherein, at one end of the adaptor, the first strand terminates with a 3'"'"'-hydroxyl blocking group, and, at the same end of the adaptor, the second strand terminates with a 5'"'"'-phosphate group;
(b) ligating said one end of the target polynucleotide end to said one end of the adaptor such that the second strand of the target polynucleotide becomes ligated to the second strand of the adaptor, to form a singly ligated target polynucleotide-adaptor adduct;
(c) converting said 3'"'"'-hydroxyl blocking group in the singly ligated adduct to a hydroxyl group, and converting said 5'"'"'-hydroxyl group in the singly ligated adduct to a 5'"'"'-phosphate group, wherein the conversions are effected simultaneously or sequentially in either order; and
p1 (d) ligating said first strand of the target polynucleotide to said first strand of the adaptor to form a doubly ligated target polynucleotide-adaptor adduct.
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Abstract
An improvement in adaptor-based sequence analysis is provided that addresses the problems created by self-ligation of target polynucleotides that have complementary ends. The improvement includes preparation of target polynucleotides with dephosphorylated 5'"'"' strands and the use of adaptors having a 3'"'"' blocking group. In a preferred embodiment, adaptors are ligated to target polynucleotides by a single strand, 3'"'"' blocking groups are removed, the adjacent 5'"'"' hydroxyl of the target polynucleotide is phosphorylated, and the ligation of the adaptor is completed by treatment with a ligase.
148 Citations
36 Claims
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1. In a method of adaptor-based sequencing of one or more nucteotides in a target polynucleotide, which method comprises forming a target polynucleotide-adaptor adduct, the improvement comprising the steps of;
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(a) providing (i) a double-stranded target polynucleotide comprising a first strand and a second strand, wherein, at one end of the polynucleotide, the first strand terminates with a 5'"'"'-hydroxyl group, and, at the same said polynucleotide end, the second strand terminates with a 3'"'"'-phosphate group, and (ii) a double-stranded polynucleotide adaptor comprising a first strand and a second strand, wherein, at one end of the adaptor, the first strand terminates with a 3'"'"'-hydroxyl blocking group, and, at the same end of the adaptor, the second strand terminates with a 5'"'"'-phosphate group; (b) ligating said one end of the target polynucleotide end to said one end of the adaptor such that the second strand of the target polynucleotide becomes ligated to the second strand of the adaptor, to form a singly ligated target polynucleotide-adaptor adduct; (c) converting said 3'"'"'-hydroxyl blocking group in the singly ligated adduct to a hydroxyl group, and converting said 5'"'"'-hydroxyl group in the singly ligated adduct to a 5'"'"'-phosphate group, wherein the conversions are effected simultaneously or sequentially in either order; and
p1 (d) ligating said first strand of the target polynucleotide to said first strand of the adaptor to form a doubly ligated target polynucleotide-adaptor adduct. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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29. A method for ligating a double-stranded polynucleotide adaptor to an end of a target polynucleotide, the method comprising the steps of:
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(a) providing (i) a double-stranded target polynucleotide comprising a first strand and a second strand, wherein, at one end of the polynucleotide, the first strand terminates with a 5'"'"'-hydroxyl group, and, at the same said polynucleotide end, the second strand terminates with a 3'"'"'-phosphate group, and (ii) a double-stranded polynucleotide adaptor comprising a first strand and a second strand, wherein, at one end of the adaptor, the first strand terminates with a 3'"'"'-hydroxyl blocking group, and, at the same end of the adaptor, the second strand terminates with a 5'"'"'-phosphate group; (b) ligating said one end of the target polynucleotide end to said one end of the adaptor such that the second strand of the target polynucleotide becomes ligated to the second strand of the adaptor, to form a singly ligated target polynucleotide-adaptor adduct; (c) converting said 3'"'"'-hydroxyl blocking group in the singly ligated adduct to a hydroxyl group, and converting said 5'"'"'-hydroxyl group in the singly ligated adduct to a 5'"'"'-phosphate group, wherein the conversions are effected simultaneously or sequentially in either order; and (d) ligating said first strand of the target polynucleotide to said first strand of the adaptor to form a doubly ligated target polynucleotide-adaptor. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36)
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Specification