Method of making a paired tag library for nucleic acid sequencing
First Claim
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1. A method of forming a paired tag comprising a first tag sequence and a second tag sequence that together comprise at least part of a polynucleotide of interest, said method comprising:
- (a) ligating a first end and a second end of a double stranded polynucleotide of interest to one or more double stranded nucleic acid adaptors to form a circular double stranded nucleic acid molecule having a first nick and a second nick on different strands of the circular nucleic acid molecule wherein a first strand of the first end of the double stranded polynucleotide of interest is joined to one strand of the one or more double stranded nucleic acid adaptors so as to form a first nick, and wherein a second strand of the second end of the double stranded polynucleotide of interest is joined to one strand of the one or more double stranded nucleic acid adaptors so as to form a second nick; and
(b) performing a nick translation reaction to move the first and the second nick to new positions within the double stranded polynucleotide of interest.
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Abstract
The present disclosure relates to methods and compositions for making paired tags and paired tag libraries.
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Citations
72 Claims
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1. A method of forming a paired tag comprising a first tag sequence and a second tag sequence that together comprise at least part of a polynucleotide of interest, said method comprising:
- (a) ligating a first end and a second end of a double stranded polynucleotide of interest to one or more double stranded nucleic acid adaptors to form a circular double stranded nucleic acid molecule having a first nick and a second nick on different strands of the circular nucleic acid molecule wherein a first strand of the first end of the double stranded polynucleotide of interest is joined to one strand of the one or more double stranded nucleic acid adaptors so as to form a first nick, and wherein a second strand of the second end of the double stranded polynucleotide of interest is joined to one strand of the one or more double stranded nucleic acid adaptors so as to form a second nick; and
(b) performing a nick translation reaction to move the first and the second nick to new positions within the double stranded polynucleotide of interest. - 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, 29, 30, 31, 32, 33, 34, 71, 72)
- (a) ligating a first end and a second end of a double stranded polynucleotide of interest to one or more double stranded nucleic acid adaptors to form a circular double stranded nucleic acid molecule having a first nick and a second nick on different strands of the circular nucleic acid molecule wherein a first strand of the first end of the double stranded polynucleotide of interest is joined to one strand of the one or more double stranded nucleic acid adaptors so as to form a first nick, and wherein a second strand of the second end of the double stranded polynucleotide of interest is joined to one strand of the one or more double stranded nucleic acid adaptors so as to form a second nick; and
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35. A method of forming a released paired tag from a DNA fragment of interest, comprising:
- ligating a first adaptor to a first end of a DNA fragment of interest and ligating a second adaptor to a second end of the DNA fragment of interest, thereby producing an adaptor modified fragment, circularizing the adaptor modified fragment by attaching a third adaptor to the adaptor modified fragment, thereby forming a circular nucleic acid molecule wherein a first nick is present between the third adaptor and the first adaptor and a second nick is present between the second adaptor and the third adaptor, wherein the circular nucleic acid molecule comprises a first strand and a second strand of DNA, and wherein the first nick and the second nick are not present on the same strand of the circular nucleic acid molecule;
performing a nick translation reaction wherein a nick on each strand of the circular nucleic acid molecule is translated into the DNA fragment of interest, and cleaving the circular nucleic acid molecule at a position at a translated nick, thereby forming a released paired tag. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
- ligating a first adaptor to a first end of a DNA fragment of interest and ligating a second adaptor to a second end of the DNA fragment of interest, thereby producing an adaptor modified fragment, circularizing the adaptor modified fragment by attaching a third adaptor to the adaptor modified fragment, thereby forming a circular nucleic acid molecule wherein a first nick is present between the third adaptor and the first adaptor and a second nick is present between the second adaptor and the third adaptor, wherein the circular nucleic acid molecule comprises a first strand and a second strand of DNA, and wherein the first nick and the second nick are not present on the same strand of the circular nucleic acid molecule;
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46. A method of forming a paired tag comprising a first tag sequence and a second tag sequence that together comprise at least part of a polynucleotide of interest, said method comprising:
- ligating a first end of a double stranded polynucleotide of interest to at least one adaptor;
ligating a second end of the double stranded polynucleotide of interest to at least one adaptor;
forming a circular nucleic acid molecule comprising the double stranded polynucleotide of interest, a first nick comprising a first 3′
end on a first strand of the circular nucleic acid molecule and a second nick comprising a second 3′
end on a second strand of the circular nucleic acid molecule, wherein the first nick and the second nick are on different strands of the circular nucleic acid molecule;
extending the first 3′
end of the first strand of the circular nucleic acid molecule into a sequence of the double stranded polynucleotide of interest; and
extending the second 3′
end of the second strand of the circular nucleic acid molecule into a sequence of the double stranded polynucleotide of interest. - View Dependent Claims (47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65)
- ligating a first end of a double stranded polynucleotide of interest to at least one adaptor;
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66. A method of preparing a paired tag, said method comprising:
- providing a nicked linking polynucleotide comprising;
a first adaptor comprising a first adaptor strand that is hybridized to a second adaptor strand, wherein the first adaptor strand lacks a phosphate group on its 5′
end;
a second adaptor comprising a third adaptor strand that is hybridized to a fourth adaptor strand, wherein the third adaptor strand lacks a phosphate group on its 5′
end; and
a linking polynucleotide comprising a first linking strand hybridized to a second linking strand, wherein the linking polynucleotide comprises a first end and a second end, wherein the first adaptor is attached to the first end of the linking polynucleotide such that a nick is present where the first adaptor strand lacks a phosphate group on its 5′
end, wherein the second linking strand is attached to the second adaptor strand, wherein the second adaptor is attached to the second end of the linking polynucleotide such that a nick is present where the third adaptor strand of the second adaptor lacks a phosphate group on its 5′
end, wherein the second strand of the linking polynucleotide is attached to the fourth adaptor strand;
ligating a first end of a double stranded polynucleotide of interest to the first adaptor and ligating a second end of the double stranded polynucleotide of interest to the second adaptor; and
moving the first and second nicks into the double stranded polynucleotide of interest. - View Dependent Claims (67, 68, 69, 70)
- providing a nicked linking polynucleotide comprising;
Specification