Compositions and methods for intramolecular nucleic acid rearrangement
First Claim
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1. A method of sample analysis, comprising:
- a) annealing a reflex sequence in a polynucleotide strand in a sample with a complement of the reflex sequence present in the polynucleotide strand, wherein the polynucleotide strand comprises the following in a 5′
to 3′
orientation;
a first domain comprising i. a primer site and ii. a multiplex identifier (MID) comprising a unique nucleotide sequence that distinguishes said polynucleotide strand from other polynucleotide strands in said sample;
the reflex sequence;
a polynucleotide of interest that is from 100 bases to 30 kilobases in length, wherein said polynucleotide of interest has a desired site that is distal to the first domain; and
the complement of the reflex sequence, wherein the complement of the reflex sequence is present at the 3′
end of the polynucleotide strand;
b) extending from the 3′
end of the complement of the reflex sequence through the first domain to produce a double stranded region comprising a complement of the first domain at the 3′
end of the extended polynucleotide strand, thereby moving the first domain into proximity with the desired site; and
c) sequencing the MID and the desired site using a primer that is complementary to the primer site in the complement of the first domain.
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Abstract
Aspects of the present invention are drawn to processes for moving a region of interest in a polynucleotide from a first position to a second position with regard to a domain within the polynucleotide, also referred to as a “reflex method”. In certain embodiments, the reflex method results in moving a region of interest into functional proximity to specific domain elements present in the polynucleotide (e.g., primer sites and/or MID). Compositions, kits and systems that find use in carrying out the reflex processes described herein are also provided.
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18 Claims
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1. A method of sample analysis, comprising:
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a) annealing a reflex sequence in a polynucleotide strand in a sample with a complement of the reflex sequence present in the polynucleotide strand, wherein the polynucleotide strand comprises the following in a 5′
to 3′
orientation;a first domain comprising i. a primer site and ii. a multiplex identifier (MID) comprising a unique nucleotide sequence that distinguishes said polynucleotide strand from other polynucleotide strands in said sample; the reflex sequence; a polynucleotide of interest that is from 100 bases to 30 kilobases in length, wherein said polynucleotide of interest has a desired site that is distal to the first domain; and the complement of the reflex sequence, wherein the complement of the reflex sequence is present at the 3′
end of the polynucleotide strand;b) extending from the 3′
end of the complement of the reflex sequence through the first domain to produce a double stranded region comprising a complement of the first domain at the 3′
end of the extended polynucleotide strand, thereby moving the first domain into proximity with the desired site; andc) sequencing the MID and the desired site using a primer that is complementary to the primer site in the complement of the first domain. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification