Restriction Enzyme-Free Target Enrichment
First Claim
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1. A method comprising:
- (a) hybridizing randomly sheared genomic DNA to a halo probe to produce a first circular complex, wherein said halo probe comprises;
(i) a first oligonucleotide comprising flanking sequences that hybridize to different regions in a fragment of the randomly sheared genomic DNA and a central sequence; and
(ii) one or more second oligonucleotides that are complementary to the central sequence of the first oligonucleotide;
(b) enzymatically digesting the overhanging ends of the genomic fragment in the first circular complex to provide a second circular complex in which 5′ and
3′
ends of the one or more second oligonucleotide are ligatably adjacent to the 3′ and
5′
ends of the digested genomic fragment; and
(c) ligating the ends of the digested genomic fragment of (c) to the ends of the one or more second oligonucleotide to produce a circular DNA molecule.
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Abstract
Provided herein are various methods for enriching a target fragment that is present in randomly sheared genomic DNA. In some embodiments, the method may involve hybridizing randomly sheared genomic DNA to a halo probe to produce a first circular complex, and then enzymatically digesting the overhanging ends of the genomic fragment. Other embodiments may include hybridizing randomly sheared genomic DNA to an RNA oligonucleotide that comprises a region that hybridizes to a fragment of the randomly sheared genomic DNA to produce an RNA/DNA duplex. The overhanging ends of the genomic fragment in the RNA/DNA duplex can then be enzymatically digested.
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Citations
20 Claims
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1. A method comprising:
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(a) hybridizing randomly sheared genomic DNA to a halo probe to produce a first circular complex, wherein said halo probe comprises; (i) a first oligonucleotide comprising flanking sequences that hybridize to different regions in a fragment of the randomly sheared genomic DNA and a central sequence; and (ii) one or more second oligonucleotides that are complementary to the central sequence of the first oligonucleotide; (b) enzymatically digesting the overhanging ends of the genomic fragment in the first circular complex to provide a second circular complex in which 5′ and
3′
ends of the one or more second oligonucleotide are ligatably adjacent to the 3′ and
5′
ends of the digested genomic fragment; and(c) ligating the ends of the digested genomic fragment of (c) to the ends of the one or more second oligonucleotide to produce a circular DNA molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method comprising:
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(a) hybridizing randomly sheared genomic DNA to an RNA oligonucleotide comprising a region that hybridizes to a fragment of the randomly sheared genomic DNA to produce an RNA/DNA duplex; (b) enzymatically digesting the overhanging ends of the genomic fragment in the RNA/DNA duplex to provide a duplex comprising a digested genomic fragment that has defined ends; (c) digesting the RNA oligonucleotide of the duplex of (b) to release the digested genomic fragment; (d) hybridizing the digested genomic fragment of (c) with a halo probe comprising; (i) a first oligonucleotide comprising flanking sequences that hybridize to the ends of the digested genomic fragment and a central sequence; and (ii) one or more second oligonucleotides that are complementary to the central sequence of the first oligonucleotide, to provide a second complex in which 5′ and
3′
ends of the second oligonucleotide are ligatably adjacent to the 3′ and
5′
ends of the digested genomic fragment;(e) ligating the ends of the digested genomic fragment to the second oligonucleotide to produce a circular DNA molecule. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20)
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Specification