Capture and characterized co-localized chromatin (4C) technology
First Claim
1. A method for analyzing the frequency of interaction of a target nucleotide sequence with one or more nucleotide sequences of interest comprising the steps of:
- (a) providing a sample of cross-linked DNA;
(b) digesting the cross-linked DNA with a primary restriction enzyme;
(c) ligating the cross-linked nucleotide sequences;
(d) reversing the cross linking;
(e) digesting the nucleotide sequences with a secondary restriction enzyme;
(f) ligating one or more DNA sequences of known nucleotide composition to the available secondary restriction enzyme digestion site(s) that flank the one or more nucleotide sequences of interest;
(g) amplifying the one or more nucleotide sequences of interest using at least two oligonucleotide primers, wherein each primer hybridizes to the DNA sequences that flank the nucleotide sequences of interest;
(h) hybridizing the amplified sequence(s) to an array; and
(i) determining the frequency of interaction between the DNA sequences.
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Accused Products
Abstract
The present invention relates in one aspect to a method for analyzing the frequency of interaction of a target nucleotide sequence with one or more nucleotide sequences of interest (e.g., one or more genomic loci) comprising the steps of: (a) providing a sample of cross-linked DNA; (b) digesting the cross-linked DNA with a primary restriction enzyme; (c) ligating the cross-linked nucleotide sequences; (d) reversing the cross linking; (e) digesting the nucleotide sequences with a secondary restriction enzyme; (f) ligating one or more DNA sequences of known nucleotide composition to the available secondary restriction enzyme digestion site(s) that flank the one or more nucleotide sequences of interest; (g) amplifying the one or more nucleotide sequences of interest using at least two oligonucleotide primers, wherein each primer hybridises to the DNA sequences that flank the nucleotide sequence of interest; (h) hybridising the amplified sequence(s) to an array; and (i) determining the frequency of interaction between the DNA sequences.
16 Citations
30 Claims
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1. A method for analyzing the frequency of interaction of a target nucleotide sequence with one or more nucleotide sequences of interest comprising the steps of:
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(a) providing a sample of cross-linked DNA; (b) digesting the cross-linked DNA with a primary restriction enzyme; (c) ligating the cross-linked nucleotide sequences; (d) reversing the cross linking; (e) digesting the nucleotide sequences with a secondary restriction enzyme; (f) ligating one or more DNA sequences of known nucleotide composition to the available secondary restriction enzyme digestion site(s) that flank the one or more nucleotide sequences of interest; (g) amplifying the one or more nucleotide sequences of interest using at least two oligonucleotide primers, wherein each primer hybridizes to the DNA sequences that flank the nucleotide sequences of interest; (h) hybridizing the amplified sequence(s) to an array; and (i) determining the frequency of interaction between the DNA sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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11. A method for analyzing the frequency of interaction of a target nucleotide sequence with one or more nucleotide sequences comprising the steps of:
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(a) providing a sample of cross-linked DNA; (b) digesting the cross-linked DNA with a primary restriction enzyme; (c) ligating the cross-linked nucleotide sequences; (d) reversing the cross linking; (e) digesting the nucleotide sequences with a secondary restriction enzyme; (f) circularizing the nucleotide sequences; (g) amplifying the one or more nucleotide sequences that are ligated to the target nucleotide sequence; (h) optionally hybridizing the amplified sequences to an array; and (i) determining the frequency of interaction between the DNA sequences.
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12. A method for preparing a circularized nucleotide sequence comprising the steps of:
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(a) providing a sample of cross-linked DNA; (b) digesting the cross-linked DNA with a primary restriction enzyme; (c) ligating the cross-linked nucleotide sequences; (d) reversing the cross linking; (e) digesting the nucleotide sequences with a secondary restriction enzyme; and (f) circularizing the nucleotide sequences.
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Specification