Circular chromosome conformation capture (4C)
First Claim
1. A method for analysing the frequency of interaction of two or more target nucleotide sequences 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, wherein the ligation reaction results in the formation of DNA circles;
(d) reversing the cross linking;
(e) optionally 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 wherein the ligation reaction results in the formation of DNA circles;
(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 sequences of interest using multiplex PCR;
(h) hybridising 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 (eg. 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) optionally digesting the nucleotide sequences with a secondary restriction enzyme; (f) optionally 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 sequences of interest; (h) hybridising the amplified sequence(s) to an array; and (i) determining the frequency of interaction between the DNA sequences.
32 Citations
49 Claims
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1. A method for analysing the frequency of interaction of two or more target nucleotide sequences 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, wherein the ligation reaction results in the formation of DNA circles; (d) reversing the cross linking; (e) optionally 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 wherein the ligation reaction results in the formation of DNA circles; (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 sequences of interest using multiplex PCR; (h) hybridising 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, 11, 12, 13, 14, 15, 16, 17, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 47)
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- 18. The method according to claim wherein the primary restriction enzyme is a restriction enzyme that recognises a 6-8 bp recognition site.
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23. A method for analysing the frequency of interaction of a target nucleotide sequence with one or more DNA 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 DNA sequences; (d) reversing the cross linking; (e) optionally digesting the DNA sequences with a secondary restriction enzyme; circularising the nucleotide sequences; (g) amplifying one or more DNA sequences obtained in step (f) that are ligated to the target nucleotide sequence; (h) optionally hybridising the amplified sequences to an array or analysing the amplified sequences by sequencing; and (i) determining the frequency of interaction between the DNA sequences.
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45. A method for analysing the frequency of interaction of two or more target nucleotide sequences 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, wherein the ligation reaction results in the formation of DNA circles; (d) reversing the cross linking; and (e) sequencing the ligated nucleotide sequences. - View Dependent Claims (48, 49)
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46. A method for determining the presence of a genomic rearrangement in a sample by determining the frequency of interaction of two or more target nucleotide sequences with one or more nucleotide sequences of interest comprising the steps of:
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(a) providing a sample of nucleic acid, wherein said nucleic acid comprises a nucleotide sequence of known sequence adjacent to the location of the suspected genomic rearrangement; (b) digesting the DNA with a primary restriction enzyme to form a plurality of restriction fragments; (c) optionally, purifying the restriction fragments; (d) ligating the restriction fragments to form circularised DNA; (e) optionally, purifying the circularised DNA; (f) digesting the circularised DNA with a secondary restriction enzyme to form a plurality of restriction fragments; (g) ligating the restriction fragments to form circularised DNA; (h) amplifying the suspected genomic rearrangement using one or more primers that hybridise to the nucleotide sequence of known sequence; and (i) sequencing the suspected genomic rearrangement.
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Specification