4c

Abstract

The present invention relates in one aspect to a method for analysing 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) 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 sequences of interest; (h) hybridising the amplified sequence(s) to an array or sequencing the amplified sequences; and (i) determining the frequency of interaction between the DNA sequences.

1 . A method for analysing 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.    
     
     
         2 . The method according to  claim 1 , wherein the ligation reaction in step (f) results in the formation of DNA circles.  
     
     
         3 . The method according to  claim 1  for analysing the frequency of interaction of two or more target nucleotide sequences with one or more nucleotide sequences of interest, comprising the use of multiplex PCR in step (g).  
     
     
         4 . The method according to  claim 1 , wherein the target nucleotide sequence is selected from the group consisting of a genomic rearrangement, promoter, an enhancer, a silencer, an insulator, a matrix attachment region, a locus control region, a transcription unit, an origin of replication, a recombination hotspot, a translocation breakpoint, a centromere, a telomere, a gene-dense region, a gene-poor region, a repetitive element and a (viral) integration site.  
     
     
         5 . The method according to  claim 1 , wherein the target nucleotide sequence is a nucleotide sequence that is associated with or causes a disease, or is located up to or greater than 15 Mb on a linear DNA template from a locus that is associated with or causes a disease.  
     
     
         6 . The method according to  claim 1 , wherein the target nucleotide sequence is selected from the group consisting of: AML1, MLL, MYC, BCL, BCR, ABL1, IGH, LYL1, TAL1, TAL2, LMO2, TCRα,δ, TCRβ and HOX or other loci associated with disease.  
     
     
         7 . The method according to  claim 1 , wherein the target sequences are distributed along the linear genome template such that the interacting sequences cover an entire chromosome or the genome.  
     
     
         8 . The method according to  claim 1 , wherein the primary restriction enzyme is a restriction enzyme that recognises a 6-8 bp recognition site.  
     
     
         9 . The method according to  claim 8 , wherein the primary restriction enzyme is selected from the group consisting of BglII, HindIII, EcoRI, BamHI, SpeI, PstI and NdeI.  
     
     
         10 . The method according to  claim 1 , wherein the secondary restriction enzyme is a restriction enzyme that recognises a 4 or 5 bp nucleotide sequence recognition site.  
     
     
         11 . The method according to  claim 1 , wherein the secondary restriction enzyme recognition site is located at greater than about 350 bp from the primary restriction site in the target nucleotide sequence.  
     
     
         12 . The method according to  claim 1 , wherein the nucleotide sequence is labelled.  
     
     
         13 . A method for analysing the frequency of interaction of a target nucleotide sequence with one or more nucleotide sequences (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) optionally digesting the nucleotide sequences with a secondary restriction enzyme;    (f) circularising the nucleotide sequences;    (g) amplifying the one or more nucleotide sequences that are ligated to the target nucleotide sequence;    (h) optionally hybridising the amplified sequences to an array; and    (i) determining the frequency of interaction between the DNA sequences.    
     
     
         14 . A circularised nucleotide sequence comprising a first and a second nucleotide sequence, wherein each end of the first and a second nucleotide sequences are separated by different restriction enzyme recognition sites, and wherein said first nucleotide sequence is a target nucleotide sequence and said second nucleotide sequence is obtainable by cross-linking genomic DNA.  
     
     
         15 . A method for preparing a circularised nucleotide sequence 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; and    (f) circularising the nucleotide sequences.    
     
     
         16 . A method for analysing the frequency of interaction of a target nucleotide sequence with one or more nucleotide sequences (e.g. one or more genomic loci) comprising the use of a nucleotide sequence according to  claim 14 .  
     
     
         17 . An array of probes immobilised on a support comprising one or more probes that hybridise or are capable of hybridising to a nucleotide sequence according to  claim 14 .  
     
     
         18 . A set of probes complementary in sequence to the nucleic acid sequence adjacent to each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA.  
     
     
         19 . The set of probes according to  claim 18 , wherein the probes are complementary in sequence to the nucleic acid sequence adjacent to each side of each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA.  
     
     
         20 . The set of probes according to  claim 18 , wherein said probes are complementary in sequence to the nucleic acid sequence that is less than 300 base pairs from each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA.  
     
     
         21 . The set of probes according to  claim 18 , wherein the probes are complementary to the sequence that is less then 300 bp from each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA.  
     
     
         22 . The set of probes according to  claim 18 , wherein the probes are complementary to the sequence that is between 200 and 300 bp from each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA.  
     
     
         23 . The set of probes according to  claim 18 , wherein the probes are complementary to the sequence that is between 100 and 200 bp or 0 to 100 bp from each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA.  
     
     
         24 . The set of probes according to  claim 18 , wherein two or more probes are capable of hybridising to the sequence adjacent each primary restriction enzyme recognition site of a primary restriction enzyme in the genomic DNA.  
     
     
         25 . The set of probes according to  claim 24 , wherein the probes overlap or partially overlap.  
     
     
         26 . The set of probes to  claim 25 , wherein the overlap is less than 10 nucleotides.  
     
     
         27 . The set of probes according to  claim 18 , wherein the probe sequence corresponds to all or part of the sequence between each one of the primary restriction enzyme recognition sites of a primary restriction enzyme and each one of the first neighbouring secondary restriction enzyme recognition sites of a secondary restriction enzyme.  
     
     
         28 . The set of probes according to  claim 18 , wherein each probe is at a least a 25 mer.  
     
     
         29 . The set of probes according to  claim 18 , wherein each probes is a 25-60 mer.  
     
     
         30 . A process for preparing a set of probes comprising the steps of: 
 (a) identifying each one of the primary restriction enzyme recognition sites for a primary restriction enzyme in genomic DNA;    (b) designing probes that are capable of hybridising to the sequence adjacent each one of the primary restriction enzyme recognition sites in the genomic DNA;    (c) synthesising the probes; and    (d) combining the probes together to form a set of probes or substantially a set of probes.    
     
     
         31 . The process according to  claim 30 , wherein the probes are Pck amplification products.  
     
     
         32 . A set of probes or substantially a set of probes obtained or obtainable by the process according to  claim 30 .  
     
     
         33 . An array comprising the array of probes immobilised on a support comprising one or more probes that hybridise or are capable of hybridising to a nucleotide sequence according to  claim 14  or substantially the set of probes complementary in sequence to the nucleic acid sequence adjacent to each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA or obtained or obtainable by the process comprising the steps of: (a) identifying each one of the primary restriction enzyme recognition sites for a primary restriction enzyme in genomic DNA, 
 (b) designing probes that are capable of hybridising to the sequence adjacent each one of the primary restriction enzyme recognition sites in the genomic DNA; (c) synthesising the probes; and (d) combining the probes together to form a set of probes or substantially a set of probes.    
     
     
         34 . An array comprising the set of probes complementary in sequence to the nucleic acid sequence adjacent to each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA or obtained or obtainable by the process comprising the steps of: (a) identifying each one of the primary restriction enzyme recognition sites for a primary restriction enzyme in genomic DNA; (b) designing probes that are capable of hybridising to the sequence adjacent each one of the primary restriction enzyme recognition sites in the genomic DNA; (c) synthesising the probes; and (d) combining the probes together to form a set of probes or substantially a set of probes.  
     
     
         35 . The array according to  claim 33 , wherein the array comprises about 300,000-400,000 probes.  
     
     
         36 . The array according to  claim 34 , wherein the array comprises about 300,000-400,000 probes.  
     
     
         37 . The array according to  claim 33 , wherein the array comprises about 385,000 or more probes, preferably, about 750,000 probes, more preferably, 6×750,000 probes.  
     
     
         38 . The array according to  claim 34 , wherein the array comprises about 385,000 or more probes, preferably, about 750,000 probes, more preferably, 6×750,000 probes.  
     
     
         39 . The array according to  claim 33 , wherein the array comprises or consists of a representation of the complete genome of a given species at lower resolution.  
     
     
         40 . The array according to  claim 34 , wherein the array comprises or consists of a representation of the complete genome of a given species at lower resolution.  
     
     
         41 . The array according to  claim 39 , wherein one out of every 2, 3, 4, 5, 6, 7, 8, 9 or 10 probes as ordered on a linear chromosome template is contained in the array.  
     
     
         42 . the array according to  claim 40 , wherein one out of every 2, 3, 4, 5, 6, 7, 8, 9 or 10 probes as ordered on a linear chromosome template is contained in the array.  
     
     
         43 . A process for preparing an array comprising the step of immobilising on a solid support substantially the array of probes according to  claim 15  or substantially the set of probes complementary in sequence to the nucleic acid sequence adjacent to each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA or obtained or obtainable by the process comprising the steps of: (a) identifying each one of the primary restriction enzyme recognition sites for a primary restriction enzyme in genomic DNA; (b) designing probes that are capable of hybridising to the sequence adjacent each one of the primary restriction enzyme recognition sites in the genomic DNA; (c) synthesising the probes; and (d) combining the probes together to form a set of probes or substantially a set of probes.  
     
     
         44 . A process for preparing an array comprising the step of immobilising on a solid support the array of probes according to  claim 16  or the set of probes complementary in sequence to the nucleic acid sequence adjacent to each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA or obtained or obtainable by the process comprising the steps of: (a) identifying each one of the primary restriction enzyme recognition sites for a primary restriction enzyme in genomic DNA; (b) designing probes that are capable of hybridising to the sequence adjacent each one of the primary restriction enzyme recognition sites in the genomic DNA; (c) synthesising the probes; and (d) combining the probes together to form a set of probes or substantially a set of probes.  
     
     
         45 . An array obtained or obtainable by the method according to  claim 43 .  
     
     
         46 . An array obtained or obtainable by the method according to  claim 44 .  
     
     
         47 . A method for identifying one or more DNA-DNA interactions that are indicative of a particular disease state comprising the step of performing steps (a)-(i) of  claim 1 , wherein in step (a) a sample of cross-linked DNA is provided from a diseased and a non-diseased cell, and wherein a difference between the frequency of interaction between the DNA sequences from the diseased and non-diseased cells indicates that the DNA-DNA interaction is indicative of a particular disease state.  
     
     
         48 . A method of diagnosis or prognosis of a disease or syndrome caused by or associated with a change in a DNA-DNA interaction comprising the step of performing steps (a)-(i) of  claim 1 , wherein step (a) comprises providing a sample of cross-linked DNA from a subject; and wherein step (i) comprises comparing the frequency of interaction between the DNA sequences with that of an unaffected control; wherein a difference between the value obtained from the control and the value obtained from the subject is indicative that the subject is suffering from the disease or syndrome or is indicative that the subject will suffer from the disease or syndrome.  
     
     
         49 . The method according to  claim 48 , wherein a transition from low to high interaction frequencies is indicative of the location of a balanced and/or unbalanced genetic rearrangement.  
     
     
         50 . The method according to 48, wherein an inversed pattern of DNA-DNA interaction frequencies for the subject sample as compared to the control is indicative of an balanced and/or unbalanced inversion.  
     
     
         51 . The method according to  claim 48 , wherein a reduction in the DNA-DNA interaction frequency for the subject sample as compared to the control, in combination with an increase in DNA-DNA interaction frequency for more distant regions, is indicative of a balanced and/or unbalanced deletion.  
     
     
         52 . The method according to  claim 48 , wherein an increase or a decrease in DNA-DNA interaction frequency for the subject sample as compared to the control is indicative of a balanced and/or unbalanced duplication or insertion.  
     
     
         53 . A method according to  claim 48 , wherein spectral karyotyping and/or FISH is used prior to performing said method.  
     
     
         54 . The method according to  claim 48 , wherein the disease is a genetic disease.  
     
     
         55 . The method according to  claim 48 , wherein the disease is cancer.  
     
     
         56 . A method of diagnosis or prognosis of a disease or syndrome caused by or associated with a change in a DNA-DNA interaction comprising the step of: performing steps (a)-(i) of  claim 1 , wherein step (a) comprises providing a sample of cross-linked DNA from a subject; and wherein said method comprises the additional step of: (j) identifying one or more loci that have undergone a genomic rearrangement that is associated with a disease.  
     
     
         57 . The method according to  claim 56 , wherein the two or more amplified sequences are differentially labelled.  
     
     
         58 . The method according to  claim 56 , wherein the two or more amplified sequences are identically labelled when the sequences reside on different chromosomes.  
     
     
         59 . The method according to  claim 56 , wherein the two or more amplified sequences are identically labelled when the sequences reside on the same chromosome at a distance that is far enough for minimal overlap between DNA-DNA interaction signals.  
     
     
         60 . An assay method for identifying one or more agents that modulate a DNA-DNA interaction comprising the steps of: 
 (a) contacting a sample with one or more agents; and    (b) performing steps (a) to (i) of  claim 1 , wherein step (a) comprises providing cross-linked DNA from the sample;    wherein a difference between (i) the frequency of interaction between the DNA sequences in the presence of the agent and (ii) the frequency of interaction between the DNA sequences in the absence of the agent is indicative of an agent that modulates the DNA-DNA interaction.    
     
     
         61 . A method for detecting the location of a balanced and/or unbalanced rearrangement (eg. a translocation) comprising the step of: 
 (a) performing steps (a) to (i) of  claim 1;  and    (b) comparing the frequency of interaction between the DNA sequences with that of a control;    wherein a transition from low to high DNA-DNA interaction frequency in the sample as compared to the control is indicative of the location of a breakpoint.    
     
     
         62 . A method for detecting the location of a balanced and/or unbalanced inversion comprising the steps of: 
 (a) performing steps (a) to (i) of  claim 1;  and    (b) comparing the frequency of interaction between the DNA sequences with that of a control;    wherein an inversed pattern of DNA-DNA interaction frequencies for the sample as compared to the control is indicative of an inversion.    
     
     
         63 . A method for detecting the location of a deletion comprising the steps of: 
 (a) performing steps (a) to (i) of  claim 1;  and    (b) comparing the frequency of interaction between the DNA sequences with that of a control;    wherein a reduction in the DNA-DNA interaction frequency for the sample as compared to the control is indicative of deletion.    
     
     
         64 . A method for detecting the location of a duplication comprising the steps of: 
 (a) performing steps (a) to (i) of  claim 1;  and    (b) comparing the frequency of interaction between the DNA sequences with that of a control;    wherein an increase or a decrease in DNA-DNA interaction frequency for the subject sample as compared to the control is indicative of a duplication or insertion.    
     
     
         65 . A method according to  claim 1 , wherein nucleotide sequences interacting with two or more target sequences are amplified.  
     
     
         66 . A method according to  claim 65 , wherein the target sequences are positioned at or near genomic loci known to be associated with a diseased state.  
     
     
         67 . A method according to  claim 66 , wherein the target sequences are selected without prior knowledge on the location of a rearrangement and are spaced such that the interacting sequences cover an entire chromosome or the genome.  
     
     
         68 . A method according to  claim 65 , wherein the amplified sequences are labelled.  
     
     
         69 . A method according to  claim 68 , wherein the amplified sequences are differentially labelled according to their position in the genome.  
     
     
         70 . A method according to  claim 65 , for the detection of a balanced and/or unbalanced rearrangement, translocation, inversion, deletion, duplication or insertion.  
     
     
         71 . An agent obtained or obtainable by the assay method according to  claim 60 .  
     
     
         72 . A method of identifying one or more DNA-DNA interactions in a sample comprising the use of the nucleotide sequence according to  claim 14 .  
     
     
         73 . A method of determining the diagnosis or prognosis of a disease or syndrome caused by or associated with a change in a DNA-DNA interaction comprising the use of the nucleotide sequence according to  claim 14 .  
     
     
         74 . A method of identifying one or more DNA-DNA interactions in a sample comprising use of the array of probes according to  claim 17  or the set of probes complementary in sequence to the nucleic acid sequence adjacent to each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA or obtained or obtainable by the process comprising the steps of: (a) identifying each one of the primary restriction enzyme recognition sites for a primary restriction enzyme in genomic DNA; 
 (b) designing probes that are capable of hybridising to the sequence adjacent each one of the primary restriction enzyme recognition sites in the genomic DNA; (c) synthesising the probes;    and (d) combining the probes together to form a set of probes or substantially a set of probes.    
     
     
         75 . A method of determining the diagnosis or prognosis of a disease or syndrome caused by or associated with a change in a DNA-DNA interaction comprising the use of the array of probes according to  claim 17  or the set of probes complementary in sequence to the nucleic acid sequence adjacent to each one of the primary restriction enzyme recognition sites of a primary restriction enzyme in genomic DNA or obtained or obtainable by the process comprising the steps of: (a) identifying each one of the primary restriction enzyme recognition sites for a primary restriction enzyme in genomic DNA; (b) designing probes that are capable of hybridising to the sequence adjacent each one of the primary restriction enzyme recognition sites in the genomic DNA; (c) synthesising the probes; and (d) combining the probes together to form a set of probes or substantially a set of probes.  
     
     
         76 . A method of identifying one or more DNA-DNA interactions in a sample comprising use of the array according to  claim 33 .  
     
     
         77 . A method of identifying one or more DNA-DNA interactions in a sample comprising use of the array according to  claim 45 .  
     
     
         78 . A method of identifying one or more DNA-DNA interactions in a sample comprising use of the array according to  claim 46 .  
     
     
         79 . A method of determining the diagnosis or prognosis of a disease or syndrome caused by or associated with a change in a DNA-DNA interaction comprising the use of the array according to  claim 33 .  
     
     
         80 . A method of determining the diagnosis or prognosis of a disease or syndrome caused by or associated with a change in a DNA-DNA interaction comprising the use of the array according to  claim 45 .  
     
     
         81 . A method of determining the diagnosis or prognosis of a disease or syndrome caused by or associated with a change in a DNA-DNA interaction comprising the use of the array according to  claim 46 .  
     
     
         82 . The method of  claim 74 , wherein the diagnosis or prognosis is prenatal diagnosis or prognosis.  
     
     
         83 . The method of  claim 76 , wherein the diagnosis or prognosis is prenatal diagnosis or prognosis.  
     
     
         84 . The method of  claim 77 , wherein the diagnosis or prognosis is prenatal diagnosis or prognosis.  
     
     
         85 . The method of  claim 78 , wherein the diagnosis or prognosis is prenatal diagnosis or prognosis.  
     
     
         86 . The method of  claim 79 , wherein the diagnosis or prognosis is prenatal diagnosis or prognosis.  
     
     
         87 . The method of  claim 80 , wherein the diagnosis or prognosis is prenatal diagnosis or prognosis.  
     
     
         88 . The method of  claim 81 , wherein the diagnosis or prognosis is prenatal diagnosis or prognosis.  
     
     
         89 . A method for analysing the frequency of interaction of one or more target nucleotide sequences 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; and    (e) sequencing the ligated nucleotide sequences.    
     
     
         90 . A method or use according to  claim 1 , wherein the array hybridisation step is replaced with a sequencing step, wherein optionally both the target nucleotide sequence and the nucleotide sequence of interest are identified by sequencing, and wherein optionally adapter sequences are ligated to the PCR products.  
     
     
         91 . (canceled)  
     
     
         92 . (canceled)  
     
     
         93 . (canceled)  
     
     
         94 . (canceled)  
     
     
         95 . A method or use according to  claim 13 , wherein the array hybridisation step is replaced with a sequencing step, wherein optionally both the target nucleotide sequence and the nucleotide sequence of interest are identified by sequencing, and wherein optionally adapter sequences are ligated to the PCR products.  
     
     
         96 . A method or use according to  claim 14 , wherein the array hybridisation step is replaced with a sequencing step, wherein optionally both the target nucleotide sequence and the nucleotide sequence of interest are identified by sequencing, and wherein optionally adapter sequences are ligated to the PCR products.  
     
     
         97 . A method or use according to  claim 15 , wherein the array hybridisation step is replaced with a sequencing step, wherein optionally both the target nucleotide sequence and the nucleotide sequence of interest are identified by sequencing, and wherein optionally adapter sequences are ligated to the PCR products.  
     
     
         98 . A method or use according to  claim 18 , wherein the array hybridisation step is replaced with a sequencing step, wherein optionally both the target nucleotide sequence and the nucleotide sequence of interest are identified by sequencing, and wherein optionally adapter sequences are ligated to the PCR products.  
     
     
         99 . A method or use according to  claim 30 , wherein the array hybridisation step is replaced with a sequencing step, wherein optionally both the target nucleotide sequence and the nucleotide sequence of interest are identified by sequencing, and wherein optionally adapter sequences are ligated to the PCR products.