CIRCULAR CHROMOSOME CONFORMATION CAPTURE (4C)

• US 20100062947A1
• Filed: 01/10/2008
• Published: 03/11/2010
• Est. Priority Date: 01/11/2007
• Status: Active Grant

• Current Assignee: Erasmus University Medical Center Rotterdam
• Original Assignee: Erasmus University Medical Center Rotterdam
• Inventors: Grosveld, Frank, De Laat, Wouter
• Granted Patent: US 8,642,295 B2
• Time in Patent Office: Days
• US Class Current: 506/8
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen

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) 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.

Claims

1. A method for analysing the frequency of interaction of a two 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;

(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 using multiplex PCR;

(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 (c) or (f) results in the formation of DNA circles.

3. The method according to claim 2 where step (h) comprises the analysis of ligation products between target sequences and cross-linked sequences of interest by means of sequencing (eg. high-throughput sequencing).

5. The method according to claim 1, further comprising pooling of some or all of the PCR products obtained for each of the target sequences in step (g) and subsequent simultaneous analysis of their DNA interactions.

6. The method according to claim 5, wherein the two or more amplified sequences are differentially labelled prior to pooling and analysis by hybridisation to an array.

7. The method according to claim 5, wherein the two or more amplified sequences are identically labelled and analysed by hybridisation to an array when the sequences reside on different chromosomes.

8. The method according to claim 5, 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.

9. The method according to claim 1, wherein high throughput sequencing is used to analyse the ligation junctions formed between target sequences and captured sequences of interest.

10. The method according to claim 9, wherein sequencing is directed to the ligation junctions formed between target sequences and captured sequences of interest by the addition of adapter sequences required for sequencing to the ends of the amplified sequences.

11. The method according to claim 10, wherein sequencing is directed to the ligation junctions formed between target sequences and captured sequences of interest by the addition of the complete, or part of the, adapter sequences required for sequencing as 5′

overhangs to the oligonucleotide primers used to amplify the one or more nucleotide sequences of interest.

12. The method according to claim 10, wherein sequencing is directed to the ligation junctions formed between target sequences and captured sequences of interest by the conjugation of a biotin substance or other moiety to the oligonucleotide primers used to amplify the one or more nucleotide sequences of interest, followed by streptavidin or otherwise mediated purification of the PCR amplified material.

13. The method according to claim 9, wherein sequencing is directed to the ligation junctions between target sequences and captured sequences of interest by designing the oligonucleotide primers used to amplify the one or more nucleotide sequences of interest within 400, 300, 200, 150, 100, 90, 80 70,60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nucleotides from the primary and/or secondary restriction enzyme recognition site(s) analysed.

14. The method according to claim 9, wherein sequencing is directed to the ligation junctions between target sequences and captured sequences of interest by designing the oligonucleotide primers used to amplify the one or more nucleotide sequences of interest such that they partially or completely overlap with the primary and/or secondary restriction enzyme recognition site analysed.

15. The method according to claim 9, wherein sequences are read across the ligation junction such that when multiplexed or pooled PCR samples are analysed, sufficient sequence information (eg. 12 nucleotides or more) is obtained on either side of the ligation junction to unambiguously identify each target sequence and each captured sequence of interest.

16. 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.

17. 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.

18. 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 as described in “

Catalogue of Unbalanced Chromosome Aberrations in Man”

2nd edition. Albert Schinzel. Berlin;

Walter de Gruyter, 2001. ISBN 3-11-011607-3.

19. 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.

20. The method according to claim 1, wherein the primary restriction enzyme is a restriction enzyme that recognises a 6-8 bp recognition site.

21. The method according to claim 20, wherein the primary restriction enzyme is selected from the group consisting of BglII, HindIII, EcoRI, BamHI, SpeI, PstI and NdeI.

22. The method according to claim 20, wherein the primary restriction enzyme is selected based on its absence from, or under representation in repetitive sequences.

23. 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.

24. 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.

26. 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 21claim 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 a difference in the linear organisation of the chromosome templates (eg. a genomic rearrangement), which is indicative of a particular trait or disease state.

27. 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) 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.

28. The method according to claim 27, wherein a transition from low to high interaction frequencies is indicative of the location of a balanced and/or unbalanced genetic rearrangement.

29. The method according to 27, wherein an inversed pattern of DNA-DNA interaction frequencies for the subject sample as compared to the control is indicative of a balanced and/or unbalanced inversion.

30. The method according to claim 27, 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.

31. The method according to claim 27, 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.

32. A method according to claim 27, wherein spectral karyotyping and/or FISH is used prior to performing said method.

33. The method according to claim 27, wherein the disease is a genetic disease.

34. The method according to claim 27, wherein the disease is cancer.

35. 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.

36. 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.

37. 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.

38. 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.

39. 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.

40. 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.

41. A method according to claim 1, wherein nucleotide sequences interacting with two or more target sequences are amplified.

42. A method according to claim 41, wherein the target sequences are positioned at or near genomic loci known to be associated with a diseased state.

43. A method according to claim 42, 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, and wherein the identified interacting sequences allow reconstructing linear chromosome maps and genomic rearrangements that occurred within and between chromosomes.

44. A method according to claim 41, wherein the amplified sequences are labelled.

45. A method according to claim 44, wherein the amplified sequences are differentially labelled according to their position in the genome.

46. A method according to claim 41, for the detection of a balanced and/or unbalanced rearrangement, translocation, inversion, deletion, duplication or insertion.

47. An agent obtained or obtainable by the assay method according to claim 35.

50. A method according to claim 1, wherein the array hybridisation step is replaced with a sequencing step.

4. (canceled)

25. A method for analysing the frequency of interaction of a target nucleotide sequence with one or more nucleotide sequences (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) 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 or analysing the amplified sequences by sequencing (eg. high-throughput sequencing); and

(i) determining the frequency of interaction between the DNA sequences.

48. 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.

51. A method according to claim 48, wherein both the target nucleotide sequence and the nucleotide sequence of interest are identified by sequencing.

52. A method according to claim 48, wherein adapter sequences are ligated to the PCR products.

49. A method for determining the presence of a genomic rearrangement in a sample comprising the steps of:

(a) providing a sample of nucleic acid (eg. genomic DNA), 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.

53. A database of nucleic acid sequences of about 6-50 basepairs that directly flank, and optionally include, the primary restriction enzyme recognition site or the secondary restriction enzyme recognition site of each target sequence.

55. Use of the database of nucleic acid sequences in claim 53 for determining the genomic position of each of the captured sequences identified.

54. A database of nucleic acid sequences of about 12-50 basepairs that directly flank all relevant primary and secondary restriction enzyme recognition sites in the genome.

56. A method or an agent or a database or a use substantially as described herein and with reference to any of the Examples or Figures.