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Circular chromosome conformation capture (4c)

一种染色体、核苷酸序列的技术，应用在环状染色体构象捕获(4C)领域

Inactive Publication Date: 2010-03-31
ERASMUS UNIV MEDICAL CENT ROTTERDAM ERASMUS MC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Loops can still form if multiple restriction fragments are cross-linked together, but they can contain more than one captured fragment and will therefore be larger

Method used

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  • Circular chromosome conformation capture (4c)
  • Circular chromosome conformation capture (4c)
  • Circular chromosome conformation capture (4c)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0567] and figure 2 , 13 , 14, 15, 16, 17, 19 matching materials and methods

[0568] 4C technology

[0569]The initial steps of the 3C technology process were performed as described previously (Splinter et al. (2004). Methods Enzymol 375, 493-507 (2004)), generating ligation products between HindIII fragments. The HindIII-ligated 3C template (~50 μg) was digested overnight at 100 ng / μl with 50 U of the second, frequently cutting restriction enzyme, DpnII (HS2, Rad23A) or NlaIII (β-major). To avoid restriction of DNA loop formation (Rippe et al. (1995) Trends Biochem Sci 20, 500-6), care is taken to select a second restriction enzyme that is not at a distance from the HindIII restriction enzyme site demarcating the restriction fragment of interest (i.e. the 'bait') Point about 350-400bp within the cut. After digestion with the second restriction enzyme, the DNA was extracted with phenol, precipitated with ethanol, and then ligated at a low concentration (200 U ligase (R...

Embodiment 2

[0592] The 3C procedure (i.e. fixation with formaldehyde, digestion with (first) restriction enzyme, religation of cross-linked DNA fragments and DNA purification), resulting in a DNA mixture ('3C template') containing restriction fragments that were ligated due to their original proximity in the nuclear space.

[0593] Inverse PCR is performed to amplify all nucleotide sequences associated with a given restriction fragment ("bait"; as it contains a promoter, enhancer, insulator, matrix attachment region, origin of replication, or any other primary (target) nucleotide sequence while being picked) connected fragments.

[0594] To this end, the 3C template is generated by digesting the 3C template with a second restriction enzyme (preferably one that recognizes frequent cleavage of sequences of four or five nucleotides), followed by ligation under dilute conditions that favor intramolecular interactions. DNA circle. In order to minimize the bias in loop formation due to topolo...

Embodiment 3

[0609] Detecting translocations using 4C techniques

[0610] The frequency of interaction against a given sequence X occurring on a given chromosome A in cells from healthy subjects and in cells from patients with a single sequence between chromosomes A and B was measured using the 4C technique Reciprocal translocations in which the breakpoint is close to sequence X (eg Figure 8 shown).

[0611] In normal cells, this analysis revealed an enhanced hybridization signal (i.e., frequent interaction with X) for (almost) every probe located within 0.2-10 Mb of sequence X on chromosome A (showing strong cross-linking The actual size of the chromosomal region of the signal depends largely on the complexity of the sample hybridized to the array). Elsewhere on the same chromosome A, as well as on other chromosomes, no such large probe regions (on linear DNA templates) with increased hybridization signals were observed.

[0612] However, in patient cells, the hybridization signal was...

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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 usingat 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.

Description

field of invention [0001] The present invention involves analyzing the frequency with which two or more nucleotide sequences interact in nuclear space. Alterations in interactions are used as a means of detecting genomic rearrangements for diagnosis and prediction. Background of the invention [0002] The study of mammalian nuclear structure aims to understand how 2-meter-long DNA folds into a 10-μm-diameter nucleus while allowing the precise expression of genes for a given cell type and how they faithfully proliferate during each cell cycle. Much of the progress in this field has come from microscopy studies that have revealed that genomes are non-randomly arranged in nuclear space. For example, tightly packed heterochromatin is segregated from more expanded euchromatin, and chromosomes occupy different regions in nuclear space 2 . There is a complex relationship between nuclear localization and transcriptional activity. While transcription occurs throughout the interior...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6823C12Q2523/101
Inventor 沃特·德拉特弗兰克·格罗斯维尔德
Owner ERASMUS UNIV MEDICAL CENT ROTTERDAM ERASMUS MC
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