Chromosome conformation capture techniques are a set of molecular biology approaches used to analyze the spatial organization of chromatin and interaction of genomic regions in a cell.
3. Chromosome conformation
capture techniques are a set of molecular
biology approaches used to analyze the
spatial organization of chromatin and
interaction of genomic regions in a cell.
3C
4C
5C
Hi-C
The Hi-C method allows unbiased, genome-
wide identification of chromatin interactions.
What Is CCC family ?
6. Cells are fixed in 1% final concentration
of formaldehyde
After crosslinking, the remaining
formaldehyde is isolated with an excess
of glycine.
Then the cells are harvested by
centrifugation
1. Cell Culture and Crosslinking of
Chromatin
8. Restriction Enzyme Digestion
Accessible chromatin is then
digested with a type II restriction
endonuclease, e.g. HindIII, at 37 C
overnight.
The HindIII enzyme recognizes the
sequence:5’-AAGCTT-3’and cleaves
the DNA, leaving a 5’ overhang of:
5’-AGCT-3’.
Filling Overhangs
The 5’ overhang is filled in by the
DNA polymerase I using equimolar
amounts of all deoxyribonucleotides
9. This cleavage provides a template for
labeling the restriction fragments with
biotin-14-dCTP
The ligation of two completely filled-in HindIII
sites forms a NheI site: 5’ -GCTAGC-3’
3.Biotin marking of DNA ends
and blunt end ligation
10. The chromatin complexes containing
the biotin-labeled ligation products
are degraded by incubation with
Proteinase K at 65 C.
5.Streptavidin pull-down
Molecules with internal biotin
incorporation are pulled down with
streptavidin coated magnetic beads and
modified for deep sequencing.
4.DNA Purification
11. 6.Paired-End Adapter Ligation
and Library Amplification
Ligation of the Illumina Paired-end
Adapters is while the Hi-C library
is bound to the streptavidin
beads.
The adsorption of the DNA to the
beads increases the efficiency of
adapter ligation by decreasing the
mobility of the DNA fragments.
PCR amplify the library
13. Advantages of HI-C
1. High resolution compared to 3C
2. Characterize the structure of the entire genome.
3. It uncovers large blocks of multiple interactions between
one chromosome and another.
4. Quantify interactions between all possible pairs of
fragments simultaneously.
5. High throughput sequencing .
6. Data produced with hi-c is more comprehensive then
other 3-c type methods.
7. Correlated genome architecture with several genomic
features like replication timing.
14. Cont.
8. Interactions can be detected even over relatively large
genomic-distances.
9. Exhibit a high cis-/trans-interaction ratio
10. Hi-C can applied to single cells
11. Detect both known and novel, balanced and unbalanced
chromosomal rearrangements from cell lines and human
tumour samples
12. Due to high sequence coverage not being required, Hi-C
costs significantly less than deep WGS
13. Hi-C does not require dividing cells and can be used on all
nucleated cell types
15. Disadvantages of HI-C
1. Less resolution then 4C.
2. Workload increase compared to 4C and 5C.
3. Complexity of HI-C library is very high.
4. Hi-C technique cannot distinguish whether interactions are
stable and present in some cells, and non-existent in others
5. Background noise is one of the major disadvantage.
6. Evaluation of the random ligation noise in the final library is
expensive and time consuming.
7. HI-C has relatively few biases.
