User:Kristenabreu/sandbox/new

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Methodology[edit]
The ChIA-PET method combines ChIP-based methods (Kuo & Allis, 1999), and Chromosome conformation capture (3C), to extend the capabilities of both approaches. ChIP-Sequencing (ChIP-Seq) is a popular method used to identify TFBS while 3C has been used to identify long-range chromatin interactions (Dekker et al., 2002). However, both suffer from limitations when used independently to identify de-novo long-range interactions genome wide. While ChIP-Seq is typically used for genome-wide identification of TFBS (Barski et al., 2007; Wei et al., 2006), it provides only linear information of protein binding sites along the chromosomes (but not interactions between them), and can suffer from high genomic background noise (false positives).

While 3C is capable of analyzing long-range chromatin interactions, it cannot be used genome wide and, like ChIP-Seq, also suffers from high levels of background noise. Since the noise increases in relation to the distance between interacting regions (max 100kb), laborious and tedious controls are required for accurate characterization of chromatin interactions (Dekker et al., 2006).

The ChIA-PET method successfully resolves the issues of non-specific interaction noise found in ChIP-Seq by sonicating the ChIP fragments in order to separate random attachments from specific interaction complexes. The next step, which is referred to as enrichment, reduces complexity for genome-wide analysis and adds specificity to chromatin interactions bound by pre-determined TFs (transcription factors). The ability of 3C approaches to identify long-range interactions is based on the theory of proximity ligation. In regards to DNA inter-ligation, fragments that are tethered by common protein complexes have greater kinetic advantages under dilute conditions, than those freely diffusing in solution or anchored in different complexes. ChIA-PET takes advantage of this concept by incorporating linker sequences onto the free ends of the DNA fragments tethered to the protein complexes. In order to build connectivity of the fragments tethered by regulatory complexes, the linker sequences are ligated during nuclear proximity ligation. Therefore, the products of linker-connected ligation can be analyzed by ultra-high-throughput PET sequencing and mapped to the reference genome. Since ChIA-PET is not dependent on specific sites for detection as 3C and 4C are, it allows unbiased, genome-wide de-novo detection of chromatin interactions (Fullwood et al., 2009). edits below

Methodology[edit]
The ChIA-PET method combines ChIP-based methods, and Chromosome conformation capture (3C) based methods, to extend the capabilities of both approaches. ChIP-Sequencing (ChIP-Seq) is a popular method used to identify TFBS while 3C has been used to identify long-range chromatin interactions. Independently, both suffer from limitations in identifying de-novo long-range interactions genome wide. While ChIP-Seq is able to identify TFBS genome-wide, it provides only linear information of protein binding sites along the chromosomes (but not interactions between them), and can suffer from high genomic background noise (false positives). While 3C is capable of analyzing non-linear, long-range chromatin interactions, it cannot be used genome wide and, like ChIP-Seq, also suffers from high levels of background noise. Since the noise increases in relation to the distance between interacting regions (max 100kb), laborious and tedious controls are required for accurate characterization of chromatin interactions. Unlike 3C which is a loci specific interaction profiling method, alternative methods such as Hi-C have been established to profile interactions genome wide. Despite whole genome profiling methods for both TFBS and long range interactions, combining approaches with the ChIA-PET method allows for identification of genomic areas in which the protein of interest is bound as well as the genomic region which it interacts with.

The ChIA-PET method successfully resolves the issues of non-specific interaction noise found in ChIP-Seq by sonicating the ChIP fragments in order to separate random attachments from specific interaction complexes. The next step, which is referred to as enrichment, reduces complexity for genome-wide analysis and adds specificity to chromatin interactions bound by pre-determined TFs (transcription factors). The ability of 3C approaches to identify long-range interactions is based on the theory of proximity ligation. In regards to DNA inter-ligation, fragments that are tethered by common protein complexes have greater kinetic advantages under dilute conditions, than those freely diffusing in solution or anchored in different complexes. ChIA-PET takes advantage of this concept by incorporating linker sequences onto the free ends of the DNA fragments tethered to the protein complexes. In order to build connectivity of the fragments tethered by regulatory complexes, the linker sequences are ligated during nuclear proximity ligation. Therefore, the products of linker-connected ligation can be analyzed by ultra-high-throughput PET sequencing and mapped to the reference genome. Since ChIA-PET is not dependent on specific sites for detection as 3C and 4C are, it allows unbiased, genome-wide de-novo detection of chromatin interactions. Compared to Hi-C, the use of an antibody pulldown limits the number of sequenced fragments to chromatin interactions bound by the protein of interest which also can ease the data analysis.