基本的 3C 技术需要五步实验步骤:
步骤一: 交联: 加入甲醛可以使 DNA 与蛋白或蛋白与蛋白之间相互粘结, 这样会导致相互作用的 DNA 片段被交联在一起. (for example cis located promoters to trans located promoters, reveals interactions like the interaction between H enhancer and odorant receptor promoters).
步骤二: 限制酶消化: 加入过量限制性内切酶将未交联的 DNA 与交联的 DNA 相互分离. 限制酶的选择取决于需要分析的基因座位的情况. 限制序列较短 (4 bp) 的内切酶切点密集, 用于研究较短的座位 (< 10~20 kb), 而限制序列较长 (6 bp) 的内切酶用于研究较长的座位.
在使用低剂量 DNA 底物的情况下, DNA 末端链接反应更偏好于将临近的 DNA 片段连接而非随机进行连接. 这种情况下, 两类连接反应会更频繁出现: 其一是限制酶未完全消化的 DNA 切口被重新连接, 这种情况大致占全部接口的 20% 到 30%, 此类连接与染色质构象捕获实验无关, 我们可以通过降低第一步交联反应的紧密程度来减少这类反应; 另一类频繁发生的连接反应是同一分子内的 DNA 的末端由于距离较近被连接, 这样的接口占到了全部接口的 30% 左右, 这类连接也会发生在交联后形成的蛋白与 DNA 复合物中不同 DNA 链之间 (图中展示的连接为复合物中相同来源 (红色) 的 DNA 末端被连接, 此外不同来源的 DNA 也可能被连接 (红色与蓝色之间). 
步骤四: 去交联: 步骤一中的交联可以通过高温去除, 所得到的 DNA 将在其序列两端与当中含有限制酶识别序列, 将这些 DNA 建成文库 (3C 库).
步骤五: 定量: 使用连接位点两端的引物进行聚合酶链式反应, 其结果可以半定量的表示 DNA 片段之间的相互作用. Quantitative PCR using Taqman probes (3C-qPCR) provides a more quantitative measurement of the fragment of interest. The Taqman probe and a constant primer hybridize to the restriction fragment that contains the site of contact and one test primer is designed against each neighboring restriction fragments. Together the probe and primers allow for a specific fluorescent signal to be emitted during amplification.
The 4C strategy has a significant advantage over 3C in that only the sequence of one of the site of interest needs to be known. The fragment, known as the “bait”, contains the site that associates with other chromosomal regions. The 4C procedure follows the same steps as in 3C, except additional processing is needed before quantification of the fragments of interest.
Steps 1-4: See procedure listed in 3C. 6-bp-cutters are preferred in step 2.
Step 5a Second Restriction Digest: After the reversal of the cross-linked DNA, the restriction fragments are subjected to another round of restriction digest, this time with a frequent cutter that will result in smaller fragments with restriction ends that differ from the central restriction site (ligation junction).
Step 5b Self-circularization: Self-circularization of the DNA fragments is more favored now that they are not bound to other proteins or fragments. Intramolecular ligation occurs to induce the formation of the circular fragments. The pool of circular fragments becomes the 4C library.
Step 5c Inverse PCR and Quantitation: Primers are designed against the outer restriction sites of the “bait” sequence, which result in the amplification of the small unknown captured fragment. Large-scale sequencing can be used to sequence the 4C library. Custom microarrays can also be made using probes designed against the adjacent upstream and downstream regions of all genomic sites of the restriction enzyme used in step 2.
The 5C technique expands from 3C and allows for the parallel analysis of interactions between many selected loci.
Steps 1-4: Same as in 3C.
Step 5 Ligation-mediated amplification and Quantitation: Performing multiplex ligation-mediated amplification (LMA) after the construction of the 3C library leads requires using multiplex primers that consist of universal primer sequences like T7 and T3 and the ligation junction sequences. They anneal to the 3C fragments and get ligated together with a DNA ligase. Perfect alignment with the 3C template ensures the success of the ligation. The ligated primers serve as templates of which get amplified to generate the 5C library. The use of universal primer sequences mean these 5C fragments can be analyzed on microarrays. The small size (~100 bp) of the 5C fragments is also compatible for analysis using high-throughput sequencing.
This method is slightly different from the previous techniques in that the interaction formed between two chromosomal regions is mediated by a bound protein. Like in the 5C methodology, a single DNA site is often considered to interact with multiple other sites. After the cross-linking and digestion, ChIP is performed to pull down the protein bound to the site of interest. Normal 3C procedures are conducted after this step.
A significant confounding factor in the 3C technology is the frequent random collisions of chromosomal regions to one another, which means that the detection of a product does not always mean a specific interaction has occurred between two regions. Therefore, a specific interaction between two regions is only confirmed when the interaction occurs at a higher frequency than with neighboring DNA.
The ChIP-loop may be useful in identifying long-range cis-interactions and trans interaction mediated through proteins since frequent DNA collisions will not occur.
The 5C technique overcomes the junctional problems at the intramolecular ligation step and is useful for constructing complex interactions of specific loci of interest. This approach is unsuitable for conducting genome-wide complex interactions since that will require millions of 5C primers to be used.
In contrast to 3C and 5C, the 4C technique does not require the prior knowledge of both interacting chromosomal regions. Results obtained using 4C are highly reproducible with most of the interactions that are detected between regions proximal to one another. On a single microarray, approximately a million interactions can be analyzed.
A common problem in all of these techniques is the requirement of a great number of cells, especially in the high-throughput methodologies. A single mammalian cell only provides two copies of any given restriction fragment, which can be ligated to only one other partner in 3C. Therefore any kind of quantitative analysis requires a large number of cells due to the need to determine the specification of an interaction between two regions. Experiments using the 4C technique routinely process ten million cells for analysis on a single microarray.
3C is based on proximity ligation, which had been used previously to determine circularization frequencies of DNA in solution, and the effect of protein-mediated DNA bending on circularization. Seyfred and colleagues developed proximity ligation in nuclei: restriction enzyme digestion of unfixed nuclei and ligation in situ without diluting the chromatin, which they termed the "Nuclear Ligation Assay"。
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