The video details a procedure for extracting PCR products from chromosome confirmation capture samples using gel extraction. It outlines both commercial and homemade methods, including constructing a purification cartridge. The effectiveness of the extracted DNA is then evaluated through QPCR, revealing positive results in the majority of tested reactions.

identification of the chromosome conformation capture (3c) products

Erfassung der Chromosomenkonformation (3C) für Forschung und Lehre im Grundstudium

An organism&#39;s genome not only holds all the genes required for function but also all the instructions on how and when to use them. This makes regulating access to the genome one of the most important functions of the cell. There are many mechanisms to control gene function; however, at its base level, gene regulation comes down to the ability of regulatory transcription factors (trans-factors) to bind to their specific DNA sequences (cis-regulatory sequences). This is not an innate ability; instead, it is governed by the organization/structure of the genome in the nucleus, which controls the availability/exposure of the cis-regulatory sequences to the trans-factors1,2,3. If the trans-factors cannot find their cis-regulatory sequences, then the trans-factors cannot perform their regulatory tasks. This has made understanding how genomes are organized in the nucleus an important source of inquiry.
It is widely accepted that during interphase, eukaryotic chromosomes in the nucleus occupy their own domain anchored to the nuclear lamina and nuclear matrix (Figure 1), thus making the chromosome more like a slice of pizza, rather than a noodle on a plate of spaghetti. Chromosomes are partially condensed by protein-DNA interactions (chromatin) that twist and loop portions of the chromosome. Through electron microscopy, three-dimensional DNA fluorescence in situ hybridization (FISH), and DNA tagging techniques (i.e., fluorescent and artificial DNA methylation), inactive domains of chromatin have been found to be packed tightly along the nuclear periphery4,5,6, while portions of active, less condensed chromatin are found in the interior of the nucleus7,8,9,10. These experiments provide a wide-angle view of chromosome dynamics but do little to capture the changes that occur locally around the gene promotors observed in DNase11,12 and nucleosome13,14,15 studies.
The key to unlocking higher-resolution chromatin dynamics was the formulation of the 3D chromosome mapping technique, 3C. The 3C technique itself comprises four main steps: crosslinking of chromatin, chromatin digestion by restriction enzymes, chromatin ligation, and DNA purification (Figure 2). The new artificial DNA fragments generated by this process can then be characterized to reveal the close physical association between linearly distant pieces of DNA16. The 3C technique became the basis for the creation of multiple spin-off techniques that utilize the initial steps of 3C to ask broader genome-wide questions (e.g., Hi-C, 4C, ChIP-C). This family of 3C techniques has identified that chromosomes are organized into multiple discrete units termed topologically associated domains (TADs). TADs are encoded in the genome and are defined by chromatin loops flanked by unlooped boundaries16,17,18,19. The TAD boundaries are maintained by two evolutionarily conserved and ubiquitous factors, including CCCT binding factor (CTCF) and cohesion, which prevent loops within separate TADs from interacting16,20. The loops are mediated by the interaction of trans-factors with their regulatory sequences, as well as CTCF and cohesion21.
Though many studies using 3C technologies ask broad genome-wide questions and employ complicated sample collection techniques, the formulation of the 3C technique is based on basic molecular biology techniques. This makes 3C intriguing for deployment in both undergraduate research and teaching labs. The 3C technique can be employed for smaller focused questions and is inherently flexible to scaling up or down (single genes22, chromosomes16, and/or genomes18) depending on the focus and direction of the questions asked. This technique has also been applied to a wide range of model systems7,16,19,23 and has been proven to be versatile in its use. This makes 3C an excellent technique for undergraduates in that students can gain experience in common molecular biology techniques while also gaining valuable experience in answering directed questions.
Presented here is an adapted protocol for 3C library preparation based on previously published protocols24,25,26,27. This protocol has been optimized for approximately 1 &#215; 107 cells, though it has generated 3C libraries with as little as 1 &#215; 105 cells. This protocol has proven to be versatile and has been used to generate 3C libraries from zebrafish embryos, zebrafish cell lines, and young-adult (YA) Caenorhabditis elegans (roundworm). The protocol should also be appropriate for mammalian cell lines and, with further adaptation, yeast.
The goal of these adaptations is to make 3C more accessible for undergraduates. Care has been taken to use techniques that are similar to those that can be accomplished in an undergraduate teaching laboratory. The 3C technique provides many learning opportunities for undergraduates to learn basic molecular biology techniques that will benefit their development at the bench, in the classroom, and in their endeavors after graduation.

Autor im Rampenlicht: Mit den 3Cs eine Eins bekommen: Erfassung der Chromosomenkonformation für Studenten  

Eine Eins mit den 3Cs bekommen: Erfassung der Chromosomenkonformation für Studenten

Watch this Scientific Journal Video about Identification of the Chromosome Conformation Capture 3C Products at JoVE.com

Identification of the Chromosome Conformation Capture (3C) Products  

Identifizierung der Produkte zur Erfassung der Chromosomenkonformation (3C)

Nehmen Sie die Chromosomenbestätigungserfassung oder die 3C-Probe und die Kontrollen und führen Sie eine QPCR durch. Entfernen Sie die Produktbanden, die der erwarteten Fragmentgröße entsprechen. Um die Gelextraktion der PCR-Produkte durchzuführen, verwenden Sie ein handelsübliches Kit oder befolgen Sie das hausgemachte Protokoll.Konstruieren Sie für letzteres eine selbstgemachte Reinigungskartusche, indem Sie mit einer Nadel ein Loch in den Boden eines 0,5-Milliliter-Röhrchens stechen. Packen Sie eine kleine Menge Watte in den Boden der Tube mit dem Loch und füllen Sie nicht mehr als die Hälfte der Tube. Legen Sie das Röhrchen in ein 1,5-Milliliter-Röhrchen und achten Sie darauf, dass das kleinere Röhrchen auf dem Rand des größeren Röhrchens und nicht im Röhrchen aufliegt.Schneiden Sie das Gelfragment vorsichtig in kleinere Stücke und legen Sie es in das kleine Röhrchen der Kartusche. Legen Sie die Baugruppe für fünf Minuten in einen Gefrierschrank bei minus 20 Grad Celsius. Schleudern Sie die Baugruppe dann drei Minuten lang bei 13.000 G und Raumtemperatur.Entsorgen Sie das kleine Röhrchen mit den Agoros-Trümmern und bewahren Sie das 1,5-Milliliter-Röhrchen mit der extrahierten DNA im Puffer auf. Die Proben wurden auf das Vorhandensein von langreichweitigen Chromatinkontakten zwischen den verschiedenen genomischen Loci getestet, wobei Kombinationen aus Loci-spezifischen Primern und QPCR verwendet wurden. Die Produkthäufigkeit wird relativ zu einem Kontrollprimer-Set berechnet und zum Vergleich gepfropft.Die Daten zeigten, dass 8 der 10 Reaktionen bedingt positiv waren. Die Gelaufreinigung und die Sequenzierung des erwarteten PCR-Produkts für die acht bedingt positiven und eine negative Reaktion wurden gelgereinigt und sequenziert. Die Ergebnisse für repräsentative positive und negative Reaktionen werden dargestellt.

identification-of-the-chromosome-conformation-capture-3c-products

Research

JoVE Journal

Genetics

Identification of the Chromosome Conformation Capture (3C) Products

502 Aufrufe. Nehmen Sie die Chromosomenbestätigungserfassung oder die 3C-Probe und die Kontrollen und führen Sie eine QPCR durch. Entfernen Sie die Produktbanden, die der erwarteten Fragmentgröße entsprechen. Um die Gelextraktion der PCR-Produkte durchzuführen, verwenden Sie ein handelsübliches Kit oder befolgen Sie das hausgemachte Protokoll.Konstruieren Sie für letzteres eine selbstgemachte Reinigungskartusche, indem Sie mit einer Nadel ein Loch in den Boden eines 0,5-Milliliter-Röhrchens...

Serie: Identification of the Chromosome Conformation Capture (3C) Products  

Getting an A with the 3Cs: Chromosome Conformation Capture for Undergraduates

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C techniques) that provide detailed information of the three-dimensional organization of chromatin. The 3C techniques have been used in a wide range of studies, from monitoring the changes in chromatin organization in cancer cells to identifying enhancer contacts made with gene promoters. While many of the studies using these techniques are asking big genome-wide questions with intricate sample types (i.e., single-cell analysis), what is often lost is that the 3C techniques are grounded in basic molecular biology methods that are applicable to a broad range of studies. By addressing tightly focused questions of chromatin organization, this cutting-edge technique can be used to enhance the undergraduate research and teaching lab experience. This paper presents a 3C protocol and provides adaptations and points of emphasis for implementation at primarily undergraduate institutions in undergraduate research and teaching experiences.

Here, we present an adaption of the chromosome conformation capture (3C) technique in detail with an emphasis on undergraduate involvement and learning.

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C ...