The principle of ChIP technology
Under physiological conditions, the DNA in the cell is cross-linked with the protein, and after the chromatin is cut into small fragments by ultrasound or enzymatic treatment, the DNA fragment which binds to the target protein is precipitated by the specific recognition reaction of the antigen antibody. To enrich the DNA fragment with histone modification or transcriptional regulation, and then detect the DNA sequence of the enriched fragment by various downstream detection techniques (quantitative PCR, gene chip, sequencing, etc.) . (Detection related technical services: Real-time PCR technology services , gene chip and results validation services , high-throughput sequencing and results validation services )
ChIP technology experiment steps
Chromatin immunoprecipitation involves three separate steps, immobilization, precipitation, and detection.
Step 1 can be effectively secured <br> formaldehyde protein - protein, protein-DNA-protein crosslinking -RNA form a biological complex, to prevent redistribution of intracellular constituents. First, fix the DNA and protein complex with formaldehyde in the body, pay attention to the crosslinking time of methanol. If it is too long, the cell chromatin is difficult to be broken by ultrasonic wave, which affects the ChIP result, and the experimental material is also easily lost during centrifugation; the crosslinking time If it is too short, the cross-linking is incomplete and a false negative is produced. The crosslinking reaction can be terminated by the addition of glycine. Then, it is randomly cut into small pieces of chromatin (200-1000 bp) of a certain length by chemical (micrococcal enzyme) or mechanical (ultrasonic) means.
Step 2 <br> immunoprecipitation using specific antibody against the tag on the protein or a protein, a protein formed by the DNA- and antigen-antibody reaction - antibody complex, and this complex precipitate, enriched protein specifically binds DNA fragment.
Step 3 Purification and detection of the fragment and crosslinking <br> heat treated, the co-precipitated DNA is released; after DNA fragment was purified and then, precipitated DNA sample to be detected. There are currently three main detection methods: the first one is a common PCR experiment comparing the precipitation template with the negative and positive control PCR signal intensity, or a relatively accurate quantitative PCR method. The second is to hybridize the precipitated DNA to a DNA microarray chip (ChIP-on-ChIP) to detect the entire interaction of the polygenic trajectories. The third is high-throughput DNA sequencing analysis.
Advantages of ChIP technology
Compared to traditional methods of studying transcription factor and DNA interaction, chromatin immunoprecipitation is an ideal method for studying DNA-protein interactions in vivo. The advantage of ChIP is that it can capture the interaction between transcription factors and target genes in vivo, and can provide one or more gene regulation mechanisms at the same time, so it has great application value.
Limitations of ChIP technology
First, the technology requires highly specific antibodies against the protein of interest or a specially modified tag.
Second, false negative signals may result from ineffective antibody binding or antigen interference during cross-linking.
Third, formaldehyde fixation may be temporary or even non-specific, which may result in false positive signals from adjacent proteins.
Fourth, it is difficult to obtain information on the binding of a plurality of proteins to the same sequence at the same time.
For the first three aspects, it is recommended to draw a standard curve after cross-linking and before antibody precipitation to determine the optimal amount of chromatin required for each experiment to ensure that the starting amount of material is equal. When the detected chromatin template (chromatin immunoprecipitate of the antibody of interest) amplifies a detectable band, the chromatin sample is serially diluted to determine the key point; at this time, the control (mockChIPed) chromatin template needs to be concentrated to A visible band is amplified. In summary, the step of chromatin immunoprecipitation is required to be precise and requires a series of pre-experiments.
Application of ChIP technology
The ChIP method can study the synergistic binding of DNA methylation, chromatin structure, histone modifications, and transcription factors, or identify direct target sites from predicted target genes. The combination of ChIP technology and other molecular biology techniques (eg, general PCR, real-time quantitative PCR, gene cloning, DNA microarrays, or more direct high-throughput sequencing techniques) has been used to determine transcription factor and DNA interactions or transcription factors. New genomic target sites.
Originally, ChIP was applied to the study of mammalian, yeast, and Drosophila chromatin; later, it was applied to the study of related transcription factors and modified histone positions. ChIP technology can also be used to study the interaction between proteins and proteins, such as the interaction between DELLA and PIF3 proteins by yeast two-hybrid and ChIP.
With the further improvement of ChIP technology, it will play an increasingly important role in the study of gene expression regulation. Because the ChIP experiment involves many steps and the repeatability of the results is low, the researchers should design corresponding controls for each step of the ChIP experiment process, and the analysis of the results also requires some experience. For beginners who are just beginning to use ChIP technology, the use of mature commercial kits and related technical services can achieve twice the result with half the effort. ( Related kit: Pierce Agarose ChIP Kit , related technical services: Chromatin immunoprecipitation (CHIP) technical service )
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