Active transcription factor detecting method and kit

Abstract

The active transcription factor detecting method and kit is the improvement of ELISA detection method, and features that the new double-stranded oligonucleotide probe is constructed to overcome the demerits of available ELISA detection method in double-stranded oligonucleotide probe design. Experiment shows that the detection method of the present invention has very high sensitivity and detection specificity, and the designed probe is suitable for transcription factor detection. The combined sequence probe detection effect with one copy and that with two copies are the same. The present invention also relates to the kit for the transcription factor detection method.

Description

technical field [0001] The invention belongs to the field of biochemistry and molecular biology, more specifically it is a transcription factor activity detection method, and the invention also relates to a kit used in the transcription factor activity detection method. Background technique [0002] Nucleic acid sequences that regulate gene expression on the same nucleic acid chain are called cis-acting elements, including promoters, enhancers, attenuators, etc.; transcription factors are also called trans-acting factors, which refer to acting on cis-acting elements to A protein capable of regulating gene expression on different nucleic acid strands. For a transcription factor to function, it must have a certain activity, which depends on the number of transcription factors and their binding affinity to cis-acting elements. Detecting the activity of transcription factors is of great significance for elucidating the expression regulation mechanism of a certain gene, so it is...

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Problems solved by technology

As long as the cell transfection efficiency is high, this method is still relatively sensitive, and with the aid of a fluorescent microplate reader, large-scale detection can also be performed at the same time; but in this case, other transcription factors may also affect the expression level of the reporter gene, The specificity of the reaction is affected; moreover, since the activity of the transcription factor is reflected indirectly through the activity of the expressed luciferase, it is bound to be affected by downstream processes such as transcription, translation, etc.

3. In situ detection of nuclear translocation of transcription factors with antibodies, but this is a qualitative method and not suitable for large-scale detection

Fourth, for some transcription factors, such as NF-κB, western blot can also be used to detect the degradation of its cytoplasmic inhibitory protein (IκB), which indirectly reflects the activity of transcription factors and is not suitable for large-scale detection

The preparation process of the whole probe is more complicated and expensive (because of the difficulty of the short-chain PCR reaction, the length of the random sequence is generally about 100bp, and the length of the whole probe sequence is about 140bp, and the synthesis of the long-chain oligonucleotide probe is not only It is much more expensive, and many domestic DNA synthesis companies cannot guarantee the quality, and the purification of short-chain DNA probes still needs some imported special purification devices), and the kits developed abroad (such as Active Motif) are expensive, which limits the domestic development, Popularize this technology for scientific research

Even so, the probe is not effective in reducing non-specific binding between the transcription factor of interest and the linker sequence, which can lead to increased background in detection

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