Chat GPTに聞いてみました。
【質問】
according to manufacture's instruction, guanine at 5' of taqman probe shoud be avoided. why?
ChatGPT
【回答】
Avoiding guanine (G) at the 5' end of a TaqMan probe is recommended because it can interfere with the probe's fluorescence and overall performance. Here are the key reasons:
Quenching Effects: Guanine is known to be a strong quencher of fluorescence. When located at the 5' end of the probe, it can quench the reporter dye, reducing the fluorescence signal and thus the sensitivity and accuracy of the assay.
Energy Transfer: Guanine can affect the energy transfer between the fluorophore and the quencher. Proper energy transfer is crucial for the probe to work effectively, and the presence of guanine can disrupt this process.
Structural Stability: The presence of guanine at the 5' end might affect the structural stability of the probe. The interaction between the guanine base and the fluorophore can lead to changes in the probe's conformation, impacting its binding efficiency and specificity.
Efficiency of Cleavage: Taq polymerase needs to cleave the probe during the PCR process to separate the reporter dye from the quencher. The presence of guanine at the 5' end can hinder this cleavage, affecting the probe's overall efficiency.
By avoiding guanine at the 5' end, the performance and reliability of the TaqMan probe in real-time PCR assays are optimized. |
|
このスレッドをはてなブックマークに追加