Why are qPCR amplicon about 100bp? Can PCR products improve the sensitivity of PCR?
PS: I do amplify the 16S of environmental sample.
Because shorter products usually are amplified with higher efficiency. Higher efficiency = more stable amplification process = less variation = more precision.
To make sure (as far as possible) that the products are amplified with ideal efficiency. If the amplification efficiency were suboptimal, small variations in reaction conditions might cause small variations in the actual amplification efficiency and, hence, cause relatively large variations in ct values. Reactions running under ideal conditions are more stable/robust, and so are the results.
Why are qPCR amplicon about 100bp? Can this PCR products improve the sensitivity of PCR?
Actually not. High sensitivity = ability to detect low concentrations of the analyte (the target molecule). A PCR can amplify a single molecule to a measurable/detectable amount, and this is possible even when the reaction is not perfectly optimized. So sensitivity is not a major problem of PCR at all (there are some exceptions for sure, but these are exceptions). For qPCR, there is even another reason why sensitivity is not relevant: in regions, where the sensitivity might play a role (say less than 100 molecules per sample), the precision is very bad (-> Poisson distribution!) so that a quantification is anyway very imprecise. Additionally, the amplification of unspecific products may obscure results in these cases. Because the ct values are high, any minor variation in amplification efficiency will have a drastic effect on the ct values, too. Therefore, real-time qPCR is not a good tool to quantify such tiny amounts (few molecules), where sensitivity could be an issue.
Yes, you are right. But I want to know that can I design the primers which can amplify the more than 100bp, even 400-500bp. Some people said that the primers for qPCR which can amplify 100-200bp are better. But I do not know why.
Because shorter products usually are amplified with higher efficiency. Higher efficiency = more stable amplification process = less variation = more precision.
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