Of course they are amplified. Why shouldn't they? But qPCR is all about efficiency. You are not interested in the product but in the process of amplification that tells you something about the starting copy number. Therefore, you want to have targets that are short and are easily amplified.
Real-time polymerase chain reaction (real-time PCR) is a widely used method for amplifying and quantifying short DNA fragments, typically less than 200 base pairs (bp) in length. This method is based on the simultaneous amplification and detection of DNA during the PCR reaction, and it is commonly used in a variety of applications, including gene expression analysis, pathogen detection, and genetic typing.
Products larger than 200 bp are typically not amplified effectively in real-time PCR for several reasons:
PCR specificity: Real-time PCR is based on the selective amplification of a specific target DNA region using specific primers. Longer DNA fragments are more likely to contain non-specific primer binding sites, leading to the amplification of non-target DNA.
Efficiency of amplification: Real-time PCR is designed to amplify small DNA fragments very rapidly, and the rate of amplification decreases as the length of the DNA fragment increases. This can lead to decreased sensitivity and increased variability in the results when amplifying larger DNA fragments.
Detection limitations: Real-time PCR uses fluorescent dyes or probes to detect the amplified DNA during the reaction. These dyes and probes are optimized for short DNA fragments, and their performance may be reduced when detecting longer fragments.
For these reasons, it is generally not recommended to use real-time PCR to amplify products larger than 200 bp. If longer fragments need to be amplified, alternative methods such as standard PCR or long-range PCR may be more appropriate.