qPCR

 

Quantitative real-time PCR technologies (what is Taqman qPCR):

 

https://www.ncbi.nlm.nih.gov/probe/docs/techqpcr/

Quantitative real-time PCR (qrt-PCR) is a good option to generate ‘real-time’ data regarding the presence of harmful algal species. The major goal of a qrt-PCR project is to design and test a qrt-PCR assay that will accurately identify and estimate marine algal species. 

Quantification is performed during the exponential phase of the PCR, where amplification efficiency is maximum. In real-time PCR, amplicon formation is monitored after each cycle by measuring a fluorescence signal.

An amplicon is a piece of DNA or RNA that is the source and/or product of natural or artificial amplification or replication events. It can be formed using various methods including polymerase chain reactions (PCR) or natural gene duplication.

In qrt-PCR, the increase in fluorescence observed during the reaction will be proportional to the starting quantity of the target molecule. Fluorescence can be generated by using fluorescent probes such as TaqMan®. Since there is a correlation between the cycle number at which the amplicon is initially detected (threshold cycle, Ct) and the starting amount of target molecules, it is possible to calculate the amount of target sequence in an unknown sample by using a standard reference curve generated using DNA extracted from a known number of cultured cells.