The polymerase chain reaction can be performed using both manual and automated methods.
Manual PCR, also referred to as conventional PCR, is time consuming and requires manual manipulation. Each step of the protocol involves multiple transfers of sample, reagent, and consumables i.e. pipette tips, tubes, gloves, etc. Once the PCR product is obtained, secondary analysis, such as a gel electrophoresis, is needed for detection. Definitive identification often requires genomic sequence analysis or other alternative methods. Thus, conventional PCR is not ideal for rapid diagnostic use in most clinical laboratories.
Real-time PCR is an automated technique that allows for detection and quantification of the target material. Results are generated at the same time that the assay is being run, hence the designation "Real-time."
Though both conventional PCR and Real-time PCR follow a similar procedure, there are many advantages to real-time PCR:
- The ability to monitor the progress of the PCR reaction (amplification) as it occurs in real time.
- The ability to precisely measure the amount of amplicon at each cycle, which allows highly accurate quantification of the amount of starting material in samples.
- Provides a dynamic range of detection (from as little as a single strand of target to millions of copies in a sample).
- Amplification and detection occurs in a single tube, eliminating post-PCR manipulations.
