Question

1.How is QPCR able to quantify the amount of template copies in a patient sample? Contrast to End Point PCR?

2. What are the necessary components for a PCR reaction?

3. How can you tell if there is contamination of your PCR samples?

Answer 1

1. QPCR uses fluorescent dyes that bind to double-stranded DNA and emit a light signal. This signal is quantified and used to measure the initial concentration of the template DNA in the sample. It is a real-time experiment. It measures the fluorescence values at each step enabling us to determine the concentration of the template below the saturation level. In normal endpoint PCR, we can not differentiate initial template concentrations if the reactions are saturated.

2. Components of PCR: dNTPs, Taq, Template, Primers, buffer, and Water.

3. If there is a contamination in the PCR reaction mix, we would observe non-specific bands in the gel.