The PCR technique is one of the most powerful technologies available to molecular biologists. Targets within DNA or cDNA templates are amplified many thousands or millions of times using PCR. This allows the researcher to isolate a portion of a nucleic acid of interest in sufficient quantities for experimental analysis.
The PCR process is carried out through repetition of the three steps that define a single PCR cycle: DNA template denaturation; annealing of primer to DNA template; enzymatic extension of primer to produce copies that can serve as template in subsequent cycles (see figure below).
The PCR process
What products are used during the amplify step?
Problems with PCR?
PCR is not perfect. Each step of the cycle should be optimized for each template and primer pair combination. The most important considerations for any PCR reaction are:
- PCR enzymes—enzyme selection can affect the yield and specificity of PCR products. Get the basics on how to select the right PCR enzyme for your application
- Primer design—optimize your primer design using our custom oligo design tool
- Magnesium concentration—the correct magnesium concentration needs to be determined empirically for each primer and template combination you have; view this webinar on PCR optimization to help you determine the magnesium concentration
- Annealing temperature—the appropriate annealing temperature is based on the calculated Tm of your primers; watch a video on better than gradient to optimize your annealing temperature