What factors affect the specificity of PCR?

Factors that affect the specificity of PCR include:

• Length of the primer
• Purity of the template DNA
• Low concentrations of template DNA
• Purity and sequence of the primer
• Annealing temperature of the PCR reaction
• Mg²⁺ concentration
• Additives such as dimethyl sulfoxide (DMSO)
• Amounts of PCR inhibitors in the sample such as excessive proteins, SDS, phenol/ethanol, or hemoglobin
• Inaccurate sample or reagent pipetting particularly when doing serial dilutions