Optimizing Blocking Primer Effectiveness

We applied fragment analysis to test and compare the effectiveness of our blocking primers^{35 (link)}. Fragment analysis of fluorescently labeled PCR products on capillary electrophoresis can separate fragments in different sizes and can be used as a semi-quantitative method. When amplified with the universal LCO1490 and HCO2198 primers, the expected lengths of PCR products were different for amphipod hosts and Rickettsia COI, because Rickettsial COI is 6 bp longer. The FAM dye was attached to the 5′ end of the LCO1490 primer. This fluorescently labeled forward primer was added to the PCR mixture instead of the normal (unlabeled) LCO1490 primer. Various factors can affect PCR success with blocking primers: Tm of primers, the concentration of primers (relative ratio between blocking primer and regular primer), the amount of the DNA templates in a PCR mixture (concentration of DNA), and the number of PCR cycles^{35 (link)}. To optimize PCR conditions, PCR reactions were conducted under several different PCR conditions (Table 1). Fragment analyses were carried out with a 1,200 LIZ size marker on an ABI 3730xl System (Applied Biosystems) at Macrogen (Korea). Results were analyzed with Peak Scanner Software 1.0 (Applied Biosystems; https://www.thermofisher.com/).