Products can appear in non-template control (NTC) wells due to the ability of some primer sequences to form structures that can be used as substrates by DNA polymerases. The most common version of this problem is the “primer dimer”, wherein some small regions of complementarity in the two qPCR primers permit the production of an amplifiable structure. Many primer design tools will aim to eliminate this possibility, and to ensure that no primer can form a secondary structure within its own sequence. This type of amplification can be discerned by evaluating melt curve profiles, as a primer dimer or nonspecific type amplification will result in DNA products with a melt peak different from that of the correct qPCR product.
A second explanation for amplification in NTC wells is the presence of contaminating PCR products amplified in earlier qPCR experiments. This is a significant concern when the same assay is used repeatedly, and in particular when the reaction vessels are opened after the qPCR is complete. General sterile procedures can avoid this problem, such as not opening vessels after reactions or doing so in a satellite laboratory area with separate equipment and routine decontamination of laboratory space and equipment. Melt curves can be used to determine if the NTC amplification is caused by carryover contamination. If this is the case, the products in the NTC reactions will be the same species as the intended target and the melt profiles will match accordingly. If contamination is detected, then all reagents (primers, water, qPCR mix) should be replaced, equipment should be decontaminated with a 10% bleach solution, and sterile procedures should be used for future experiments. Use of 0.2 U/μL Antarctic Thermolabile UDG (NEB #M0372) can help mitigate carryover contamination for situations where it is a persistent problem or in particularly sensitive applications.
