Journal: BMC Research Notes
Article Title: False negative results from using common PCR reagents
Figure Lengend Snippet: Primer-dimers and UNG degradation products can inhibit PCR amplification . a) The negative control reactions (water and PCR reagents, but no DNA) from the mouse mitochondrial DNA PCR and the PSMA transgene PCR were diluted from 10 -2 to 10 -5 . Both sets of dilutions were used to contaminate a PCR for the PSMA transgene. All reactions were amplified with the ABI Gene Expression Master-mix (containing UNG). b) The same experiment was conducted except the NEB Taq Polymerase and buffer was used (no UNG in the PCR for both generation of the primer-dimers and subsequent PCR). NEB Taq is less efficient than ABI Taq at amplifying the target for the lower band in the PSMA transgene PCR, however inhibition by contaminating primer-dimers is the same regardless of PCR mix. c) The PSMA transgene PCR product and negative control were amplified with the ABI Gene Expression Master-mix and gel purified to remove primer-dimers. These products as well as non-purified PSMA transgene PCR product and negative control (that contain primer-dimers) were used to contaminate a subsequent PSMA transgene PCR. The results indicate that amplification of legitimate PCR target is significantly inhibited by contamination with previously generated negative control reactions using the same primers, regardless of the presence or absence of UNG. It also indicates that the UNG degradation products can weakly inhibit amplification and the UNG degradation products and primer-dimers can completely inhibit PCR amplification.
Article Snippet: Sixty nanograms of transgenic mouse DNA was amplified in 25 μL reaction containing 0.5 U Taq DNA Polymerase, 10× ThermoPol Reaction Buffer (New England BioLabs, Inc.), 0.4 mM dNTP (dATP, dTTP, dCTP, and dGTP), 1 μM S49, 1 μM INTA, 1 μM S1368, and 1 μM AS2015.
Techniques: Polymerase Chain Reaction, Amplification, Negative Control, Expressing, Inhibition, Purification, Generated