K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific to nonspecific primer-template binding over a wide temperature range.
The PyroMark PCR Kit ensures greater amplification efficiency with high specificity. This results in [A] higher Pyrogram peaks and more reliable Pyrosequencing results, compared with [B] standard PCR.
Template DNA (10 ng) was amplified with the PyroMark PCR Kit in parallel with PCR master mixes from Suppliers R, I, and AII. Primers specific for Cyclin A1, BARHL1, MINT1, or DNMT3b were designed with the PyroMark Assay Design Software 2.0, and an annealing temperature gradient ranging from 48.4°C to 60.1°C was used. The PyroMark PCR Kit consistently produced higher amplicon yields and fewer nonspecific PCR products than the other master mixes, ensuring reliable results from downstream Pyrosequencing analyses. M: 50 bp ladder
[A] CoralLoad Concentrate contains [B] 2 gel-tracking dyes, enabling immediate gel loading of PCR products for easy visualization of DNA migration.