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Amplification of Epstein-Barr Virus Exon C with Difficult Primers ,,, Using Eppendorf Mastercycler gradient

of Eppendorf Mastercycler. A: DNA template was amplified using the primers CF and CR over an annealing temperature gradient from 48C to 68C. Specific band corresponding to the 825 bp product of Exon C was detected at 56C. No bands were observed at higher temperatures while spurious product of lower molecular weight product began to generate at temperatures below 54C due to unspecific primer binding. M: Generuler 100 bp Molecular Ladder Plus (Fermentas, Vilnius, Lithuania). B: Amplification of Exon C at annealing temperature of 56C using conventional thermal cycler (Lane 2) and Eppendorf Mastercycler (Lanes 3 and 4). Lane 1: Generuler 100 bp Molecular Ladder Plus (Fermentas). Lane 5: Generuler 50 bp molecular ladder (Fermentas).

At temperatures below 54C, lower molecular weight spurious product became evident. Amplification of Exon C was shown to be reproducible at the optimized annealing temperature of 56C using the Mastercycler gradient but not with the conventional thermal cycler (Fig. 2B). The optimized annealing temperature was far from that predicted using the standard formula for Tm. Although the annealing temperature of 56C was tried with the conventional thermal cycler using the same cycling parameters, no specific product was obtained. This suggests that the interaction between oligonucleotide primers and DNA polymers is a complex one, and factors such as temperature ramping are crucial. In this case, ramping was proven to be more efficient with the Mastercycler gradient.

References
  1. Kieff E. Epstein-Barr virus and its replication. In: Fields, B, Knipe D, and Howley P, eds. Fields Virology. Vol 2
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