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

ollowing final concentration of each component: 200 M of each dNTP, 100 ng template DNA, 1X reaction buffer, 2 mM MgCl2 and 1.25 U Taq Polymerase. Amplification was carried out using the Mastercycler gradient under the following conditions: Initial denaturation at 94C for 3 minutes followed by 30 cycles of denaturation at 94C for 30 seconds, annealing temperatures ranging from 48C to 68C for 30 seconds, extension at 72C for 1 minute. Final extension at 72C was carried out for 3 minutes. Analysis of the amplified product was performed with two tenths of the reaction mixture on a 0.8% (w/v) TBE agarose gel stained with ethidium bromide.

Results

As mentioned by the experts in Current Protocols in Molecular Biology, primer selection is a factor that is least predictable and most difficult to troubleshoot. Simply put, some primers just do not work.6

The CR primer (Table 1) contains stretches of polypurines and polypyrimidines that are prone to form secondary structures that inhibit proper primer function. This, coupled with the rarity of total EBV genome in the total cellular DNA extract and the fact that the LMP-1 gene constitutes only 1% of the viral genome, made the task of amplifying Exon C difficult if not impossible using a conventional thermal cycler. With a switch to the Eppendorf Mastercycler gradient and applying its temperature gradient capability, the specific band (825 bp) for LMP-1 Exon C was successfully amplified at an annealing temperature of 56C (Fig. 2A). No amplimers were detected at 54C and at temperatures above 56C.



Fig. 2: Amplification of EBV LMP-1 Exon C using the gradient function
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