Epstein-Barr virus (EBV) is associated with various types of malignancy, in particular, nasopharyngeal carcinoma (NPC). The viral oncogene, latent-membrane protein 1 (LMP-1), is known to transform B-lymphocytes and rodent fibroblast in vitro. It is thought to protect the infected cells from apoptosis by up-regulating the bcl2 and A20 genes.1 LMP-1 gene consists of three exons separated by short introns (Fig. 1).2 One of our investigation efforts attempted to study the possibility of using the LMP-1 protein as an antigenic marker for early progression of NPC.
Fig. 1: Schematic representation of LMP-1 gene. The three exons (EA, EB and EC) are separated by two short introns (I-1 and I-2). Arrows indicate the position of the forward and reverse primers for each exon.
In vitro analysis of LMP-1 requires cloning and expression of this protein for use in antibody detection using ELISA. Each of the three exons of the LMP-1 gene was amplified separately using PCR3 for TA-cloning reactions. For this reason, primers flanking exactly the 5'-terminal and the 3'-terminal of each exon were synthesized in order that the cloned amplimer will be in-framed during subsequent clonings (Fig.1). In so doing, we were restricted to only one choice for each primer. Exons A and B were successfully amplified by their respective primers at the calculated annealing temperature using a conventional thermal cycler. However, this was not the case for Exon C. The properties of primers used for amplifying Exon C are tabulated in Table 1.
During optimization for primers CF and CR, we applied the general rule of starting with annealing temperatures around 5C below the calculated melting temperature (Tm ) and titrating for optimal MgCl2 concentrations. The Tm of both primers were recommended by the manufacturer (Operon, Inc., Alameda, CA, USA) calculated using the standard formula. Using a conventional thermal cycler, we were unable to amplify Exon C despite attempts over a range of annealing temperatures (52C to 65C) and MgCl2 concentrations (1 mM to 4 mM). This problem was solved when we used the temperature gradient function of the Eppendorf Mastercycler gradient.4,5
By performing a wild search for the correct annealing temperature, we subjected our amplification reaction using primers CF and CR (Table 1) over a temperature gradient of 20C (48C to 68C) at a constant MgCl2 concentration of 2 mM. Template EBV DNA was extracted from B95.8 marmoset lymphocytes cell line. PCR was carried out in 50 l reactions with the following 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.
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 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.