Simultaneous amplification of exons 18 to 21 of the EGFR gene using 5' tailed primers and a two-stage protocol.

OBJECTIVE: Reduction of non-specific amplification and achievement of efficient amplification of multiple gene fragments under the same reaction condition is the basic goal of PCR diagnosis; however, this is often difficult. This study was conducted to establish a highly specific and effective amplification of the epidermal growth factor receptor (EGFR) gene's exons, 18-21, simultaneously.

METHODS: The 5'-tailed primers were synthesized by adding 10 to 20 bp of a non-specific sequence to the 5'-terminus of sequence-specific primers (tailless primers). The two-stage protocol consisted of 5-10 cycles of a conventional 3-step cycling, which was then followed by 30-35 cycles of two-step cycling. The exons 18-21 of EGFR gene were amplified in 28 non-small cell lung cancer (NSCLC) patients using an optimized PCR that combined 5' tailed primers with a two-stage protocol.

RESULTS: The 5' tailed primers exhibited a wider range of suitable annealing temperatures, similar range of primer concentration, similar sensitivity, specificity, and reproducibility, as well as a reduced, non-specific amplification compared with the corresponding tailless primers. The amplification of exons 18-21 of EGFR gene in NSCLC patients revealed that a combination of 5' tailed primers with two-stage protocol (optimized PCR) had a similar PCR success rate (P = 0.873) but had significantly reduced non-specific amplification (P <0.001) compared to conventional PCR.

CONCLUSION: 5' tailed primers exhibited a wider range of suitable annealing temperatures and improved specificity compared with conventional PCR primers. An optimized PCR was established with 5' tailed primers and a two-stage protocol to amplify exons 18-21 of the EGFR gene in NSCLC patients.