Fig. 1.

Diagram of the proposed two-step strategy. In the first step, template DNA (A) is subjected to PCR amplification (panel 1) by the use of target-specific primers (red), one of which is modified to contain a “linker” sequence (dark blue). The product of the first step (B) is cleaned to remove the initial primers and subjected to a short period of PCR amplification (panel 2) by the use of a primer composed of the linker sequence and a nucleotide tag (purple). The resulting product (C) is then ready for sequencing. In this specific instance, 454 sequencing technology was used, and appropriate adapter sequences (light blue and yellow) were included in the primers as required.