Figure 1.

Design methodology. A: Each synthetic reference standard is designed to be amplified with a single primer pair that anneals to each primer tag region. Because the oligonucleotides are single stranded, the first round of PCR amplification generates a complementary strand for each synthetic oligonucleotide. B: After this initial cycle, the remaining PCR cycles amplify the DNA fragments in a standard manner. Reference oligonucleotides are designed for each mutation of interest. In some cases, more than one mutation may be incorporated within the same reference oligonucleotide (red circles). C: Assorted mixtures of the various reference oligonucleotides are combined and amplified using a single primer pair to produce synthetic controls carrying one or dozens of mutations that may be visualized in a single reaction tube.