<p>In presence of K103N-AAC (A1) and K103-AAT (A2) mutants, only the specific mutant amplicons will be amplified when using mutant specific forward (MSFP) and a common reverse primer (CRP), respectively. The total population of sequences in the reaction are amplified using non-specific forward primer (NSFP) and the CRP (A3). An intentional mismatch at the penultimate base (indicated with I) was introduced in the allele-specific real-time PCR primers in order to increase the specificity and minimize the risk for amplification of the wild type allele. Mutant specific (Sp) and non-specific (NSp) standard curves of K103N AAC allele (B1), K103N AAT allele (B2) and Y181C TGT allele (B3) are in parallel with each experiment. The copy number of each mutant specific and total population of sequences amplifications of clinical samples were determined using such standard curves that has been run in duplicate, parallel with each sample. The quantity of the patients' mutant specific and the total population of sequences (amplified with non-specific primer) was then determined by comparing the samples Ct value with those of the specific and non-specific standard curves derived from the standard plasmid controls using the corresponding primers. The percentage of mutant specific sequences was then determined by dividing the quantity of mutant specific sequence by the quantity of the total sequences and multiplying by 100. Positive samples were repeated at least twice. Correlation coefficients (r<sup>2</sup>) were higher than 0.99. Sp: mutant specific amplification. MSFP, mutant-specific forward primer; CRP, common reverse primer; NSFP, non-specific forward primer. NSp: non-specific amplification (amplify the total population of sequences).</p
