Figure 1.
Design of primers used in the study.
A WT allele-specific primer forward primer (Upper), a mutant allele-specific forward primer (Lower), and a common primer were designed. The 3′ end of the forward mutant primer was specific to the mutant site (G to T) and an internal mismatch at the second nucleotide from 3′ end (G to A) was introduced to improve specificity.
Table 1.
Sequence of allele-specific primers used for this study.
Table 2.
Analysis of genomic DNA samples.
Table 3.
Primer sequences for making PCR amplicons of FFPE samples.
Figure 2.
A. Melting curve constructed using WT allele-specific primers. B. Melting curve constructed using mutant allele-specific primer set.
Figure 3.
Standard curve showing linearity of quantitative allele-specific PCR.
A standard curve was generated by serial dilution of WT or G17V cDNA that had been subcloned into pBluescript. A. Serial dilution of pBS/mutRHOA. Black dots correspond to 1.0×10−9∼1.0 unit of mutant plasmid (duplicate samples). The titration slope is −3.550 and R2 is 0.996. B. pBS/mutRHOA was mixed with pBS/wtRHOA at 100%, 10%, 1.0%, 0.1%, 0.01% and 0%. Mix concentrations were adjusted to 1.0 ng/well and diluted 1∶10 4 times for quantitative PCR analysis with allele-specific mutant primers. Horizonal axis indicates the amount of DNA per well. Vertical axis indicates unit for each sample. Black dot, MUT 100%; open dot, MUT 10%; square, MUT 1%; open square, MUT 0.1%; diamond, MUT 0.01%; triangle, MUT 0% (WT 100%) C. Serial dilution of pBS/wtRHOA. Black dots correspond to 1.0×10−6∼1.0 unit of WT cDNA (duplicate samples). The titration slope is −4.256, and R2 is 0.998. D. pBS/wtRHOA was mixed with pBS/mutRHOA at 100%, 10%, 1.0%, 0.1%, 0.1% and 0%. Mix concentrations were adjusted to 1.0 ng/well and diluted 1∶10 4 times for quantitative PCR analysis with WT allele-specific primers. Black dot, WT 100%; open dot, WT 10%; square, WT 1%; open square, WT 0.1%; triangle, WT 0% (MUT 100%).
Figure 4.
qAS-PCR of AITL and PTCL-NOS samples.
A, Shown are [mut]/([wt]+[mut]) values for each sample. N, mutation negative determined by MiSeq; P, mutation positive determined by MiSeq; Amp, amplified; PLP, periodate/lysine/paraformaldehyde-fixed; FFPE, formalin-fixed/paraffin-embedded. B, Comparison of [mut]/([wt]+[mut]) values by qAS-PCR and mutant allele frequencies as determined by MiSeq for 95 original or whole genome-amplified DNA samples, including 43 AITL and 52 PTCL-NOS. Cut-off values were determined as 1.5×10−2 for [mut]/([wt]+[mut]) by qAS-PCR and as 0.02 for mutant allele frequencies as determined by MiSeq. C, Comparison of [mut]/([wt]+[mut]) values by qAS-PCR and mutant allele frequencies as determined by MiSeq for 95 DNA samples in a log scales. D, Comparison of [mut]/([wt]+[mut]) values by qAS-PCR and mutant allele frequencies as determined by MiSeq for 13 FFPE PCR-amplicon samples.
Table 4.
Correlation between qAS-PCR and MiSeq.
Figure 5.
qAS-PCR for 275 tumor and control samples.
qAS-PCR was performed for tumor samples, including 43 AITL (a), 52 PTCL-NOS (b), 5 T-cell lymphoma other than AITL and PTCL-NOS (c), 19 B-cell lymphomas (d), 129 myeloid malignancies (e) and 27 control samples (f).