Genotyping mutations in RET exon 10 at codons 618 and 620. a: Derivative melting curve plot for the primary WT10B probe data from Figure 1e. Three wild-type controls in duplicate (WT, black traces) are shown with 10 heterozygous mutant samples in duplicate, five at codon 618 (thin traces) and five at codon 620 (thick traces). The colors used for mutant codon DNA sequences and traces are consistent throughout the figure. Graph traces for both the codon 618 and 620 mutation sequences are gray for AGC, light blue for CGC, red for GGC, dark blue for TAC, orange for TCC, green for TTC, and pink for TGG. The ΔTm range values used to generate the Tm guidelines are listed next to each Tm guideline. Wild-type allele Tms are within the black Tm guidelines, ±0.25°C of control wild-type sample. The mutant alleles detected by the primary probe were divided into two ΔTm ranges by the red Tm guidelines. The mutation sequences that are predicted to be grouped into each ΔTm range are listed below the traces. The secondary assay data for the MS probe sets, MS10B set 1 or MS10B set 2, are displayed for the two mutation groups. b–e: The mutation group with ΔTm values of 3.3°C to 5°C was tested with the four probes in MS10B set 1: MS 618/620 TAC (b), MS 618/620 TCC (c), MS 618/620 TTC (d), and MS 618/620 TGG (e). f–h: The mutation group with ΔTm values of 0.25°C to 3.3°C was tested with the three probes in MS10B set 2: MS 618/620 AGC (f), MS 618/620 CGC (g), and MS 618/620 GGC (h). On each mutation-specific probe graph (b–h), the codon mutation DNA sequence that complements the MS probe is listed. i: All mutant alleles (primary WT10B probe data, ΔTm of 0.25°C to 5°C) will also use the location probe L10B 620mask to locate codon position of the detected mutations. Two derivative melting temperature ranges are indicated, with the codon 618 mutant alleles (618 MUT) labeled as well as the WT allele and masked codon 620 mutant alleles (620 MASK).