Table 2.

Primers and Probes

RET exon	Primers*	Amplicon (bp)	Codon of variation†	Probes‡	Probe sequence§
10	0.275 μmol/L, 1F:9R	146	609	WT10A	GGCTATGGCACCTGCAACTGCTTCCCTGAG
	GGGCAGCATTGTTGGGGGAC		611	L10A 611mask¶	GGCTATGGCACCTGCAAC---TTCCCTGAG
	TGGTGGTCCCGGCCGCCA			MS 609/611 TAC∥	GGCTATGGCACCTACAACTACTTCCCTGAG
			618	WT10B	GGAGAAGTGCTTCTGCGAGCCCGAAGACATC
			620	L10B 620mask¶	GGAGAAGTGCTTC---GAGCCCGAAGACATC
				MS 618/620 TAC∥	GGAGAAGTACTTCTACGAGCCCGAAGACATC
11	0.55 μmol/L, 5F:1R	109	630	WT11	TGCGATCACCGTGCGGCACAGCTCGTCGCAC
	TGCCAAGCCTCACACCAC		631	L11 634mask¶	TGCGATCACCGTGCG---CAGCTCGTCGCAC
	GACAGCAGCACCGAGAC		634	MS 630/634 TAC∥	TGCGATCACCGTGCGGTACAGCTCGTCGTAC
13	1.1 μmol/L, 1F:9R	187		WT13	CCCGAGTGAGCTTCGAGACCTGCTGTCAGA
	CTGCATTTCAGAGAACGCCTC		768	Mask 1bp 769**	CCCGAGTGAGCT-CGAGACCTGCTGTCAGA
	GAACAGGGCTGTATGGAGC		769	MS 768 GAC + mask**	CCCGAGTGACCT-CGAGACCTGCTGTCAGA
				MS 768 GAT + mask**	CCCGAGTGATCT-CGAGACCTGCTGTCAGA
14	0.55 μmol/L, 1F:5R	235		WT14A	CTCCTCCTCATCGTGGAGTACGCCAAA
	CAGGGCCCCCTCTCTCCGC		804	MS 804 TTG	CTCCTCCTCATCTTGGAGTACGCCAAA
	TCTCGGCCAGATACTGCAT		806	MS 804 ATG	CTCCTCCTCATCATGGAGTACGCCAAA
				MS 804 CTG	CTCCTCCTCATCCTGGAGTACGCCAAA
			844	WT14B	GAGCGGGCCCTCACCATGGGCGACCTCATCTCA
			852	MS 844 CTG	GAGCTGGCCCTCACCATGGGCGACCTCATCTCA
				MS 852 ATG	GAGCGGGCCCTCACCATGGGCGACCTCATGTCA
16	0.55 μmol/L, 1F:9R	154	918	MS 918 ACG††	TCCAGTTAAATGGACGGCAATTGAATCCCT
	ATAGGGCCTGGGCTTCTC		922
	ACACATCACTTTGCGTGGTG

^*Final total primer concentration and forward (F) to reverse (R) primer ratio is displayed above the primer sequences. Primer sequences are listed 5′ to 3′, with the forward primer above the reverse primer. 

^†Codon of variation under each probe set. The underlined codons contain a polymorphism or sequence variant, whereas the other codons contain mutations. 

^‡Mask, masking deletion of one or three nucleotides. Wild-type probe sequence (WT exon #) is used in the primary assay for each exon, except for exon 16. If two wild-type probes are used for one exon, they are labeled as A and B probes. 

^§Probe sequences are listed 5′ to 3′. RET exons 10, 13, 14, and 16 have forward probes, whereas exon 11 has reverse probes. The known mutation locations are highlighted in bold, and the possible polymorphism or sequence variant locations are underlined. 

^¶Location (L) probes have a three nucleotide masking deletion (−−−) of one pathogenic codon from the wild-type probe sequence. 

^∥Example for the MS probes used for genotyping the mutations in exons 10 and 11. The same mutation-specific sequence is at both pathogenic codon positions within the probe. These probes have the mutation-specific sequence in the name, with the changed sequence in bold. There are seven possible mutant sequence changes from the ‘TGC’ wild-type cysteine codon sequence to another amino acid, therefore seven mutation-specific probes (AGC, CGC, GGC, TAC, TCC, TTC, TGG) for a total of 21 MS probes for exon 10 and 11 mutations. The MS probe sets used to genotype each mutation are diagrammed in the figures and Table 3 as well as in Supplemental Tables 1 to 5. 

^**Exon 13 used masking probes in the primary and secondary assays. The ‘Mask 1bp 769’ primary probe and the exon 13 mutation-specific probes have a single nucleotide deletion (−) at the polymorphism position within codon 769 (deletion of the underlined ‘T’ in the wild-type probe sequence). 

^††Exon 16 used a codon 918(ATG→ACG) mutation-specific probe for the primary assay.