Table 2.

Expected Molecular Mass of Extension Primers and Products of Multiplex Single-Base Extension (SBE) and Very Short Extension (VSET) Assay Employed for Detection of HBB Mutations

Primer No	Mutation site in HBB gene	Primer sequence	Primer strand	Primer conc. (nmol/L)	Primer mass (Da)	Normal product mass (Da)	Mutant product mass (Da)	Mass difference
Panel 1: Multiplex SBE–for detection of 21 HBB mutations (using 19 extension primers)
1.1	c.19G>A (Hb C)	5′-GCATCTGACTCCT-3′	Sense	425	3885.6	+ddG = 4198.8	+ddA = 4182.8	−16
1.2	c.20A>T (Hb S)	5′-CCGGCAGACTTCTCC-3′	Antisense	200	4489.0	+ddT = 4777.2	+ddA = 4786.2	+9
	c.20A>C (Hb G Makassar)						+ddG = 4802.2	+25
1.3	c.216_217insA	5′-TGCTCGGTGCCTTTA-3′	Sense	300	4550.0	+ddG = 4863.2	+ddA = 4847.2	−16
1.4	c.410G>A (Hb Hope)	5′-GAGGGCATTAGCCACA-3′	Antisense	150	4915.3	+ddC = 5188.5	+ddT = 5203.5	+15
1.5	c.79G>A (Hb E)	5′-CCACCTGCCCAGGGCCT-3′	Antisense	100	5092.3	+ddC = 5365.6	+ddT = 5380.5	+15
1.6	c.52A>T	5′-GTACTGCGCTGTGGGGC-3′	Sense	385	5258.4	+ddA = 5555.6	+ddT = 5546.6	−9
1.7	c.-78A>G	5′-ATGGCTCTGCCCTGACTT-3′	Antisense	225	5441.6	+ddT = 5729.8	+ddC = 5714.8	−15
1.8	c.170G>A (Hb J-Bangkok)	5′-CTCCTGATGCTGTTATGG-3′	Sense	175	5496.6	+ddG = 5809.8	+ddA = 5793.8	−16
1.9	c.59A>G (Hb Malay)	5′-ACCACCAACTTCATCCACG-3′	Antisense	100	5661.7	+ddT = 5949.9	+ddC = 5934.9	−15
1.10	c.-80T>C	5′-CATTAGCCAGGGCTGGGCA-3′	Sense	150	5853.8	+ddT = 6142.0	+ddC = 6127.0	−15
1.11	c.441_442insAC (Hb Tak)	5′-GCAAGAAAGCGAGCTTAGTG-3′	Antisense	100	6215.1	+ddA = 6512.3	+ddT = 6503.3	−9
1.12	c.380T>G (Hb Dhonburi)	5′-ACTTTCTGATAGGCAGCCTGC-3′	Antisense	87.5	6397.2	+ddA = 6694.4	+ddC = 6670.4	−24
1.13	c.130G>T	5′-GGGACAGATCCCCAAAGGACT-3′	Antisense	150	6449.0	+ddC = 6722.2	+ddA = 6746.2	+24
1.14	c.2T>G	5′-ACTAGCAACCTCAAACAGACACCA-3′	Sense	150	7252.8	+ddT = 7541.0	+ddG = 7566.0	+25
1.15	c.251G>A (Hb Pyrgos)	5′-TGGGTCACCTGGACAACCTCAAGG-3′	Sense	125	7362.8	+ddG = 7676.0	+ddA = 7660.0	−16
1.16	c.316–197C>T	5′-ACAGTGATAATTTCTGGGTTAAGG-3′	Sense	150	7446.9	+ddC = 7720.1	+ddT = 7735.1	+15
1.17	c.126delC	5′-GTTGCGTACCCTTGGACCCAGAGGTT-3′	Sense	350	7978.2	+ddC = 8251.4	+ddT = 8266.4	+15
	c.124_127delTTCT						+ddG = 8291.4	+40
1.18	c.364G>C (Hb D Punjab)	5′-CTCTCTCCTCGCCCATCACTTTGGCAAA-3′	Sense	450	8395.5	+ddG = 8708.7	+ddC = 8668.7	−40
1.19	c.92 + 5G>C	5′-TCCTTAAACCTGTCTTGTAACCTTGATA-3′	Antisense	225	8488.6	+ddC = 8761.8	+ddG = 8801.8	+40
Panel 2: Multiplex SBE–for detection of 16 HBB mutations (using 14 extension primers)
2.1	c.19G>A (Hb C)	5′-GCATCTGACTCCT-3′	Sense	425	3885.6	+ddG = 4198.8	+ddA = 4182.8	−16
2.2	c.52A>T	5′-ATCCACGTTCACCT-3′	Antisense	385	4158.8	+ddT = 4447.0	+ddA = 4456.0	+9
2.3	c.20A>T (Hb S)	5′-CCGGCAGACTTCTCC-3′	Antisense	140	4489.0	+ddT = 4777.2	+ddA = 4786.2	+9
	c.20A>C (Hb G Makassar)						+ddG = 4802.2	+25
2.4	c.27_28insG	5′-GCTCCTGAGGAGAAG-3′	Sense	250	4642.1	+ddT = 4930.3	+ddG = 4955.3	+25
2.5	c.126delC	5′-CCTTGGACCCAGAGGTT-3′	Sense	125	5186.4	+ddC = 5460.0	+ddT = 5475.0	+15
	c.124_127delTTCT						+ddG = 5500.0	+40
2.6	c.108C>A	5′-AGGCTGCTGGTGGTCTA-3′	Sense	125	5257.5	+ddC = 5530.7	+ddA = 5554.7	+24
2.7	c.441_442insAC (Hb Tak)	5′-GAGAAAGCGAGCTTAGTG-3′	Antisense	75	5612.7	+ddA = 5909.9	+ddT = 5900.9	−9
2.8	c.46delT	5′-CATTGCCGTTACTGCCCTG-3′	Sense	125	5730.7	+ddT = 6018.9	+ddG = 6043.9	+25
2.9	c.−81A>G	5′-GGCAGGAGCCAGGGCTGGGC-3′	Sense	125	6249.1	+ddA = 6546.3	+ddG = 6562.3	+16
2.10	c.84_85insC	5′-GATGAAGTTGGTGGTGAGGCCC-3′	Sense	150	6871.5	+ddT = 7159.7	+ddC = 7144.7	−15
2.11	c.−137C>A	5′-AGACCTCACCCTGTGGAGCCACAC-3′	Sense	100	7267.8	+ddC = 7541.0	+ddA = 7565.0	+24
2.12	c.370_377del ACCCCACC	5′-ACTTTCTGATAGGCAGCCTGCACTG-3′	Antisense	150	7633.0	+ddG = 7946.2	+ddA = 7930.2	−16
2.13	c.92 + 1G>T	5′-CTCCTGTCTTGTAACCTTGATACCAA-3′	Antisense	125	7856.2	+ddC = 8129.4	+ddA = 8153.4	+24
2.14	c.5T>C (Hb Raleigh)	5′-ACTAGCAACCTCAAACAGACACCATGG-3′	Sense	200	8215.4	+ddT = 8503.6	+ddC = 8488.6	−15
Panel 3: Multiplex VSET–for confirmation of three HBB mutations (using three extension primers)
3.1	c.20A>T (Hb S)	5′-TGCATCTGACTCCTG-3′	Sense	150	4519	+ddA = 4816.2	+dT+ ddG = 5136.4	320.2
3.2	c.52A>T	5′-GTACTGCGCTGTGGGGC-3′	Sense	375	5258.4	+ddA = 5555.6	+dT+ ddA = 5859.8	303.8
3.3	c.441_442insAC (Hb Tak)	5′-GCAAGAAAGCGAGCTTAGTG-3′	Antisense	100	6215.1	+ddA = 6512.3	+dT+ ddG = 6832.5	320.2

Bold bases are modifications for mass adjustment.

Underlined-bold bases are modifications for removal of self-complementary or hairpin formation.

Italic-bold bases are positions where there may be mismatches of bases due to the presence of other closely-located HBB mutations or SNPs.