Table 1. Primers and conditions of PCR... | American Society of Hematology

Table 1.

Primers and conditions of PCR assays

Primers	Specificity	Localization	Sequence 5′-3′	PCR assays	PCR products	^†PCR conditions
P1-s	CE	5′UT	CTCCATAGACAGGCCAGCACAG	P1-P3	1537 bp	1 min 95°C, 1 min 62°C, 1 min 30 sec 72°C: 30 cycles
P2-s	D-CE	5′UT	ATGCCTGGTGCTGGTGGAACCCC	P2-P3¹⁸	1427 bp	1 min 95°C, 1 min 62°C, 1 min 30 sec 72°C: 30 cycles
P3-a	CE	3′UT	CTGTCTCTGACCTTGTTTCATTATAC
P4-s	D	5′UT	CTCCATAGAGAGGCCAGCACAA	P4-P5	1590 bp	1 min 95°C, 1 min 62°C, 1 min 30 sec 72°C: 30 cycles
P5-a	D	3′UT	ATGGTGAGATTCTCCTCAAAGAGTG	P2-P5	1480 bp	1 min 95°C, 1 min 62°C, 1 min 30 sec 72°C: 30 cycles
P6-s	D-CE	In 2	tcagtcatcctggctctcc
P7-a	D-CE	In 3	aggtccctcctccagcac	P6-P7¹⁸	210 bp	30 sec 95°C, 30 sec 62°C, 30 sec 72°C: 25 cycles
P8-s	D	Ex 4	GACTACCACATGAACATGAT
P9-a	D-CE	In 5	aatatgtgtgctagtcctgt	P8-P9	790 bp	1 min 95°C, 1 min 60°C, 1 min 30 sec 72°C: 30 cycles
P10-s	CE	Ex 4	ACTACCACATGAACCTGAG	P10-P9¹⁸	1460 bp	1 min 95°C, 1 min 60°C, 1 min 30 sec 72°C: 30 cycles
P11-s	CE	Ex 5	CCCAAAGGAAGATCAGCAT
P12-a	D-CE	In 6-Ex 6	tgtctagtttcttacCGGCA	P11-P12	1807 bp	30 sec 95°C, 3 min 68°C: 32 cycles
P13-s	D	Ex 5	CCCAAGGGAAGATCAGCAA	P13-P12	1807 bp	30 sec 95°C, 3 min 68°C: 32 cycles
P14-a	CE	In 6-Ex 6	agtttcttacCGGCAGGC	P13-P14	1807 bp	30 sec 95°C, 3 min 68°C: 32 cycles
P15-s	D-CE	In 6	tgttagaaatgctgttagacc
P16-a	D	In 7-Ex 7	cacATGCCATTGCCGGCT	P15-P16	435 bp	1 min 95°C, 1 min 60°C, 1 min 30 sec 72°C: 30 cycles
P17/P17^*-a	D/CE^*	Ex 5	GCATAGTAGGTGTTGAACAC(T^*)	P18-P17/17^*	1400 bp	30 sec 95°C, 30 sec 62°C, 30 sec 72°: 30 cycles
P18-s	CE	In 4	gcaacagagcaagagtcca
P19/P19^*-a	D/CE^*	Ex 5	TGTCACCACACTGACTGCTAC(G^*)	P18-P19/19^*¹⁷	428 bp	30 sec 95°C, 30 sec 65°C, 30 sec 72°C: 26 cycles
P20/P20^*-s	1006T/G^*	Ex 7	ACTCCATCTTCAGCTTGCTGT(G^*)
P21-a	CE	In 7-Ex 7	acccacATGCCATTGCCGTTC	P20/20^*-P21¹⁷	94 bp	30 sec 95°C, 30 sec 65°C, 30 sec 72°C: 26 cycles
P22-s	D	Ex 3	TCGGTGCTGATCTCAGTGGA
P23/P23^*-a	CE/D^*	Ex 3	ACTGATGACCATCCTCAGGG(T^*)	P22-P23/23^*¹⁷	110 bp	30 sec 95°C, 30 sec 65°C, 30 sec 72°C: 26 cycles
P24/P24^*-s	D/CE^*	Ex 5	TGGATGTTCTGGCCAAGTT(G^*)
P25-a	CE	In 5-Ex 5	tcacCATGCTGATCTTCCT	P24/24^*-P25¹⁷	150 bp	30 sec 95°C, 30 sec 60°C, 30 sec 72°C: 25 cycles
P26/P26^*-s	340T/C^*	In 2-Ex 3	ccttctcacccccagTATTT(C^*)
P27-a	CE	Ex 3	CTGATGACCATCCTCAGGG	P26/26^*-P27²⁷	152 bp	30 sec 95°C, 30 sec 63°C, 30 sec 72°C: 30 cycles
P28-s	D-CE	5′UT	ATAGTCCCTCTGCTTCCG
P29-a	D-CE	In 1-Ex 1	ccaatgaactctcacCTTG	P28-P29²⁷	340 bp	Internal control: variable conditions

Primers	Specificity	Localization	Sequence 5′-3′	PCR assays	PCR products	^†PCR conditions
P1-s	CE	5′UT	CTCCATAGACAGGCCAGCACAG	P1-P3	1537 bp	1 min 95°C, 1 min 62°C, 1 min 30 sec 72°C: 30 cycles
P2-s	D-CE	5′UT	ATGCCTGGTGCTGGTGGAACCCC	P2-P3¹⁸	1427 bp	1 min 95°C, 1 min 62°C, 1 min 30 sec 72°C: 30 cycles
P3-a	CE	3′UT	CTGTCTCTGACCTTGTTTCATTATAC
P4-s	D	5′UT	CTCCATAGAGAGGCCAGCACAA	P4-P5	1590 bp	1 min 95°C, 1 min 62°C, 1 min 30 sec 72°C: 30 cycles
P5-a	D	3′UT	ATGGTGAGATTCTCCTCAAAGAGTG	P2-P5	1480 bp	1 min 95°C, 1 min 62°C, 1 min 30 sec 72°C: 30 cycles
P6-s	D-CE	In 2	tcagtcatcctggctctcc
P7-a	D-CE	In 3	aggtccctcctccagcac	P6-P7¹⁸	210 bp	30 sec 95°C, 30 sec 62°C, 30 sec 72°C: 25 cycles
P8-s	D	Ex 4	GACTACCACATGAACATGAT
P9-a	D-CE	In 5	aatatgtgtgctagtcctgt	P8-P9	790 bp	1 min 95°C, 1 min 60°C, 1 min 30 sec 72°C: 30 cycles
P10-s	CE	Ex 4	ACTACCACATGAACCTGAG	P10-P9¹⁸	1460 bp	1 min 95°C, 1 min 60°C, 1 min 30 sec 72°C: 30 cycles
P11-s	CE	Ex 5	CCCAAAGGAAGATCAGCAT
P12-a	D-CE	In 6-Ex 6	tgtctagtttcttacCGGCA	P11-P12	1807 bp	30 sec 95°C, 3 min 68°C: 32 cycles
P13-s	D	Ex 5	CCCAAGGGAAGATCAGCAA	P13-P12	1807 bp	30 sec 95°C, 3 min 68°C: 32 cycles
P14-a	CE	In 6-Ex 6	agtttcttacCGGCAGGC	P13-P14	1807 bp	30 sec 95°C, 3 min 68°C: 32 cycles
P15-s	D-CE	In 6	tgttagaaatgctgttagacc
P16-a	D	In 7-Ex 7	cacATGCCATTGCCGGCT	P15-P16	435 bp	1 min 95°C, 1 min 60°C, 1 min 30 sec 72°C: 30 cycles
P17/P17^*-a	D/CE^*	Ex 5	GCATAGTAGGTGTTGAACAC(T^*)	P18-P17/17^*	1400 bp	30 sec 95°C, 30 sec 62°C, 30 sec 72°: 30 cycles
P18-s	CE	In 4	gcaacagagcaagagtcca
P19/P19^*-a	D/CE^*	Ex 5	TGTCACCACACTGACTGCTAC(G^*)	P18-P19/19^*¹⁷	428 bp	30 sec 95°C, 30 sec 65°C, 30 sec 72°C: 26 cycles
P20/P20^*-s	1006T/G^*	Ex 7	ACTCCATCTTCAGCTTGCTGT(G^*)
P21-a	CE	In 7-Ex 7	acccacATGCCATTGCCGTTC	P20/20^*-P21¹⁷	94 bp	30 sec 95°C, 30 sec 65°C, 30 sec 72°C: 26 cycles
P22-s	D	Ex 3	TCGGTGCTGATCTCAGTGGA
P23/P23^*-a	CE/D^*	Ex 3	ACTGATGACCATCCTCAGGG(T^*)	P22-P23/23^*¹⁷	110 bp	30 sec 95°C, 30 sec 65°C, 30 sec 72°C: 26 cycles
P24/P24^*-s	D/CE^*	Ex 5	TGGATGTTCTGGCCAAGTT(G^*)
P25-a	CE	In 5-Ex 5	tcacCATGCTGATCTTCCT	P24/24^*-P25¹⁷	150 bp	30 sec 95°C, 30 sec 60°C, 30 sec 72°C: 25 cycles
P26/P26^*-s	340T/C^*	In 2-Ex 3	ccttctcacccccagTATTT(C^*)
P27-a	CE	Ex 3	CTGATGACCATCCTCAGGG	P26/26^*-P27²⁷	152 bp	30 sec 95°C, 30 sec 63°C, 30 sec 72°C: 30 cycles
P28-s	D-CE	5′UT	ATAGTCCCTCTGCTTCCG
P29-a	D-CE	In 1-Ex 1	ccaatgaactctcacCTTG	P28-P29²⁷	340 bp	Internal control: variable conditions

Coding sequences are given in capital letters, noncoding sequences in capital italics, and intron sequences in lowercase letters. When already published, references are given for PCR assays.

s indicates sense; a, antisense; UT, untranslated region; In, intron; and Ex, exon.

Wild-type mutation.

†

PCR conditions: a denaturation step of 4 minutes at 95°C and an elongation step of 5 minutes at 72°C were performed for all PCR assays.

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