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

Table 1.

Primers and PCR amplification conditions for factor VIII mutation screening

Exon	5′ Primer	3′ Primer	Size (base pairs)	Annealing time (sec), °C	Extension,^* sec at 72°
Exon	(primers listed from 5′ to 3′ sequence)		Size (base pairs)	Annealing time (sec), °C	Extension,^* sec at 72°
20	ACG TTG AGT ACA GTT CTT GG	ACT AAT AGA AGC ATG GAG ATG	320	30, 52°	50
21	GAA TTT AAT CTC TGA TTT CT	GAG TGA ATG TGA TAC ATT TC	279	30, 52°	50
22	AAA TAG GTT AAA ATA AAG TGT TAT	TTA ATG GTA TGT AAT TAG TCA TTT A	218	35, 43°	50
23	ACT CTG TAT TCA CTT TCC AT	AAC TAG AAC AGT TAG TCA CC	258	40, 55°	50
24	ATA ACT GAG GCT GAA GCA TG	CTC TGA GTC AGT TAA ACA GT	270	30, 54°	60
25	GAG TGA GAA GTG CTG TGG	TTG CTC TGA AAA TTT GGT CAT A	381	30, 58°	50
26	GCT TTG CAG TGA CCA TTG TC	AGC TGA GGA GGG AGA GGT GA	265	40, 60°	50

Exon	5′ Primer	3′ Primer	Size (base pairs)	Annealing time (sec), °C	Extension,^* sec at 72°
Exon	(primers listed from 5′ to 3′ sequence)		Size (base pairs)	Annealing time (sec), °C	Extension,^* sec at 72°
20	ACG TTG AGT ACA GTT CTT GG	ACT AAT AGA AGC ATG GAG ATG	320	30, 52°	50
21	GAA TTT AAT CTC TGA TTT CT	GAG TGA ATG TGA TAC ATT TC	279	30, 52°	50
22	AAA TAG GTT AAA ATA AAG TGT TAT	TTA ATG GTA TGT AAT TAG TCA TTT A	218	35, 43°	50
23	ACT CTG TAT TCA CTT TCC AT	AAC TAG AAC AGT TAG TCA CC	258	40, 55°	50
24	ATA ACT GAG GCT GAA GCA TG	CTC TGA GTC AGT TAA ACA GT	270	30, 54°	60
25	GAG TGA GAA GTG CTG TGG	TTG CTC TGA AAA TTT GGT CAT A	381	30, 58°	50
26	GCT TTG CAG TGA CCA TTG TC	AGC TGA GGA GGG AGA GGT GA	265	40, 60°	50

The first step is 93°C for 2 min for each exonic fragment followed by 35 cycles each of 1 min at 93°, annealing, and then extension.