Table 4. Primers and reaction conditions for GAPDH gene amplifications used to verify quality of BLV DNA extracted from human breast tissue samples, human cell lines, and animal cell lines*.

Gene	Primer pair sequences, 5′ → 3′†	Location in bp‡	Product length, bp	Annealing temperature, °C§	Extension time, s§
Human GAPDH	F: GAGTCAACGGATTTGGTCGT	194–213	237	50	22
	R: TTGATTTTGGAGGGATCTCG	431–412
Mouse GAPDH	F: AGCTTGTCATCAACGGGAAG	246–265	796	58	60
	R: ATGTAGGCCATGAGGTCCAC	1041–1022
Bovine GAPDH	F: CCTTCATTGACCTTCACTACATGGTCTA	172–199	857	59	60
	R: GCTGTAGCCAAATTCATTGTCGTACCA	1028–1002

*GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BLV, bovine leukemia virus; F, forward primer; R, reverse primer.
†Reverse sequences are reversed and complementary to the published genomic sequences. Primers were synthesized by Operon Biotechnologies, Huntsville, AL, USA.
‡Sequence bp numbering according to GenBank no. NM 002046.4 (human), XM 001476707.3 (mouse), and NM 001034034.2 (bovine). 
§Cycling conditions were 1 cycle at 95°C for 2 min; 35 cycles at 95°C for 30 s; XX°C for 30 s, 72°C for XX sec; and 1 cycle at 72°C for 5 min, where XX represents annealing temperature and extension times, respectively, for primer pairs.