Strategy for determination of the nucleic acid sequence of the rhesus macaque factor IX cDNA. (Above) 1% agarose gel electrophoresis of 5′ and 3′ RACE PCR fragments obtained by amplification of adapter-ligated first-strand DNA template with adapter primer AP1 (Clonetech) and various factor IX–specific oligonucleotides. Lane 5′8: amplimer obtained with AP1 and H8 primers; lane 5′9: amplimer obtained with AP1 and H9 primers; lane 5′10: amplimer obtained with AP1 and H10 primers; lane 3′2: amplimer obtained with H2 and AP1 primers; lane 3′4: amplimer obtained with H4 and AP1 primers. Note the double bands observed for 3′ RACE PCR fragments 3′2 and 3′4, due to the two different polyadenylation signal sequences, separated by 224 base pairs in the cDNA. (Below) Schematic diagram of rhesus macaque factor IX cDNA depicting the position of oligonucleotide primers used to amplify overlapping RACE PCR fragments and determine the nucleic acid sequence. H series denotes oligonucleotides completely homologous to human factor IX coding sequence. R series denotes oligonucleotides with sequence unique to the rhesus macaque factor IX cDNA sequence. Not shown is the adapter primer that is ligated to each end of the cDNA, to which oligonucleotide AP1 is complementary. M, Methionine translation initiation start sites; pA, AATAAA polyadenylation signal sequence. Drawing not to scale for clarity in depicting oligonucleotide primer positions.