Anaplastic lymphoma kinase (ALK) is abnormally expressed in
anaplastic large cell lymphoma (ALCL) and its expression associated with
chromosomal translocations involving the ALK gene at 2p23. These translocations lead to the synthesis of novel chimeric
proteins that retain the C-terminal portion of ALK, where the
tyrosine kinase domain is located. In most of these
tumors, the t(2;5)(
p23;q35) translocation causes fusion of the ALK gene to the 5' region of the
nucleophosmin (NPM) gene, but other different ALK partners have been identified, including nonmuscle
tropomyosin (TPM3), TRK-fused gene (TFG), 5' aminoimidazole-4-carboxamide
ribonucleotide formyltranferase/
IMP cyclohydrolase (ATIC),
clathrin heavy chain gene (CLTC), and
moesin (MSN). The characterization of these ALK partners has been performed using different molecular methods, including the 5' Rapid Amplification of complementary
deoxyribonucleic acid (
cDNA) Ends (5'RACE) polymerase chain reaction (PCR)-based technique. This approach allows the potential amplification and identification of either 5' or 3'
mRNA ends from an internal known sequence. In ALK translocations, identification of the 5' gene involved has been performed using primers designed within the known 3' catalytic domain of the ALK. Initial reaction consists in a first-strand
cDNA synthesis primed using a gene-specific antisense primer (ALK1), performing the
cDNA conversion of specific messenger
ribonucleic acids, and maximizing the potential for complete extension to the 5'-end of the message. After
cDNA synthesis, the first-strand product is purified from unincorporated dNTPs and ALK1.
Terminal deoxynucleotidyl transferase is used to add homopolymeric tails to the 3' ends of the
cDNA. Tailed
cDNA is then amplified by PCR using a nested gene-specific primer (ALK2), which anneals 3' to ALK1, and a complementary homopolymer containing an anchor primer (i.e.,
AAP), which permits amplification from the homopolymeric tail. This allows amplification of unknown sequences between the ALK2 and the 5' end of the
mRNA. Further, nested PCRs usually are required to confer an adequate level of specificity to the process to permit the characterization of RACE products. The reamplification is achieved by using a nested gene-specific primer (ALK3), which anneals 3' to ALK2, and a universal amplification primer, which anneals to the 5' sequence previously introduced by the
AAP primer.