Cloning of the HLA-G 5′URR haplotypes into the pGL3-basic vector

DNA samples from seven Brazilian individuals carrying the ten main HLA-G 5′URR haplotypes (Table ​(Table11)^{20,24,27,29,30} were selected for the cloning procedure. DNA was amplified using the forward 5′-AAGCTTCACAAGAATGAGGTGGAGC and reverse 5′-CGCGGATCCTTGGCGTCTGG primers, generating a 1438 bp fragment. Cycling conditions consisted of 35 cycles of 30 s at 95 °C, 30 s at 60 °C and 1 min at 72 °C. PCR-amplified fragments were first inserted into pUCm-T vector (Bio Basic, Ontario, Canada) and confirmed by HindIII (Invitrogen, Carlsbad, CA) digestion. Ten constructions carrying each of the main haplotypes were selected and identified by Sanger’s sequencing analysis (Applied Biosystems, Foster City, CA). A 1525 bp KpnI/BamHI (Invitrogen) fragment obtained from each selected clone was subcloned into pGL3-Basic vector (Promega, Madison, WI) upstream of the firefly luciferase gene.