Reconstitution of the complete Tat RNA sequences. A. Organization of the splicing donor and acceptor sites in the HIV-1 pNL4.3 genome. B. Reconstitution of the complete Tat1 and Tat2 DNA sequences by PCR. "Hybridization PCR" can associate two different exons. Each Tat1 and Tat2 exon located in the pNL4.3 plasmid sequence (top panel) was independently amplified with specific oligonucleotides (Table 1). In fact, the antisense oligonucleotides used for exon amplification were designed in such a way that their 5' extremity is complementary to the 5' extremity of the next exon sense strand (see the colour codes). With this first PCR, the exon1 sense strand partially hybridizes with the exon2 antisense strand and the exon1 antisense strand with the exon2 sense strand. Then "Amplification PCR" resulted in the accumulation of DNA corresponding to exon1 + exon2. All further steps needed to completely reconstitute the Tat1 and Tat2 sequences were performed using this procedure (Additional file 1). The only difference between Tat1 and Tat2 sequences corresponds to exon EX' (see bottom lane).