RT-PCR for Autol DC Therapy Figure 1. Amplification of autologous HIV sequences using multiplex PCR. Panel A. Sequence alignment of multiple HIV isolates, revealed a region of relative conservation with variable residues in positions 7847 and 7848. Primer REVF7830 is perfectly complimentary to consensus sequence B, whereas primers REVF7830.1 and REVF7830.2 encode compensatory mutations in the 39 region of the primer, indicated in bold. ???denotes deletions, -sequence identity, letters indicate alternative bases in the corresponding positions relative to consensus sequence B. Consensus sequences for common HIV clades as well as less frequent isolates are denoted in bold. Panel B. Schematic overview of the Rev RNA amplification strategy. Open bar denotes regions outside of open reading frame of interest, hatched bar denotes RNA region exon 2 Rev, grey bar represent DNA intermediate products durig amplification process. For details on primer design and amplification refer to Method section. doi:10.1371/journal.pone.0001489.g001 More interestingly however is the observation that the number of the Nef variable sequences differed in each subject. At the nucleotide level (Figure 3, Panel A) the subject HTM 344 displayed greater diversity where out of 15 clones analyzed, 14 clones were unique, followed by subject HTM 367 with 13 unique clones and for subject HTM 349 only 6 unique clones. The subject-specific sequence clustering together with the variable number of unique clones between patients eliminates the possibility that the mutations are random mutational artifacts introduced during RT-PCR. Not every nucleotide mutation leads to an amino acid substitution, so the diversity is lower at the level of amino acid sequence (Figure 3, Panel B) with the same order of diversity trend for the three subjects. Similar analyses were performed for cDNAs encoding Gag, Rev and Vpr cDNA amplified from various subjects (data not shown). These data indicate that, as predicted, the multiplex RT-PCR is capable of capturing various quasispecies within each individual subject. We next performed further analysis of the Nef sequences and scored individual primers on the level of productive secondary PCR reactions. Since the same formulation of primers was used with all samples independent of clade, we were interested to learn which primers within the formulated groups were leading to productive amplification and analysis of preferential use of forward and reverse primers was performed (Table 4). The forward primer utilization of sample HTM 344 demonstrated that 12 out of 15 clones were formed by primer F8343.1, however in sample HTM 367 a different primer, F8343 formed the majority of clones. Interestingly, a novel primer annealing sequence was identified. The sequence was termed ‘‘new’’ and differed from either F 8343 or F8343.1 primer sequence by 2 nucleotides in the most 39 position. We believe that the new sequence was formed due to the 39 exonuclease proof-reading activity of the PCR enzyme. Similarly, different preferences for use of reverse primers were observed although no new reverse primers were identified in all three groups analyzed. The 39 end of the PCR fragment is defined by the cDNA synthesis step during the RT reaction, and the lack of ‘‘repaired’’ primers is most likely due to lack of proofreading activity in the RT enzyme. Similar analyses were performed on multiple Rev clones and confirmed the original observation (data not shown). Since the sequence within regions of interest varies from patient to patient, the preferred utilization of the primers in the PCR reactions differ as well. PLoS ONE | www.plosone.org 3 January 2008 | Issue 1 | e1489