File S1 - Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

Supporting figures and tables. Figure S1. Sequence comparison between templates. Two different HIV-1 strains 89.6 and NL4-3 (A) and two genetic variants 1B7 and 1D1 of HIV-1 strain WEAU (B) were compared to each other. The identical nucleotides are indicated by dash. The nucleotides that were used in the PASS assay to distinguish both templates from each other are indicated by boxes. The positions of each nucleotide used for PASS assay in the PCR amplicon are indicated above the base. The PCR primer sequences are indicated by underline. Figure S2. Detection of homoduplexes and heteroduplexes by PASS. The NL4-3 (A) and 89.6 (B) were either amplified individually or as a mixture (C) by PCR. The PCR products were then subjected to PASS analysis. Each polony was annealed to primer 729 and SBE was performed with the Cy3-labeled NL4-3-specific nucleotide (green) and the Cy5-labeled 89.6-specific nucleotide (red). No heteroduplexes were detected in the PCR products amplified from 89.6 or NL4-3 template alone, while a high proportion of the polonies were heteroduplexes (indicated by arrow) in the PCR products amplified from the mixture of 89.6 and NL4-3. Table S1. Recombination patterns and frequencies of the PCR amplicons with different thermocycles. Table S2. Recombination patterns and frequencies of the PCR amplicons with different input numbers of templates. Table S3. Recombination patterns and frequencies of the PCR amplicons with different extension time.
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