Bulk PCR Research Articles

Deciphering Human Immunodeficiency Virus Type 1 Transmission and Early Envelope Diversification by Single-Genome Amplification and Sequencing

Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 x 10(-5). Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 x 10(-5) substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the results of the SGA approach with those of more-conventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.

Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines.

The complete nucleotide sequences of the human papillomavirus type 16 (HPV-16) variants present in the CaSki and SiHa cervical carcinoma cell lines and the primary subgenomic HPV-18 variant present in the HeLa cervical carcinoma cell line were determined using overlapping bulk PCR products as templates. PCR-based methods were also used to characterize five previously unreported CaSki HPV-16 genomic disruptions and the 5' cellular-viral junction common to all HeLa HPV-18 subgenomic structures.

From ABI sequence data to LASERGENE's EDITSEQ.

This chapter describes the analysis and assembly of sequence data generated by the Applied Biosystems (ABI, Foster City, CA) automated sequencer after sequencing PCR products using Taq dye terminators (e.g., with the PRISM DyeDeoxy Terminator cycle sequencing kit). We use Taq dye terminator chemistry rather than dye primer sequencing because we are currently interested in sequencing PCR products of viral genomes amplified from clinical specimens (,). We prefer to sequence PCR products directly rather than after cloning them to avoid seeing unrepresentative sequences. To be able to look at the distribution of sequences in a population (the quasispecies), we dilute the starting specimen until amplification is achieved from a single molecule (). Alternatively, if the sequence population is relatively homogeneous, sequencing from a bulk PCR amplification will be accurate since Taq misincorporation errors will be effectively diluted out.