Quantitation of mixed-base populations of HIV-1 variants by automated DNA sequencing with BODIPY dye-labeled primers.

TitleQuantitation of mixed-base populations of HIV-1 variants by automated DNA sequencing with BODIPY dye-labeled primers.
Publication TypeJournal Article
Year of Publication1998
AuthorsMetzker, ML, Ansari-Lari, MA, Liu, XM, Holder, DJ, Gibbs, RA
JournalBiotechniques
Volume25
Issue3
Pagination446-7, 450-2, 454, passim
Date Published1998 Sep
ISSN0736-6205
KeywordsBoron Compounds, DNA Primers, DNA, Viral, DNA-Directed DNA Polymerase, Genes, env, Genes, pol, Genetic Heterogeneity, Genetic Variation, Genome, Viral, HIV Envelope Protein gp120, HIV-1, Humans, Reproducibility of Results, Sequence Analysis, DNA
Abstract

Direct DNA sequencing of human immunodeficiency virus type 1 (HIV-1) pol and env gene regions was characterized for accuracy and precision. Restricted maximum likelihood (REML) analysis of molecular clone reconstruction experiments using a primer-walking strategy showed that the BODIPY and BODIPY energy-transfer (BET) dye primers sets were significantly more accurate in quantitating heterogenous base mixtures than the fluorescein/rhodamine dye primers. Of the three sets examined, the BET dye primers were the most accurate. The improved accuracy correlated with the reduced emission band-widths of BODIPY and BET dye primers and the more uniform signal intensities of BET dye primers. However, comparing % coefficients of variation (CV) for the three dye primer sets, revealed that BODIPY dye primers gave better precision than both BET and fluorescein/rhodamine dye primer sets. Several sequence-dependent motifs were identified that showed specific nucleotide-biased incorporation and were determined to be the major variable component of the total %CV. Taken together, these results show that BODIPY and BET direct DNA sequencing can accurately and precisely characterize complex mixed-base populations.

Alternate JournalBioTechniques
PubMed ID9762443