%0 Journal Article %J Biotechniques %D 1998 %T Quantitation of mixed-base populations of HIV-1 variants by automated DNA sequencing with BODIPY dye-labeled primers. %A Metzker, M L %A Ansari-Lari, M A %A Liu, X M %A Holder, D J %A Richard A Gibbs %K Boron Compounds %K DNA Primers %K DNA, Viral %K DNA-Directed DNA Polymerase %K Genes, env %K Genes, pol %K Genetic Heterogeneity %K Genetic Variation %K Genome, Viral %K HIV Envelope Protein gp120 %K HIV-1 %K Humans %K Reproducibility of Results %K Sequence Analysis, DNA %X

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.

%B Biotechniques %V 25 %P 446-7, 450-2, 454, passim %8 1998 Sep %G eng %N 3 %1 https://www.ncbi.nlm.nih.gov/pubmed/9762443?dopt=Abstract