Quantitation of mixed-base populations of HIV-1 variants by automated DNA sequencing with BODIPY dye-labeled primers.
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.
Baylor College of Medicine, Houston, TX, USA.
M L
ML
Metzker
M A
MA
Ansari-Lari
X M
XM
Liu
D J
DJ
Holder
R A
RA
Gibbs
D001896:Boron Compounds / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015752:Genes, env / Q000235:genetics; D015751:Genes, pol / Q000235:genetics; D018740:Genetic Heterogeneity; D014644:Genetic Variation; D016679:Genome, Viral; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D017931:DNA Primers; D004279:DNA, Viral; D015699:HIV Envelope Protein gp120; D004259:DNA-Directed DNA Polymerase