Metzker M L, Ansari-Lari M A, Liu X M, Holder D J, Gibbs R A
Baylor College of Medicine, Houston, TX, USA.
Biotechniques. 1998 Sep;25(3):446-7, 450-2, 454, passim.
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.
对人类免疫缺陷病毒1型（HIV-1）的pol和env基因区域进行直接DNA测序，以评估其准确性和精密度。使用引物步移策略对分子克隆重建实验进行限制最大似然（REML）分析，结果表明，与荧光素/罗丹明染料引物相比，BODIPY和BODIPY能量转移（BET）染料引物组在定量异质碱基混合物时显著更准确。在所检测的三组引物中，BET染料引物最准确。准确性的提高与BODIPY和BET染料引物发射带宽的减小以及BET染料引物信号强度更均匀有关。然而，比较三组染料引物的变异系数（CV）百分比发现，BODIPY染料引物比BET和荧光素/罗丹明染料引物组具有更好的精密度。鉴定出了几个序列依赖性基序，这些基序显示出特定的核苷酸偏向性掺入，并被确定为总CV百分比的主要可变成分。综上所述，这些结果表明，BODIPY和BET直接DNA测序可以准确、精确地表征复杂的混合碱基群体。
