DNA sequencing by capillary electrophoresis with four-decay fluorescence detection.
DNA sequencing by capillary electrophoresis with four-decay fluorescence detection.
复制标题
通过毛细管电泳和四衰变荧光检测进行 DNA 测序。
DOI：
10.1021/ac000952n
复制
发表时间：
2000
影响因子：
7.4
通讯作者：
McGown,LB
中科院分区：
文献类型：
--
作者：
He,H;McGown,LB
描述了一种在DNA测序中用于染料标记的DNA片段的多重检测的方案，其中在单泳道CE分离中使用即时频域荧光寿命检测来区分四个末端碱基的染料标记的片段。两个四染料系统进行了评估，一个激发在488 nm和其他，在514 nm。488 nm系统被证明是成功的四衰变检测。直接从运行中的寿命或从从测序反应产物的混合物的电泳图中回收的每个碱基的寿命分辨电泳图中实现碱基识别。后一种方法被发现更准确（两个碱基为99%，三个碱基为98.5%），并且可以实现更长的读取长度，但对所有四个碱基的测序都不成功。第一种方法在41 - 220个碱基的片段长度范围内，四碱基测序的碱基识别准确率为96%。
A scheme for multiplex detection of dye-labeled DNA fragments in DNA sequencing is described in which on-the-fly, frequency-domain fluorescence lifetime detection is used to discriminate among the dye-labeled fragments of the four terminal bases in a single-lane CE separation. Two four-dye systems were evaluated, one excited at 488 nm and the other, at 514 nm. The 488 nm system proved successful for four-decay detection. Base calling was achieved either directly from on-the-fly lifetimes or from lifetime-resolved electropherograms recovered for each base from the electropherogram of the mixture of sequencing reaction products. The latter method was found to be more accurate (99% for two bases and 98.5% for three bases) and could achieve longer read lengths, but it was unsuccessful for sequencing of all four bases. The first method gave a base-calling accuracy of 96% for four-base sequencing over the fragment length range of 41−220 bases.