High-throughput flow cytometric DNA fragment sizing.
High-throughput flow cytometric DNA fragment sizing.
复制标题
高通量流式细胞术 DNA 片段大小测定。
作者：
A. Van Orden;R. Keller;W. P. Ambrose
在传统的单分子流式细胞术（SMFC）中，单个荧光标记的DNA片段的检测和大小确定的速率受到光饱和、光子计数统计和片段重叠的限制，大约为100个片段/s。我们已经增加了SMFC中的DNA片段大小的检测速率约2000片段/秒，通过从单个DNA分子的荧光平行成像，用荧光嵌入染料染色，因为它们通过激发激光的平面片，导致在测量速度和样品吞吐量的数量级改进相比，传统的SMFC。荧光猝发从DNA片段的fM溶液中测量，所述DNA片段的大小范围为7至154个双酶对。仅几秒钟的数据采集时间就足以确定DNA片段大小分布。确认了每次爆发检测到的光子数与DNA片段大小之间的线性关系。这种并行荧光成像方法的应用将导致涉及单分子、染色体、细胞等研究的其他类型的基于流动的分析的速度、通量和灵敏度的改进。
The rate of detection and sizing of individual fluorescently labeled DNA fragments in conventional single-molecule flow cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to approximately 100 fragments/s. We have increased the detection rate for DNA fragment sizing in SMFC to approximately 2000 fragments/s by parallel imaging of the fluorescence from individual DNA molecules, stained with a fluorescent intercalating dye, as they passed through a planar sheet of excitation laser light, resulting in order of magnitude improvements in the measurement speed and the sample throughput compared to conventional SMFC. Fluorescence bursts were measured from a fM solution of DNA fragments ranging in size from 7 to 154 kilobase pairs. A data acquisition time of only a few seconds was sufficient to determine the DNA fragment size distribution. A linear relationship between the number of detected photons per burst and the DNA fragment size was confirmed. Application of this parallel fluorescence imaging method will lead to improvements in the speed, throughput, and sensitivity of other types of flow-based analyses involving the study of single molecules, chromosomes, cells, etc.