Huang Z, Petty J T, O'Quinn B, Longmire J L, Brown N C, Jett J H, Keller R A
Chemical Science and Technology Division, Los Alamos National Laboratory, NM 87545, USA.
Nucleic Acids Res. 1996 Nov 1;24(21):4202-9. doi: 10.1093/nar/24.21.4202.
A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation.
一种基于流式细胞术的超灵敏荧光检测技术被用于测定长度达167 kb的单个DNA片段的大小。本文描述了该技术在线性和超螺旋形式的P1人工染色体（PAC）大小测定中的应用。结果表明，该方法非常适合于表征PAC/BAC克隆，并且对于大插入片段文库的分析将非常有用。当单个、经染料染色的DNA片段穿过低功率、聚焦的连续激光束时，记录荧光脉冲。荧光脉冲的大小与DNA片段的长度呈线性比例关系。在不到3分钟的时间内，使用不到1 pg的DNA即可生成脉冲大小的直方图。线性片段的结果与脉冲场凝胶电泳获得的结果一致。与脉冲场凝胶电泳相比，用这种方法测定大DNA片段的大小更准确、速度更快、所需DNA更少，并且与DNA构象无关。
