Huang Z, Jett J H, Keller R A
Chemical Science and Technology Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Cytometry. 1999 Feb 1;35(2):169-75. doi: 10.1002/(sici)1097-0320(19990201)35:2<169::aid-cyto9>3.3.co;2-b.
A flow cytometry-based, ultrasensitive fluorescence detection technique has been developed that demonstrates unique advantages in the analysis of large DNA fragments over the currently most widely used technology, pulsed-field gel electrophoresis (PFGE). The technique described herein is used to characterize the restriction fingerprints of the bacteria genome Staphylococcus aureus in this study.
The isolation of the bacterial genomic DNA and the subsequent complete digestion by a restriction endonuclease were performed inside an agarose plug. Electroelution was used to move the DNA fragments out-of the agarose plug into a solution containing low concentrations of spermine and spermidine, added to stabilize the large DNA fragments. DNA was stained with the bisintercalating dye thiazole orange homodimer (TOTO-1) and subsequently introduced into our ultrasensitive flow cytometer from a capillary.
Individual DNA fragments up to 351 kbp were successfully handled and sized. The histograms of the burst sizes were generated from signals associated with individual fragments in <7 min with <2 pg of DNA. The sizing accuracy was better than 98%. In contrast, standard PFGE takes approximately 20 h and requires approximately 1 microg of DNA with a sizing accuracy of approximately 90%.
With the demonstrated success and advantages, our approach has the potential of being applied to fast, accurate bacteria species and strain identification.
已开发出一种基于流式细胞术的超灵敏荧光检测技术，与目前使用最广泛的技术——脉冲场凝胶电泳（PFGE）相比，该技术在分析大DNA片段方面具有独特优势。本文所述技术用于在本研究中表征金黄色葡萄球菌细菌基因组的限制性指纹图谱。
细菌基因组DNA的分离以及随后用限制性内切酶进行的完全消化在琼脂糖块内进行。采用电洗脱法将DNA片段从琼脂糖块转移到含有低浓度精胺和亚精胺的溶液中，添加这些物质以稳定大DNA片段。DNA用双插入染料噻唑橙同二聚体（TOTO-1）染色，随后通过毛细管引入我们的超灵敏流式细胞仪。
成功处理并测定了长达351kbp的单个DNA片段大小。在不到7分钟内，使用不到2pg的DNA，从与单个片段相关的信号生成了脉冲大小直方图。大小测定准确度优于98%。相比之下，标准PFGE大约需要20小时，需要大约1μg的DNA，大小测定准确度约为90%。
鉴于已证明的成功和优势，我们的方法有潜力应用于快速、准确的细菌种类和菌株鉴定。
