A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry
A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry
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DOI：
10.1128/aem.66.10.4258-4265.2000
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发表时间：
2000-10-01
影响因子：
4.4
通讯作者：
Brown, D
中科院分区：
文献类型：
--
作者：
Spiro, A;Lowe, M;Brown, D
描述了一种新的基于微珠的多路复用方法，该方法利用聚苯乙烯微球表面的核酸杂交来识别不同种类的DNA序列混合物中的特定序列。该方法由三部分组成：表面附着低聚物捕获探针的微珠(直径5.6微米)、用于多路检测的三个荧光团、以及如何使用细胞仪。在每个珠内以不同的量浸入两个荧光团，以产生不同类型的珠子，每个珠子与一个独特的探头相关联。第三个荧光团是记者。从环境样品中捕获荧光cDNA序列后，用流式细胞仪技术对珠子进行分析，每个捕获探针的信号强度与分析物中靶序列的数量成正比。从华盛顿州汉福德核废料场受污染的地下水中采集的微生物的16S/23S基因间隔区获得了目标序列(长度为628至728bp)。建立了一种由表面含有已知数量的荧光DNA分子的珠子组成的荧光标准，并确定了测量丰度的分辨率、灵敏度和检测下限。结果与使用相同序列的DNA微阵列的结果进行了比较。珠子方法表现出更好的序列辨别力，并具有便于准确定量的特征。
A new multiplexed, bead-based method which utilizes nucleic acid hybridizations on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences is described. The method consists of three elements: beads (5.6-mu m diameter) with oligomer capture probes attached to the surface, three fluorophores for multiplexed detection, and how cytometry instrumentation. Two fluorophores are impregnated within each bead in varying amounts to create different bead types, each associated with a unique probe. The third fluorophore is a reporter. Following capture of fluorescent cDNA sequences from environmental samples, the beads are analyzed by flow cytometric techniques which yield a signal intensity for each capture probe proportional to the amount of target sequences in the analyte, Tn this study, a direct hybrid capture assay was developed and evaluated with regard to sequence discrimination and quantitation of abundances. The target sequences (628 to 728 bp in length) were obtained from the 16S/23S intergenic spacer region of microorganisms collected from polluted groundwater at the nuclear waste site in Hanford, Wash. A fluorescence standard consisting of beads with a known number of fluorescent DNA molecules on the surface was developed, and the resolution, sensitivity, and lower detection limit for measuring abundances were determined. The results were compared with those of a DNA microarray using the same sequences. The bead method exhibited far superior sequence discrimination and possesses features which facilitate accurate quantitation.