Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations
Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations
复制标题
DOI：
10.1073/pnas.1133470100
复制
发表时间：
2003-07-22
影响因子：
11.1
通讯作者：
Vogelstein, B
中科院分区：
文献类型：
--
作者：
Dressman, D;Yan, H;Vogelstein, B
生物医学研究的许多领域依赖于对单个基因或转录本中不常见变异的分析。在这里，我们描述了一种方法，它可以在一定范围内量化这种变化，并缓解迄今无法实现的问题。这些分子的集合中的每个DNA分子都被转化为一个单一的磁性粒子，数千个与原始DNA序列相同的DNA副本被绑定在上面。然后，这一群珠子与起始DNA分子的一对一表示相对应。然后，通过流式细胞术对荧光标记的颗粒进行计数，就可以简单地评估原始DNA分子群体中的变异。这种方法被称为基于其四个主要成分(珠子、乳剂、放大和磁性)的光束。数以百万计的单个DNA分子可以用标准的实验室设备以这种方式进行评估。此外，还可以通过流动分选来分离特定的变体，并用于进一步的实验。BEAM可用于识别和量化罕见突变，以及研究特定群体或组织中基因序列或转录本的变异。
Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single magnetic particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently labeled particles via flow cytometry. This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics). Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and used for further experimentation. BEAMing can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.