Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays
Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays
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DOI：
10.1038/76469
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发表时间：
2000-06-01
影响因子：
46.9
通讯作者：
Corcoran, K
中科院分区：
文献类型：
--
作者：
Brenner, S;Johnson, M;Corcoran, K
我们描述了一种新的测序方法，它将基于非凝胶的特征测序与在直径为5微米的单独微珠上对数百万个模板进行体外克隆相结合。通过体外克隆构建DNA模板的微珠文库后，我们在流动池中以大于3×10⁶个微珠/平方厘米的密度组装了一个包含一百万个含模板微珠的平面阵列。然后使用一种基于荧光的特征测序方法同时分析每个微珠上克隆模板的游离端序列，该方法不需要DNA片段分离。通过用Ⅱs型限制性内切酶进行酶切、接头连接以及用编码的杂交探针进行序列询问的重复循环，获得了19 - 20个碱基的特征序列。通过对从一株已完全测序的酿酒酵母菌株构建的两个cDNA文库中的超过269,000个特征进行测序，并通过测量人类细胞系THP - 1中的基因表达水平，验证了该方法。这种方法提供了前所未有的分析深度，允许应用强大的统计技术来发现基因之间的功能关系，无论这些基因是事先已知的还是未知的，也无论其表达水平是高还是非常低。
We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 mu m diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3 x 10(6) microbeads/cm(2). Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 19-20 bases were obtained by repeated cycles of enzymatic cleavage with a type Ils restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.