Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays

被引：985

作者：

Brenner, S ^{[1
]}

Johnson, M ^{[1
]}

Bridgham, J ^{[1
]}

Golda, G ^{[1
]}

Lloyd, DH ^{[1
]}

Johnson, D ^{[1
]}

Luo, SJ ^{[1
]}

McCurdy, S ^{[1
]}

Foy, M ^{[1
]}

Ewan, M ^{[1
]}

Roth, R ^{[1
]}

George, D ^{[1
]}

Eletr, S ^{[1
]}

Albrecht, G ^{[1
]}

Vermaas, E ^{[1
]}

Williams, SR ^{[1
]}

Moon, K ^{[1
]}

Burcham, T ^{[1
]}

Pallas, M ^{[1
]}

DuBridge, RB ^{[1
]}

Kirchner, J ^{[1
]}

Fearon, K ^{[1
]}

Mao, J ^{[1
]}

Corcoran, K ^{[1
]}

机构：

[1] Lynx Therapeut Inc, Hayward, CA 94545 USA

来源：

NATURE BIOTECHNOLOGY | 2000年 / 18卷 / 06期

关键词：

DNA sequencing; ligation; gene expression; fluid microarray; yeast;

D O I：

10.1038/76469

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 [微生物学]; 0836 [生物工程]; 090102 [作物遗传育种]; 100705 [微生物与生化药学];

摘要：

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.

引用

页码：630 / 634

页数：5

共 17 条

[1]

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J].