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Journal of Biochemistry Advance Access published online on September 7, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm166
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© 2007 The Japanese Biochemical Society

Multiple Hybridization-Extension Sequencing (MHES) on Microarray

Zhiqiang Pan, Yanqiang Li, Pengfeng Xiao and Zuhong Lu*

State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China

*To whom correspondence should be addressed: Zuhong Lu: State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China, Tel: 86-25-83793310; Fax: 86-25-83793779, Email: zhlu{at}seu.edu.cn

Received August 6, 2007; Accepted August 17, 2007


  Abstract

Sequencing-by-synthesis (SBS) by fluorescein-labeled nucleotide incorporating into a target DNA template has been greatly concerned on microarray. The extended fluorophore-base must be required to be quenched prior to sequencing the next one. However, the low quenching efficiency has been an obstacle in length-read. Here, we present a new sequencing strategy, multiple hybridization-extension sequencing (MHES), to resolve the above problem. Firstly, the sequencing primers hybridize to the ssDNA template immobilized on microarray. The first 3-5 bases next to the primer’s end are sequenced by SBS of Cy5-dNTP. The extended primers are rapidly removed by {lambda} DNA exonuclease. Then, the same primers hybridize to the same ssDNA templates again. The sequenced bases are polished by natural dNTP. The other 3-5 bases next to the polished primer’s end are sequenced. According to this principle, the unknown sequences of a target DNA could be sequenced after primers’ hybridization-extension multiple times. Although the fluorescein-labeled nucleotides are also needed, it is unnecessary to quench the fluorophore-bases in the process of sequencing. It has been successfully demonstrated that 10bp fragment from synthetic template and 10bp fragment from DTBNP1 gene were accurately sequenced. The new method has a great potential in read-length and high-throughput sequencing on microarray.

Key Words: multiple hybridization-extension, sequencing, microarray, DTBNP1 gene


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