Multiple hybridization-extension sequencing (MHES) on microarray

Sequencing-by-synthesis (SBS) by fluorescein-labelled 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. First, 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-labelled nucleotides are also needed, it is unnecessary to quench the fluorophore-bases in the process of sequencing. It has been successfully demonstrated that 10 bp fragment from synthetic template and 10 bp fragment from DTBNP1 gene were accurately sequenced. The new method has a great potential in read-length and high-throughput sequencing on microarray.