Reverse Sequencing by Hybridization with Fluorescence Incorporation

摘要

Sequencing by hybridization, one of the next-generation DNA sequencing techniques, can greatly accelerate biological and biomedical research. However, challenges remain in accuracy and throughput. In the present study, we investigated a new strategy of DNA sequencing based on the hybridization of primers combined with fluorescence dye-tagged dideoxyribonucleotide triphosphates (ddNTPs). This simple strategy requires a universal panel of tiling primers containing multiple degenerate bases and some specific bases. The DNA sequencing involves many cycles of hybridization of primers with the immobilized template, extension with labeled ddNTPs, scanning the microarray, and removing the extended primers. Under optimal conditions, 13-16 bp of primer with 10-13 degenerate bases and 3 specific bases could improve the accuracy of sequencing; especially, a mismatch in the second position of the specified bases was favorable. Additionally, as many as 64 cycles could be performed without substantial dephasing of the templates or reduction in fluorescent signals after optimizing the sequencing conditions, including the concentration and the removal of primer. A 25-bp DNA target was accurately determined by this method. This new method, termed reverse sequencing by hybridization with fluorescence incorporation, could be used for DNA sequencing in a simple, cost-effective, robust and high-throughput format.