DNA Sequencing Using Rolling Circle Amplification and Precision Glass Syringes in a High-Throughput Liquid Handling System

Hui-Chung Wu, Jean Shieh, David J. Wright, Arezou Azarani
2003 BioTechniques  
An automated high-throughput method that employs rolling circle amplification (RCA) to generate template for large-scale DNA sequencing has been developed using liquid handling systems equipped with precision glass syringes. A protocol was designed to perform the sequencing analysis from template preparation to thermal cycle sequencing within the same vessel, thus minimizing the amount of liquid handling and transfer. The amplified DNA was directly used for cycle sequencing with no need for any
more » ... ith no need for any purification procedures. Total RCA reaction volumes as low as 500 nL generated sufficient templates for successful sequencing. Reducing the RCA total reaction volumes by a 40-fold factor, from a total of 20 µL to 500 nL, resulted in a significant reduction in cost, from $1.25/reaction to less than $0.04/reaction. Additionally, the volume of the sequencing reactions was reduced from a total of 20 to 10 µL, thus generating a further cost advantage. This high-throughput DNA sequencing protocol maximizes the speed and precision of processing while significantly reducing the cost of amplification.
doi:10.2144/03341pf01 pmid:12545561 fatcat:gf6e6plzfjbgfj4t6k5rpqihna