( Geoffrey Grove Ed Alderman ...)Scalable Automation of the TempliPhi DNA Sequencing Template Amplification and DYEnamic ET Terminator Sequencing Kits on the Sciclone ALH 3000

Geoffrey Grove, Ed Alderman
Applied Science and Technology, Caliper Life Sciences, Inc., Hopkinton, MA 01748

Monica Adams, Jason Brennan
Development Integration, Amersham BioSciences, 800 Centennial Avenue, Piscataway, NJ 08855

Overview
The TempliPhi DNA Sequencing Template Amplification Kit is designed to eliminate the requirement for extended bacterial growth prior to sequencing. The automation of this kit on the Sciclone ALH 3000 produced 3.0 μg of DNA, which is comparable to the manual technique. Using the DYEnamic ET Terminator Sequencing Kit, Phred20 scores averaging 603 bp across four 96-well plates were observed, with a maximum readlength of 810 bp. IF/THEN programming functions provided by the control software are used to create scalable methods capable of drawing enzyme reagents from various source types. An overview of the basic workflow and software is described.

Introduction

Amplification of source DNA is a basic building block technique in molecular biology research. The TempliPhi DNA Sequencing Template Amplification Kit employs bacteriophage j29 DNA polymerase applied in an isothermal rolling circle amplification (RCA)¹ to produce large amounts of product DNA (1-3 μg)² and eliminate the need for extended bacterial growth prior to sequencing. Amplification times may vary from 4-18 hours, with increased yields achieved with overnight amplification.

The ability to quickly set up overnight amplification of DNA templates ensures a ready supply of starting materials to feed a variety of research areas. The reaction uses picogram quantities of starting material, and for our work, we used pUC19 control template. The principle concerns for automation included transfers of 0.5 μL of starting template, minimizing cross-contamination by using disposable tips, and temperature control f
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