Julie Chang, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547
Capillary sequencing technology has altered the field of genomics, increasing throughput and automating the sequencing process. This high-throughput platform has become a standard in production laboratories, driving such achievements as the completion of the Human Genome Project.
One of the requirements for achieving this higherthroughput processing is high sample purity. Samples must be free of unincorporated dye terminators and other contaminants, including salt.
Achieving a High-Quality Sequence
Many factors besides sample cleanup contribute to a highquality sequence read. It is essential to start with a pure template that has been prepared using a reliable method. Poorly prepared template will result in poor sequencing results, including low signal strengths, short reads, and dye blob retention. It is also important to use the proper amounts of reagents in cycle sequencing reactions. Too much or too little template or primer may result in reads that are difficult to interpret and analyze. Another factor to be aware of is the use of optimized cycling parameters proper annealing temperatures, cycle times, cycle numbers, etc. Finally, instrument error can be minimized through proper matrix installation, fresh polymer, and clean and unclogged capillaries. These guidelines will help to increase the likelihood of a high-quality sequence.
Evaluation of Kits
Four parameters relevant to the evaluation of dye terminator cleanup kits will be discussed: signal intensity, Phred analysis, dye blob removal, and cost. A total of five 96- well