Fluorescent Sequencing Protocols for the ALF

The development of polymerase chain reaction (PCR) (1 ), fluorescent DNA labels, and automated detection systems have revolutionized the efficiency, safety, and speed of DNA sequencing as compared to the original method of Sanger et al. (2 ), who published their chain terminating inhibitor (dideoxy) method of DNA sequencing more than 20 years ago. Detection of genomic alterations by direct sequencing of PCR products using the dideoxy method is now commonplace. The Pharmacia ALF family of DNA sequencers is designed for the automated electrophoresis and analysis of such sequencing reactions by the direct detection of fluorescently-labeled DNA products. A single fluorescent dye-labeled DNA primer is used, in standard four-lane dideoxy sequencing. The system produces highly accurate, easy to interpret raw data with automatic sequence processing. The data do not require the mobility shift corrections employed by multicolored fluorescent detection systems to compensate for different sized fluorophores. Although limited to four-lane sequencing, throughput is fast and any electrophoretic anomalies, such as “smiling,” are easily redressed by the system software.