Rapid DNA Sequence Analysis Using Fluorescent Labels

Normal and disease-associated gene sequences may be rapidly and accurately characterized at the molecular level using the procedures described here. First, a modification of the polymerase chain reaction (PCR) technique (1 ,2 ) provides a simple method of template preparation starting from either genomic or cloned DNA samples. This modification, called asymmetric polymerase chain reaction (APCR), is diagrammed in Fig. 1. After a simple purification procedure, the resulting DNA is directly sequenced using an oligonuclcotide primer labeled with a fluorescent reporter group. This preparation scheme eliminates the requirement of overnight culturing of bacteria or phage and provides the user with a rapid means of purifying sufficient template DNA for several sequencing reactions. The fluorescent DNA-sequencing procedure described here has been optimized to give the best results with the high-throughput APCR technique.