Fluorescence-based cycle sequencing with primers selected from a nonamer library.

We have previously demonstrated that nonamers can prime manual T7 polymerase sequencing reactions. We next wanted to determine whether nonamers could be used to prime sequencing reactions for the Applied Biosystems Model 373A fluorescence-based sequencer. Both the Applied Biosystems T7 and Taq DNA Polymerase DyeDeoxy Terminator methodologies were tested, and successful results were obtained using a modified Taq DNA polymerase cycle sequencing procedure. The best nonamer cycle sequencing reaction conditions found, thus far, include denaturation at 96 degrees C for 30 s, primer-template annealing at 20 degrees C for 5 min, a 5-min ramp to the extension temperature, extension at 60 degrees C for 4 min and repeating this cycle 50 times. Furthermore, we found that the results were greatly improved by using linear (restriction enzyme cut) and alkaline-denatured, double-stranded DNA in combination with a doubling (2x) of the standard cycle sequencing reaction mixture containing a 2% final concentration of dimethyl sulfoxide. A total of 121 nonamer primers were tested, and approximately 50% primed successful cycle sequencing reactions. An average reading length of 257 bp was obtained from the successful reactions.