FLUORESCENCE-BASED CYCLE SEQUENCING WITH PRIMERS SELECTED FROM A NONAMER LIBRARY

We have previously demonstrated that nobamers can prime manual T7 poly merase sequencing reactions. We next wanted to determine whether nonam ers could be used to prime sequencing reactions for- the Applied Biosy stems Model 373A fluorescence-based sequencer Both the Applied Biosyst ems T7 and Tag DA TA Polymerase DyeDeoxy(TM) Terminator methodologies were tested and successful results were obtained using a modified Tag DNA polymerase cycle sequencing procedure. The best nonamer cycle sequ encing reaction conditions found, thus far include denaturation at 96 degrees C for 30 s, primer-template annealing at 20 degrees C for 5 mi n, a 5-min ramp to the extension temperature, extension at 60 degrees C for 4 min and repenting this cycle 50 times. Furthermore, we found t hat the results were greatly improved by using linear (restriction enz yme cut) and alkaline-denatured, double-stranded DNA in combination wi th a doubling (2x) of the standard cycle sequencing reaction,mixture c ontaining a 2% final concentration of dimethyl sulfoxide. A total of 1 21 nonamer primers were tested, and approximately 50% primed successfu l cycle sequencing reactions. An average reading length of 257 bp was obtained from the successful reactions.