Primer design for automated DNA sequencing in a core facility.
We assessed the quality of nine standard primers for automated fluorescent dye terminator DNA sequencing by whether their melting temperatures (Tms) were in the optimal range for DNA sequencing, and the degree to which their sequences matched the sequences of 36 common vectors. The M13F (-21/-20), M13F (-41/-40), M13R, and T7R primers showed optimal physicochemical characteristics and were not redesigned. The M13R (-41/-40), T3, and SP6 primers showed mismatches and/or Tm values outside of the optimal range and were redesigned by these two criteria. With few exceptions, the redesigned primers did not significantly improve DNA sequencing quality as assessed by Phred scores compared to the corresponding original primer. However, both redesigned T7 primers, which were also redesigned to function as pET primers, strikingly improved sequencing on pET vectors. The original and redesigned T7 primers also improved the sequence quality for the Bluescript family of vectors compared to the original and redesigned M13R (-41/-40) primers. The mismatch-redesigned T7, SP6, and M13R (-41/-40) primers marginally improved the sequence quality over the corresponding original primers for specific vector types. Within limits, decreasing the %GC and Tm below reported optimal levels did not affect the sequence quality. Commercially available standard vector primers were surveyed.
O'Shaughnessy
Jennifer Brush
JB
Biomolecular Resource Center, University California at San Francisco, 90 Medical Center Way, 104, San Francisco, CA 94143-0541, USA. kathyi@cgl.ucsf.edu
Chan
Millie
M
Clark
Katherine
K
Ivanetich
Kathryn M
KM
D001483:Base Sequence; D017931:DNA Primers / Q000138:chemical synthesis* / Q000592:standards; D015342:DNA Probes / Q000138:chemical synthesis* / Q000592:standards; D020869:Gene Expression Profiling / Q000379:methods*; D008969:Molecular Sequence Data; D011786:Quality Control; D015203:Reproducibility of Results; D012371:Robotics / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards
D017931:DNA Primers; D015342:DNA Probes