Design strategies and performance of custom DNA sequencing primers

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to r elate their preferred primer design strategies to identify primer character istics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G+C, a me lting temperature (T-m) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clam p, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 n t, primer dimers of less than 3-4 bp and secondary priming sites of less th an 3-4 bp. We provided a 300-bp rest sequence and asked participants to sub mit sequences of 1-3 optimal sequencing primers. Submitted primers ranged f rom 17-24 nt and largely conformed to the preferred parameters. Submitted p rimers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "m anually", more than by any software package Each of 69 submitted :and 95 co ntrol primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM(TM) DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators ). Approximately half of the control primers were also tested using rhodami ne dye terminator reagents ("old" rhodamine dye terminators). The results i ndicated that primer physico-chemical characteristics thought to have a str ong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G+C or T-m, strong secondary priming score s or long 3' homopolymeric stretches yielded excellent sequences with the d Rhodamine dye terminator reagents, although these characteristics had a str onger effect when the:old rhodamine reagents were used. The old rhodamine r eagents gave sequences with a similar average read length, but the number o f errors and ambiguities or "N's" was consistently higher Moreover the effe cts of the primer physico-chemical characteristics were also more evident w ith the old rhodamine dyes. We conclude that under optimal sequencing condi tions with highly pure template and primer many of the commonly applied pri mer design parameters are dispensable, particularly when using one of the n ew generation of sequencing reagents such as the dichlororhodamine dye term inators.