TABLE 1.
Sequencing Through Hairpin Structures
| BigDye‰ Terminator Mix (Q ≥ 20 Phred Scores) | |||||||||||
| BD V3.0 Treatment | BD dGTPV3.0 Treatment | BD V3.1 Treatment | |||||||||
| Hairpin Type (#) | No of GC Pairs | Overall GC Content (%) | No HD | +HD | +HD+C | No HD | +HD | +HD+C | No HD | +HD | +HD+C |
| 1 | 20 | 67 | 366 | 543 | 551 | 336 | 527 | 555 | 493 | 605 | 622 |
| 2 | 19 | 64 | 196 | 573 | 589 | 345 | 452 | 460 | 448 | 590 | 596 |
| 3 | 17 | 60 | 435 | 597 | 574 | 442 | 515 | 555 | 453 | 599 | 599 |
| 4 | 16 | 58 | 285 | 542 | 549 | 440 | 537 | 548 | NT | NT | NT |
| 5 | 15 | 55 | 288 | 512 | 605 | 333 | 520 | 551 | NT | NT | NT |
| 6 | 14 | 53 | 352 | 506 | 523 | 437 | 489 | 511 | NT | NT | NT |
| 7 | 13 | 50 | 471 | 600 | 607 | 361 | 488 | 554 | NT | NT | NT |
NT, not tested. No/+ HD, no heat-denaturation/ + 5 min heat denaturation at 98° C with reagent C prior to adding appropriate Taq mix.
Three different chemistries were evaluated for the effectiveness of reading through various hairpin structures. Only representative data for 29 bp hairpins are shown. The remaining 13 hairpins gave similar results. The BigDye 3.1 chemistry was tested only on three 29 bp hairpins, as it did not offer any advantages over BigDye 3.0 chemistry. No improvement was observed when reagent C was part of a standard sequencing protocol (not shown). All sequencing reactions for this set were run on an ABI3100 DNA sequencer equipped with a 50-cm capillary array.
The overall GC content refers to the hairpin part only.