This will drastically reduce redundancy in the shotgun approach and the postsequencing fragment-assembly process. The UWM group is also quantitating M13 sequencing-template concentrations to identify unsequenceable clones and normalize base calling. Current widely used procedures for automated DNA sequencing all involve the use of substrates synthesized by DNA polymerases and terminated by a dideoxynucleotide analog. This is critical for obtaining bands of uniform intensity in the gel electrophoresis step, increasing sequencing sensitivity and accuracy. The method is based on efficient and stable incorporation into DNA of a new class of boronated triphosphates that permit exponential amplification, unlike chain terminators.
Sequence is revealed by simple exonuclease digestion.
The method should be completely automatable and requires much less DNA template. Application of higher electric fields in electrophoretic separation of DNA fragments can increase sequencing speed and efficiency. Progress on these systems is reported below. Karger observed that mutation detection will be another important use for capillary technology. As they exit capillaries, DNA fragments with fluor labels are smoothly entrained in an optically clean fluid sheath flow in which as few as 120 fluorescein-labeled molecules can be detected.