From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

Figure 8

The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (Life Technologies). (1) Library preparation: two different adapters are ligated to sheared genomic DNA. (2) Emulsion PCR: emulsion PCR is conducted using magnetic beads to generate “bead clones,” in which each contains a single nucleic acid species. (3) Bead deposition: the beads are then attached to the surface of a glass slide. (4) Sequencing by ligation: ligase-mediated sequencing begins by annealing a universal primer to the shared adapter sequences on each amplified fragment (i), and then DNA ligase is provided along with specific fluorescently labeled 8-mers, in which the two bases at the 3′ end of the probe are encoded by the attached fluorescent group. Each ligation step is followed by fluorescence detection (ii), after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group) (iii), and concomitantly prepares the extended probe for another round of ligation (iv–vii). Since each fluorescent group on a ligated 8-mer identifies a two-base combination, the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms or single base deletions, by aligning the individual reads to a known high-quality reference sequence.