AAT Bioquest

What is sequencing by ligation approach in next-generation sequencing (NGS)?

Posted July 22, 2020


Sequencing by ligation is a DNA sequencing method that uses DNA ligase to identify the nucleotide present at a given position in a DNA sequence. Instead of creating a second strand as in sequencing-by-synthesis approach, sequencing by ligation harnesses the mismatch sensitivity of a DNA ligase to determine the underlying sequence of a target DNA molecule.

The DNA strand to be sequenced is flanked on at least one end by a known sequence, which can bind a short “anchor” strand. A mixed pool of probe oligonucleotides, which are typically labeled with fluorescent dyes according to the position that will be sequenced, are introduced and hybridize to the target DNA sequence, next to the anchor sequence. When the bases of the probe oligonucleotide match the target DNA sequence, it will be preferentially joined to the anchor sequence by the DNA ligase. Based on the fluorescence produced by the probe, the nucleotide on the unknown target sequence can thus be identified.

Additional resources

Helixyte™ Green *10,000X Aqueous PCR Solution*

6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*

Krishna, B. M., Khan, M. A., & Khan, S. T. (2019). Next-Generation Sequencing (NGS) Platforms: An Exciting Era of Genome Sequence Analysis. In Microbial Genomics in Sustainable Agroecosystems (pp. 89-109). Springer, Singapore.

Pfeifer, G. P., & Riggs, A. D. (1996). Genomic sequencing by ligation-mediated PCR. Molecular biotechnology, 5(3), 281.