AAT Bioquest

What methods are used in next-generation sequencing (NGS) to amplify templates?

Posted July 22, 2020


There are three methods commonly used in NGS for template amplification: emulsion PCR, bridge amplification, and rolling circle amplification.

  • Emulsion PCR: Single-stranded DNA fragments (templates) are attached to the surface of beads, which are then compartmentalized into water-oil emulsion droplets. Each of the droplets capturing one bead acts as a PCR microreactor, producing amplified copies of the DNA template.
  • Bridge amplification: It is a technology used by Illumina to generate hundreds of identical strands of DNA, known as “clusters”, on the surface of the flow cell. The flow cell is exposed to the reagents for polymerase-based extension, and polymerization occurs when the ligated fragment “bridges” to the complementary oligo on the surface. Repeated denaturation and extension result in localized amplification of DNA templates.
  • Rolling circle amplification: DNA fragments (templates) are ligated with adapters and followed by circularization. These single-stranded circle DNA molecules then are copied by rolling circle amplification, resulting in a long single-stranded DNA comprising several head-to-tail copies of the circular template.
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.

Ju, J., Kim, D. H., Bi, L., Meng, Q., Bai, X., Li, Z., ... & Edwards, J. R. (2006). Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proceedings of the National Academy of Sciences, 103(52), 19635-19640.

Morozova, O., & Marra, M. A. (2008). Applications of next-generation sequencing technologies in functional genomics. Genomics, 92(5), 255-264.