Nanopore-Based DNA Sequencing and DNA Motion Control - Nanopores: Sensing and Fundamental Biological Interactions - page 258

Much-lower-cost methods for DNA sequencing will be required to make
genome sequencing feasible for routine healthcare practice. Aggressive research
and development during the past decade resulted in a decrease of both cost and time
of sequencing by at least five orders of magnitude. The current faster and cheaper
sequencing methods are referred generically as “next-generation sequencing tech-
nologies” to distinguish them from the traditional Sanger sequencing.
11.1.2 Fluorescent In Situ Sequencing
Fluorescent in situ sequencing (FISSEQ), a method faster and cheaper than Sanger
Sequencing, is based on detecting the fluorescent signals of the added
deoxynucleotide triphosphates in real time. As demonstrated by Mitra et al. [ 4 ],
and illustrated in Fig. 11.3 , the target DNA molecules are first PCR amplified in a
polyacrylamide gel [ 5 ]. One strand of the amplified DNA is then covalently
Fig. 11.3 Fluorescent in situ sequencing. (a) Polony amplification. A library of linear DNA
molecules with universal priming sites is PCR amplified in a polyacrylamide gel. A single
template molecule gives rise to a polymerase colony or polony. (b) Fluorescent in situ sequencing.
Polonies are denatured, and a sequencing primer is annealed. Polonies are sequenced by serial
additions of a single fluorescent nucleotide. Adapted from [ 4 ]