Nanopore-Based DNA Sequencing and DNA Motion Control - Nanopores: Sensing and Fundamental Biological Interactions - page 257

Fig. 11.2 Principle of the chain-terminating method. Besides having the all the dNTPs, each of
four lanes has one of the four ddNTP, which, when incorporated, terminates the polymerase chain
reaction at different lengths. Adapted from [ 2 ]
sample with annealed template is loaded into four separate reaction buffers, each
containing the four standard radioactively or fluorescently labeled deoxynucleo-
tides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. Only one of the
four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) is added to each
reaction buffer at lower concentration than the standard deoxynucleotides. Since
the dideoxynucleotides can terminate DNA strand extension, the random incorporation
of dideoxynucleotides instead of the corresponding standard deoxynucleotides
results in various DNA fragments of varying lengths. The length information of the
DNA fragments is then extracted by gel or capillary electrophoresis with a resolution
of a single nucleotide. The length of each DNA fragment in a reaction buffer
corresponds to the distance between the primer to the DNA base complementary to
the added dideoxynucleotide in the target DNA sample.
Sanger sequencing has been an extremely successful technique for DNA
sequencing, and continues to be the gold standard when it comes to data quality
of DNA sequences. Through parallelization, automation and refinement of the
established Sanger's sequencing method, the Human Genome Project (HGP)
achieved a sequencing cost of $0.01/base in 2003 [ 3 ], which resulted in a cost of
about $30,000,000 to sequence the three billion bases of a human genome.