Four part reaction tubes would be necessary, each one containing radioactively labeled DNA primers, DNA Polymerase II, and an plenty quantity of all 4 dNTP (dATP, dTTP, dGTP, dCTP), each to be included into the DNA strand being synthesize as a nucleotide. In adding, each reaction tubes would contain a different ddNTP, allowing each tube to identify a dissimilar nucleotide along the strand. For example, one tube would hold a ddATP, enabling that reaction tube to identify all the A's being integrated into the synthesize strand, and thus all the T's in the balancing template strand (recall that T nucleotides are complementary and base pair with A nucleotides).
All 4 dNTPs and a different ddNTP are additional to each reaction tube in a ratio of around 300:1, and Polymerase will randomly integrate either a dNTP or a ddNTP into the synthesizing strand if the ddNTP complements with the nucleotide on the template strand. As a result, a reacting that has ddATP would integrate a dGTP, dCTP, and dTTP if the template strand's nucleotide was C, G, or A, in that order. If the template strand's nucleotide was T, however, Polymerase will arbitrarily integrate either dATP or ddATP. If it integrates dATP, the strand will keep on to synthesize, which is what generally happens 97% of the time. If it integrates a ddATP, however, the reaction for that strand of DNA is terminated immediately, and will be of that size for good. This process is frequent with three other tubes with ddGTP, ddCTP, and ddTTP.
Once this response is ran to completion, it is then put onto a flat slab of polyacrylamide gel and an electric present is practical in a process called PolyAcrylamide Gel Electrophoresis. This allows for the separation of strands of DNA one base-pair apart, allowing one to resolve the sequence of the template strand by viewing the gel in a process called Autoradiography. Because the primers added were radioactive, an image of it can be taken, where every time strands of DNA are encountered on the gel, a band appears. The end product is a gel with a banding pattern that looks similar.
In an automated sequencing process, instead of labelling the primers with radioactive labels, the ddNTP is labeled with a fluorescent label, where each ddNTP would fluoresce a different color when a laser is fired through it. Unlike the autoradiography, which will show a band of the same color regardless of the ddNTP, this method will fluoresce a different color for each of the four different nucleotides.
Thus, this allows for the sequencing reaction to occur in one tube, as each ddNTP would fluoresce a different color and identify the nucleotide in the sequence, Thus, using computers, the amount of time it takes to sequence strands of DNA is significantly shortened. Without this computing technology, large scale sequencing projects would be impossible to even initiate, much less complete. This paved the way for projects such as the Human Genome Project, which was initiated by the NIH in 1991
