Question

: Sanger sequencing is the method of dideoxy sequencing that we use in Bio 2450. It is a modified PCR reaction that has the dNTPS replaced with a mixture of dNTPs:ddNTPs. In this section, go into detail on how the addition of ddNTP alters the produced PCR fragments and describe how we ultimately get the sequence.

Answer 1

This Sanger sequencing method makes use of the mechanism of DNA synthesis by DNA polymerases. The procedure uses dideoxynucleoside triphosphate (ddNTP) analogs to interrupt DNA synthesis. When a ddNTP is inserted in the place of a dNTP, strand elongation is halted after the analog is added, because it lacks the 3'-hydroxyl group needed for the next step. The DNA to be Sequenced is used as the template strand, and a short primer, radioactively or fluorescently labeled, is annealed to it. By addition of small amounts of a single ddNTP, for example, ddCTP, to an otherwise normal reaction system, the synthesized strands will be prematurely terminated at some locations where C normally occurs. Given the excess of dCTP over ddCTP the chance that the analog will be incorporated whenever a dC is to be added is small. However, ddCTP is present in sufficient amounts to ensure that each new strand has a high probability of acquiring at least one ddC at some point during synthesis. The result is a solution containing a mixture of labeled fragments, each ending with a C residue. Each C residue in the sequence generates a set of fragments of a particular length, such that the different sized fragments, separated by electrophoresis, reveal the location of C residues. This procedure is repeated separately for each of the four ddNTPs, and the sequence can be read directly from an autoradiogram of the gel. Because shorter DNA fragments migrate faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the primer (the5' end), and the sequence is read (in the 5' to 3' direction) from bottom to top. The sequence obtained is that of the strand complementary to the strand being analyzed.