DNA Sequencing and the Evolution of the “-Omics” - Insect Molecular Genetics - page 252

Figure 7.3 Dideoxynucleoside triphosphates (ddNTPs) (bottom) act as chain terminators because
they lack a 3 ′ -OH group found on normal deoxynucleotides (dNTPs) (top).
derivatives of dNTPs that do not contain a hydroxyl group at the 3 ′ position of
the deoxyribose ring ( Figure 7.3 ).
When ddNTPs are incorporated into the growing DNA chains instead of
dNTPs, that particular DNA molecule is terminated at that point. All four
dNTPs are present in each tube with each ddNTP, but the ratio is adjusted so
that ddNTPs are less abundant than dNTPs. This makes the incorporation of a
ddNTP a random event. The newly synthesized DNA molecules in a specific reac-
tion tube therefore are a mixture of DNA fragments of different lengths, each
with a fixed starting point (determined by the primer) but with variable ending
points. Thus, for example, in the reaction in which the chain is terminated when
thymines (T) are incorporated, a ddNTP does not always get incorporated into
the first site where a T occurs. Nor does a ddNTP necessarily get incorporated
into the chain where the second T occurs. However, over the length of the DNA
being sequenced, each site where a T is incorporated will have ddNTPs incor-
porated, so that a series of DNA molecules of different length are produced