III.

Assignment
1. What is the purpose of dideoxyribonucleoside triphosphates, or ddNTPs?
Dideoxyribonucleoside triphosphates, or ddNTPs are used in DNA sequencing. Its
purpose is to act as chain terminators to create a set of DNA fragments of different lengths.The
ddNTPs lack a 3' hydroxyl group, which is essential for the formation of a phosphodiester bond
between adjacent nucleotides during DNA synthesis. This missing hydroxyl group prevents
further DNA synthesis once a ddNTP is incorporated into the growing DNA chain.
The Sanger sequencing technique involves the addition of ddNTPs with dNTPs and a
DNA polymerase in a reaction mixture to generate a set of partially elongated DNA strands.
Incorporating a ddNTP into a growing DNA chain terminates the extension reaction and
produces a DNA fragment of a defined length. These fragments can be generated by using
different concentrations of dNTPs and ddNTPs, thereby creating a ladder of DNA fragments with
varying lengths that enable the underlying DNA sequence to be deduced. The ladder is
subjected to electrophoresis for evaluation, with the sequence being read by detecting the
fluorescent labels bonded to different ddNTPs.
2. What is the difference between the first steps of DNA sequencing to PCR in terms of
the number of primers being used?
In DNA sequencing, a single primer is typically used to initiate DNA synthesis. This
primer is complementary to a specific region of the DNA template, and DNA polymerase
extends from this primer to replicate the DNA strand. On the other hand, in PCR, two primers
are used. These primers are designed to specifically bind to the DNA sequences flanking the
target region to be amplified.