Molecular biology: DNA sequencing
Author: Prof Marinda Oosthuizen
Licensed under a Creative Commons Attribution license.

What is DNA sequencing?
produce a sequence of coloured bands.
The biological information of DNA is contained in its
nucleotide sequence. DNA sequencing is the experimental
The mixture is separated on an electrophoresis gel to

process of determining the exact order of nucleotide bases
The sequence of coloured bands indicates the order of
the nucleotides in the constructed strands.
(adenine, guanine, cytosine, and thymine) in a DNA
molecule. In simple terms, the process involves making many
copies of the base sequence that match the code of a single
strand of DNA. These copies vary in length by one
nucleotide; the shortest is only one nucleotide long, and the
longest matches the entire DNA strand.
There are several methods that can be used in DNA
sequencing. The first methods were developed in the 1970s,
and are very laborious and time consuming. The most
popular and common sequencing reaction used today is
dideoxynucleotide sequencing, which can be done by hand

The sequence in the constructed strands can be
or by machines, depending on the quantity of material to be
converted to the sequence of nucleotides in the original
sequenced.
DNA strand by using the matching base-pair rules
(adenine with thymine and cytosine with guanine).

These are the steps involved:

screen as a set of coloured peaks. These peaks also
The double helix of the DNA segment is separated into
single strands.


DNA sequencing results may be viewed on a computer
indicate the order of nucleotides in the constructed
copies, and the coloured graph is interpreted in the
The single-stranded DNA is mixed with the four
same way as the coloured bands in the electrophoresis
nucleotides to construct matching strands.
results.
Some of the nucleotides are slightly different from the
normal
nucleotides
–
they
are
known
as
dideoxynucleotides. Inclusion of a dideoxynucleotide
terminates
DNA
strand
synthesis.
The
dideoxynucleotides are also fluorescent — each type of
base represents a different colour.

During the sequencing reaction, many matching strands
are constructed. Construction of any one strand stops
when a fluorescent dideoxynucleotide is added to the
sequence.
Why clone before sequencing a gene?

Sequencing large templates: Since it is only possible
to obtain 800-1000 bp from a single sequencing reaction,
it is difficult to sequence larger templates. Such larger
DNA templates must first be broken up into smaller
overlapping fragments which can be cloned into a vector
for sequencing. These subclones should overlap, so that
the individual DNA sequences will themselves overlap.
The overlaps can then be located, either by eye or using
a computer, and the master sequence gradually built up.

Mixed infections: If there is any possibility that the PCR
product might contain a mixed sequence, i.e. more than
one template (for example, if the sample contains a
mixture of similar organisms), the primers might amplify
the gene of interest from more than one organism. In this
case, it will be necessary to clone the PCR product and
sequence individual clones.
Following completion of the online module or parts thereof,
practicals will be scheduled to allow you to acquire the
necessary skills.