3.4 Sequencing of DNA 41
(a) Disease diagnosis
(b) Drug discovery
(c) Toxicological research
(d) Environmental microbiology (Zhou 2002 )
Microarrays are particularly useful in studying gene
function. A microarray works by exploiting the ability
of a given mRNA molecule to bind specifically to, or
hybridize to, the DNA template from which it origi-
nated. By using an array containing many DNA sam-
ples, it is possible to determine, in a single experiment,
the expression levels of hundreds or thousands of genes
within a cell by measuring the amount of mRNA bound
to each site on the array. With the aid of a computer, the
amount of mRNA bound to the spots on the microarray
is precisely measured, generating a profile of gene
expression in the cell. It is thus possible to determine
the bioactive potential of a particular microbial metab-
olite as a beneficial material in the form of a drug or its
deleterious effect (Madigan and Martinko 2006 ).
When a diseased condition is identified through
microarray studies, experiments can be designed which
may be able to identify compounds, from microbial
metabolites or other sources, which may improve or
reverse the diseased condition.
In the environment, it can be used to determine the
nature of the microbial population without necessarily
isolating the organisms (Hinds et al. 2002b).
3.4 Sequencing of DNA
3.4.1 Sequencing of Short DNA Fragments
DNA sequencing is the determination of the precise
sequence of nucleotides in a sample of DNA. Two
methods developed in the mid-1970s are available:
The Maxim and Gilbert method and the Sanger method.
Both methods produce DNA fragments which are
studied with gel electrophoresis. The Sanger method is
more commonly used and will be discussed here. The
Sanger method is also called the dideoxy method, the
enzymic method. The dideoxy method gets its name
from the critical role played by synthetic analogues of
nucleotides that lack the -OH at the 3¢ carbon atom
(star position): dideoxynucleotide triphosphates
(ddNTP) (see Fig. 3.6). When (normal) deoxynucle-
otide triphosphates (dNTP) are used, the DNA strand
continues to grow, but when the dideoxy analogue is
incorporated, chain elongation stops because there is
no 3¢ – ¢ -OH for the next nucleotide to be attached to.
For this reason, the dideoxy method is also called the
chain termination method.
For Sanger sequencing, a single strand of the DNA to
be sequenced is mixed with a primer, DNA polymerase
I, an excess of normal nucleotide triphosphates and a
limiting (about 5%) of the dideoxynucleotides labeled
with a fluorescent dye, each ddNTP being labeled with
a different fluorescent dye color. This primer will deter-
mine the starting point of the sequence being read, and
the direction of the sequencing reaction. DNA synthesis
begins with the primer and terminates in a DNA chain
when ddNTP is incorporated in place of normal dNTP.
Because all four normal nucleotides are present, chain
elongation proceeds normally until, by chance, DNA
polymerase inserts a dideoxy nucleotide instead of the
normal deoxynucleotide. The result is a series of frag-
ments of varying lengths. Each of the four nucleotides is
run separately with the appropriate ddNTP. The mix
with the ddCTP produces fragments with C (cytosine);
that with ddTTP (thymine) produces fragments with T
terminals, etc. The fluorescent strands are separated
from the DNA template and electrophoresed on a poly-
acrilamide gel to separate them according to their
lengths. If the gel is read manually, four lanes are pre-
pared, one for each of the four reaction mixes. The read-
ing is from the bottom of the gel up, because the smaller
the DNA fragment the faster it is on the gel. A picture of
the sequence of the nucleotides can be read from the gel
(see Fig. 3.7). If the system is automated, all four areO
P P P C Thymine
1'
3' 2'4'5'OH H
O
P P P C Thymine
1'
3' 2'4'5'H H
Deoxthymidine triphosphate
(DTTP)Dideoxythymidine triphosphate
(DDTTP)Fig. 3.6 Normal and dedeoxy nucleotides