Microbiology, 2021

490 12 • Modern Applications of Microbial Genetics
number of DNA copies, and sometimes organisms, present in a sample. In clinical settings, qRT-PCR is used to
determine viral load in HIV-positive patients to evaluate the effectiveness of their therapy.
DNA Sequencing
A basic sequencing technique is the chain termination method, also known as the dideoxy method or the
Sanger DNA sequencing method, developed by Frederick Sanger in 1972. The chain termination method
involves DNA replication of a single-stranded template with the use of a DNA primer to initiate synthesis of a
complementary strand, DNA polymerase, a mix of the four regular deoxynucleotide (dNTP) monomers, and a
small proportion of dideoxynucleotides (ddNTPs), each labeled with a molecular beacon. The ddNTPs are
monomers missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a
chain (Figure 12.21). Every time a ddNTP is randomly incorporated into the growing complementary strand, it
terminates the process of DNA replication for that particular strand. This results in multiple short strands of
replicated DNA that are each terminated at a different point during replication. When the reaction mixture is
subjected to gel electrophoresis, the multiple newly replicated DNA strands form a ladder of differing sizes.
Because the ddNTPs are labeled, each band on the gel reflects the size of the DNA strand when the ddNTP
terminated the reaction.
In Sanger’s day, four reactions were set up for each DNA molecule being sequenced, each reaction containing
only one of the four possible ddNTPs. Each ddNTP was labeled with a radioactive phosphorus molecule. The
products of the four reactions were then run in separate lanes side by side on long, narrow PAGE gels, and the
bands of varying lengths were detected by autoradiography. Today, this process has been simplified with the
use of ddNTPs, each labeled with a different colored fluorescent dye or fluorochrome (Figure 12.22), in one
sequencing reaction containing all four possible ddNTPs for each DNA molecule being sequenced (Figure
12.23). These fluorochromes are detected by fluorescence spectroscopy. Determining the fluorescence color of
each band as it passes by the detector produces the nucleotide sequence of the template strand.
Figure 12.21
A dideoxynucleotide is similar in structure to a deoxynucleotide, but is missing the 3ʹ hydroxyl group (indicated by the
shaded box). When a dideoxynucleotide is incorporated into a DNA strand, DNA synthesis stops.
Figure 12.22 Frederick Sanger’s dideoxy chain termination method is illustrated, using ddNTPs tagged with fluorochromes. Using
ddNTPs, a mixture of DNA fragments of every possible size, varying in length by only one nucleotide, can be generated. The DNA is
separated on the basis of size and each band can be detected with a fluorescence detector.
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