Gene Cloning

Forensic and Medical Applications 431
As with most diagnostic tests molecular beacons are usually used in conjunction
with a PCR reaction. Amplification is required to produce enough
DNA to hybridize with sufficient of the beacon molecules to yield a
detectable level of fluorescence. In fact the molecular beacon can be added
to the assay mixture before PCR amplification and fluorescence measured
in real time in a development of real time PCR (Section 3.18). The number
of cycles required to reach a given level of fluorescence can be used as a
measure of the amount of target sequence in the original sample. This provides
a method not only for detecting the target sequence but also for
quantification. Using this quantitative approach it is possible to distinguish
between homozygous and heterozygous individuals using a single
probe as the homozygotes will have twice as much of one allele as the heterozygote.
Molecular beacons used in conjunction with real time PCR can
also be useful in the diagnosis of infectious disease (see Section 13.11).
Molecular beacon assays offer an additional safeguard in diagnostic
testing because they require both PCR amplification and annealing of the
molecular beacon to the product in order to generate a signal. In other
words they require three oligonucleotides to anneal, the two PCR primers
and the molecular beacon, reducing the chances of a false positive resulting
from mispriming by PCR primers.
Because fluorophores are available which emit a range of different colors
of fluorescence (Box 5.2), it is possible to use molecular beacons in conjunction
with multiplex PCR in order to test for several different mutations
at the same time. Each different mutation is tagged with a different fluorophore
allowing the results of the different tests to be distinguished from
each other. Most common genetic disorders are caused by more than one
type of mutation, either in a gene or the upstream sequences regulating its
expression. In this case it is necessary to test for a range of mutations in
order to accurately diagnose the condition. For a disease like cystic fibrosis
(Box 13.2) it is possible to test for seven of the most common mutations in
a single multiplex PCR reaction using molecular beacons labeled with
seven different fluorophores.
Fluorogenic 5′ exonuclease assay (TaqMan®)
This assay also makes use of dual-labeled fluorescent probes (Figure 13.8)
which are commercially available as TaqMan® probes. The target DNA is
amplified by PCR and the TaqMan® probe is included in the reaction.
When the target DNA becomes single-stranded during the denaturation
step the probe hybridizes to its complementary sequence but cannot fluoresce
because of the proximity of the quencher. However, as PCR proceeds
the probe is degraded by the 5′ exonuclease activity of Taq polymerase,
releasing it into solution where it emits a fluorescent signal. In each successive
cycle of the PCR more probe molecules will anneal, and then be
degraded releasing the fluorophore and increasing the intensity of the