Gene Cloning

430 Gene Cloning
(a)
Target specific region
R Q
Molecular beacon
(b)
R
Q
Target
Figure 13.7 Using a molecular beacon to detect a disease gene. a) The
molecular beacon is a single-stranded DNA molecule with a fluorophore reporter
covalently linked to one end (R in the blue circle) and a quencher to the other
(Q in the gray circle). The central target specific region is an ASO
complementary to the target. Flanking this region are short sequences that are
complementary to each other. The complementary sequences cause the molecule
to adopt a hairpin loop structure which brings the reporter and quencher into
close proximity and prevents the reporter from fluorescing. b) Once mixed with
test DNA the target specific region hybridizes with the target region on the test
DNA; this puts enough distance between the reporter and quencher to allow the
reporter to fluoresce. If there is no complementary target DNA sequence the
molecular beacon will not anneal and there will be no fluorescence.
target sequence the target specific region will hybridize causing a conformational
change (Figure 13.7b). The fluorophore will now emit fluorescence.
In designing a molecular beacon the sequences are chosen so that
the perfectly complementary probe-template hybrid is more stable than the
hairpin loop structure. This means that the molecular beacons will preferentially
form the probe template hybrid in the presence of target sequence.
Like ASOs, molecular beacons are able to distinguish between alleles which
differ by as little as one nucleotide. If there is a single mismatch between
probe and target, the hybrid is less stable and the hairpin loop conformation
is favored, so no fluorescence is emitted. To distinguish between homozygous
and heterozygous individuals two molecular beacons are used, each
one specific for one or other allele and each labeled with a fluorophore of a
different color. If the test results in fluorescence of only one color then the
sample was from a homozygous individual, if both colors are produced then
the individual is heterozygous.
Q13.5. What parameters need to be considered when designing the
sequence of the molecular beacon to ensure that the probe target hybrid is
more stable than the hairpin structure?