Molecular beacons are
single stranded hairpin shaped
oligonucleotide probes. In the presence
of the target sequence, they unfold,
bind and fluoresce. The molecular beacon
chemistry is one of the chemistries
used to carry out a real time experiment.
Molecular Beacon
Structure
A molecular beacon consists
of 4 parts, namely
Loop: This is the 18-30 base pair region of the molecular beacon which is complementary to the target sequence.
Stem: The beacon stem sequence lies on both the ends of the loop. It is typically 5-7 bp long at the sequences at both the ends are complementary to each other.
5' fluorophore: Towards the 3' end of the molecular beacon, is attached a dye that fluoresces in presence of a complementary target.
3' quencher (non fluorescent): The quencher dye is covalently attached to the 3' end of the molecular beacon and when the beacon is in closed loop shape, prevents the fluorophore from emitting light.
Molecular Beacons
Functioning
Molecular beacons can
report the presence of specific nucleic
acids from a homogeneous solution. In
the presence of a complementary target,
the "stem" portion of the
beacon separates out resulting in the
probe hybridizing to the target.
As explained in the illustration above,
one end of the molecular beacon is tagged with
a fluorophore, and the other one is
tagged with a quencher. In the absence
of a complimentary target sequence,
the beacon remains closed and there
is no appreciable fluorescence. When
the beacon unfolds in the presence of
the complementary target sequence, the
fluorophore is no longer quenched, and
the molecular beacon fluoresces. The
fluorescence is easily detected in a
thermal cycler.
The amount of fluorescence at any given
cycle, or following cycling, depends
on the amount of specific product. For
quantitative PCR, molecular beacons
bind to the amplified target following
each cycle of amplification and the
resulting signal is proportional to
the amount of template.
Fluorescence is monitored and reported
during each annealing step when the
beacon is bound to its complementary
target. This information is then used
during PCR or RT-PCR (reverse transcriptase
PCR) experiments to quantify initial
copy number.
For endpoint analysis, PCR reactions
containing molecular beacons can be
run on any 96-well thermal cycler, then
read in a fluorescence reader.
Applications
of Molecular Beacons Include
SNP detection
Real-time nucleic acid detection
Real-time PCR quantification
Allelic discrimination and identification
Multiplex PCR assays
Diagnostic clinical assays
Molecular Beacons Versus Linear
Probes
While other systems use fluorescence
to detect the accumulation of PCR product,
molecular beacons add another level
of specificity due to the presence of
a distinct molecular probe apart from the primers.
In addition, the stem probe structure
of a molecular beacon makes it better
able to discriminate single base-pair
mismatches (compared to linear probes)
because the hairpin makes mismatched
hybrids thermally less stable than hybrids
between the corresponding linear probes
and their mismatched target.
Furthermore, unlike linear hydrolysis
probes, quenching of molecular beacons
has been shown to occur through a direct
transfer of energy from the fluorophore
to quencher. Consequently, a common
quencher molecule can be used, increasing
the number of possible fluorophores
that can easily be used as reporters.
This is an important advantage when
designing Real time PCR experiments in which several
molecular beacons with different colored
fluorophores are used to detect multiple
targets in the same tube (multiplexing).
The use of molecular beacons in diagnostic assays has thus been ever increasing. Diagnostic assays that aim at detecting single nucleotide polymorphisms, screening genetically diverse species and developing drugs through pharmacogenetic applications are now using molecular beacons based real time PCR assays. The ability of molecular beacons to fluoresce when bound specifically to a double stranded target and their accuracy to discriminate alleles, make them a powerful tool in the research lab armory. Molecular beacons can be successfully used to ascertain not only the presence or absence of a particular causative agent, but also in screening which antibodies will be effective against a particular strain of the causative organism.
Beacon Designer™; for designing real time PCR assays, was developed in consultation
with one of the inventors of molecular
beacon technique, Dr. Sanjay Tyagi.
Beacon Designer™ finds the best possible
primer pair, TaqMan® probes and molecular
beacons for single or multiplex real
time PCR assays. For designing primers,
Beacon Designer™ avoids cross homologies
identified by automatically interpreting
BLAST search results and template secondary
structures. The resultant primers
are highly specific and efficient. Beacon
Designer can be used to design primers
and probes for allele discrimination
in multiplex experiments and to evaluate
pre-designed assays as well. Verification
BLAST for primers and probes is also
made available.
Other than the design of molecular beacons, Beacon Designer™ also supports the following real time PCR chemistries.
TaqMan® Probes: Beacon Designer™ makes it easy to design TaqMan® probe based real time PCR assays. The TaqMan®probes that the program designs are specific and efficient.
SYBR® Green Assays: Perhaps the most commonly used real time PCR assays. Beacon Designer™ can design and evaluate SYBR® Green primers in minutes.
FRET Probes: With a single click, you can design FRET probes that are free of dimers and other secondary structures.
Scorpions®: Scorpions® primers and probes can be designed using Beacon Designer™ for specific detection of the target.
