LightCycler™ Hybridization Probes
The most direct way to monitor PCR amplification for
quantification and mutation detection.
Brian Erich Caplin 1, Randy P. Rasmussen1, Philip S. Bernard2, and Carl T. Wittwer1,2
1
ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108
2
Department of Pathology, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132
I
ntroduction
Sequence detection with adjacent oligonucleotides
and fluorescence was suggested as early as 1985
(6). However, it was not until 1997 that fluorescent
hybridization analysis was demonstrated during PCR
(11). All reagents for both amplification and detection are added before temperature cycling is begun.
Sequence-specific probe hybridization occurs
during amplification, allowing real-time product
identification, quantification, and mutation detection
(1, 2, 8, 11, 13, 14). The LightCycler is the only instrument currently available for real-time fluorescent
hybridization probe analysis. Although other fluorescent probes and dyes (including SYBR ® Green I Dye
and TaqMan® Probes) can be used on the LightCycler Instrument (11), this article will focus on the
unique characteristics and advantages of fluorescent hybridization probes.
H
ybridization probe basics
The hybridization probe system consists of two fluorescently labeled oligonucleotides. A donor probe
labeled with fluorescein at the 3' end absorbs light
from the blue LED of the LightCycler Instrument. An
adjacent acceptor probe absorbs resonance energy
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from the donor probe. The acceptor probe is labeled
with a LightCycler specific fluorophore, LightCycler
Red 640 (LC Red 640). Fluorescence from the acceptor probe will only occur when both the donor probe
and the acceptor probe have annealed to the product. This process of transferring the energy from one
fluorescent dye, to a second fluorescent dye is called
Fluorescence Resonance Energy Transfer (FRET;
Figure 1). Hybridization probes use FRET to provide
a homogeneous real-time measure of amplification
product formation.
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The polymerase chain reaction (PCR) is perhaps the
most powerful modern tool available for today's
molecular biologist. Its extraordinary sensitivity
allows for the detection of only a few molecules of
DNA. The sensitivity of PCR is often complimented by
the specificity of hybridization techniques such as
Southern blotting or oligonucleotide hybridization.
Amplification and hybridization techniques are
usually performed separately. However, with the
recent development of LightCycler™ technology, PCR
amplification and hybridization probe detection occur
simultaneously in homogeneous solution. That is,
both amplification and hybridization analysis can proceed in the same reaction. Because the LightCycler
Instrument uses rapid cycling techniques (12), the
entire process is finished in 15–30 min.
Figure 1: Hybridization probes produce fluorescence when both are annealed to
a single strand of amplification product. The transfer of resonance energy from the
donor fluorophore (3'-fluorescein) to the acceptor fluorophore (5'-LC Red 640) is a process
known as fluorescence resonance energy transfer.
Three advantages to using hybridization probes are:
■ Fluorescence is the direct result of the hybridization of two independent probes. As expected, this
results in very high specificity.
■ Fluorescence from hybridization probes does not
depend on an irreversible cleavage of the probe by
polymerase exonuclease activity. Because the
fluorescence is reversible, the strand status and
melting temperature of the probes can be followed.
The probe melting temperature is sequence dependent, providing a simple and elegant method to
genotype mutations, including single base mutations (2, 8), and multiplex mutation analysis (1).
■ Hybridization probes are easy to design, synthesize, and optimize.
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ybridization probe design
The design of hybridization probes is straightforward. Following a few general principles will
ensure success:
■ Hybridization probes should anneal adjacent to each
other on the same strand of product (Figure 1).
■ The spacing between adjacent hybridization
probes is optimally one base. However, excellent
fluorescence transfer occurs with as many as five
bases between the two probes, and some observable fluorescence can occur with up to 20 intervening bases or more.
■ One probe is best labeled at the 3' end with fluorescein, and the other at the 5' end with LC Red
640. To prevent extension of the LC Red 640
labeled probe, the 3' end must be blocked by
phosphorylation.
■ Probe T m should be approximately 5–10°C higher
than the Tm of the primers. Usually, the probes are
23–35 bases in length with a G+C content ranging
from 38–60%.
■ Probe sequences that cause secondary structures
must be avoided, as for normal PCR primers.
M
utation detection with hybridization probes
Hybridization probes provide a simple and elegant
system for real-time detection of mutations, including single-base mutations (1, 2, 8). Only one reaction
and one set of probes are required for genotyping
with the LightCycler System. A melting curve of
hybridization probe fluorescense produces a highresolution “dynamic dot blot” that can easily
discriminate even the most stable single base
mismatches (2). Unlike a standard dot blot where
hybridization occurs at a single temperature, melting
curve analysis on the LightCycler Instrument simplifies the optimization of probe hybridization with
continuous monitoring of probe hybridization status
as the temperature changes. A single base mismatch
under the probe decreases the melting temperature
by as little as 3°C for G::T mismatches, to as great as
7–10°C for A::C mismatches. Typically the probe
should be designed to produce the greatest temperature change between the wild type and mutant
melting curves. Figure 3 demonstrates a typical
derivate melting curve for single base genotyping.
The optimal Tm difference between the two probes
will depend on the type of experiment that is being
performed. For detection and quantification, the Tm of
the hybridization probes should be the same (within
2ºC of each other). For mutation detection, the best
melting curves are obtained when the difference
between probe Tm is 5–10ºC. The probe with the
lowest stability should be positioned directly over the
mutation to be detected.
Q
uantification with hybridization probes
Real-time or kinetic PCR is a powerful method for
estimating the initial template copy number (7, 11,
14). Fluorescence is acquired once each cycle and
the fluorescence is plotted against the cycle number.
A typical titration experiment on the LightCycler
Instrument with hybridization probes is shown in
Figure 2.
In addition to hybridization probes, the double
stranded DNA binding SYBR ® Green I dye can also
be used for analysis of PCR products (11, 14), even
for quantification of low-copy transcripts (9). The
Light Cycler System is also compatible with duallabeled TaqMan ® Probes that are commonly labeled
with fluorescein (FAM) and rhodamine (TAMRA).
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Figure 2: Quantification of initial template copy number with
hybridization probes. Samples of ten-fold serial dilutions of template
(human genomic DNA) were amplified using primers and hybridization
probes, specific for the human β-globin gene. Template copy numbers are
10 (30 pg), 102 (300 pg), 103 (3 ng), 104 (30 ng), and 105 (300 ng). The
cycling conditions were 95°C for 0, 55°C for 10 sec. and 72°C for 5 s.
Temperature transition rates were programmed at 20°C/s. The 45 cycle
PCR was completed in 20 min.
H
ybridization probe synthesis
Hybridization probes with a single label are easier to
synthesize and characterize than dual-labeled oligonucleotides such as exonuclease probes (TaqMan),
hairpin probes (Molecular Beacons™), or hairpin
primers (Sunrise™ Primers). The single fluorescent
label can be added during or after automated oligonucleotide synthesis.
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H
ybridization probe characterization
Probe purity can be assessed by HPLC, PAGE gels,
and/or the concentration ratio of dye to oligonucleotide. This ratio can be calculated from two experimental absorbance values:
1. The absorbance at 260 nm (A 260).
2. The absorbance at the absorbance maximum of
the dye (Adye).
For fluorescein labeling, it is easiest to start with a fluorescein-coupled CPG-support. Such supports are
prelabeled with fluorescein and the oligonucleotide is
extended in the 5'-direction during synthesis. After
cleavage and deprotection, the result is a 3'-fluorescein-labeled oligonucleotide. LightCycler Red 640
(LC Red 640) is a special dye, optimized specifically
for use as a fluorescence acceptor for hybridization
probes. It is currently available for addition to aminoderivatized oligonucleotides. The N-hydroxysuccinimide ester of LC Red 640 is reacted with a 5'-amino
linker attached to the desired oligonucleotide. The
result is a 5'-labeled LC Red 640 probe. The 5'-labeled
probe must be phosphorylated on its 3' end to prevent
extension of the probe during the thermal cycling
reaction. This is best achieved by starting the oligonucleotide synthesis on a modified CPG-support. A
3'-fluorescein labeled probe and a 5'- LC Red 640
labeled probe make up a single hybridization probe
pair. Reverse phase HPLC of the labeled oligonucleotide is highly recommended for purification.
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The predicted absorbance of the unlabeled oligonucleotide at 260 nm (nmol/A260) is calculated from
nearest neighbor values [3] or conveniently from
commercial software such as Oligo 4.0 (National
Biosciences). Finally,
[dye] = Adye/ εdye
[oligo] = [A260– (Adye x ε260(dye)/εdye)]/[106/(nmol/A260)]
The ratio [dye]/[oligo] should be about 1.0, indicating that on average, one dye molecule is present for
each oligonucleotide.
Dye
Fluorescein
LC Red 640
Absorbance
Maximum
ε dye
(nm)
(M –1cm–1)
494
622
68,000
110,000
ε 260 (dye)
(M–1cm–1)
Emission
Maximum
(nm)
12,000
31,000
524
638
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Figure 3: Derivative melting curve (-dF/dT) showing single
base genotyping. Samples are wild type (black) with a perfect
match to the hybridization probe and a melting temperature of
60°C, the mutation (red) with a C::A mismatch to the hybridization probe and a melting temperature of 54°C, and a heterozygous
(yellow) sample with both wild type and mutant alleles.
Table 1: LightCycler dye fluorescence constants*
* Spectral data obtained in 50 mM Tris, 3 mM MgCl2 , pH 8.3.
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S
ummary
Hybridization probes are simple to design and to
use. They are effective in such powerful applications
as real-time quantification and mutation detection
by high resolution melting curves. Rapid cycling and
fluorescence monitoring allow complete amplification and analysis in less than 30 min. Although this
article has focused on hybridization probes, other
fluorescent dyes and probes can be used on the
LightCycler System. For example, the use of SYBR ®
Green I for real time analysis of PCR was first introduced on the LightCycler System (11). In addition,
the most commonly used TaqMan probes can be
analyzed in real-time on this system.
The LightCyclerTM technology is licensed from Idaho Technology
Inc., Idaho, USA.
References
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