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Hybridization Probes the Most Direct Way to Monitor PCR Amplification for Quantification and Mutation Detection

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Hybridization Probes the Most Direct Way to Monitor PCR Amplification for Quantification and Mutation Detection LightCycler™ Hybridization Probes The most direct way to monitor PCR amplification for quantification and mutation detection. Brian Erich Caplin1, 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 IIntroduction The polymerase chain reaction (PCR) is perhaps the from the donor probe. The acceptor probe is labeled most powerful modern tool available for today's with a LightCycler specific fluorophore, LightCycler molecular biologist. Its extraordinary sensitivity Red 640 (LC Red 640). Fluorescence from the accep- allows for the detection of only a few molecules of tor probe will only occur when both the donor probe DNA. The sensitivity of PCR is often complimented by and the acceptor probe have annealed to the prod- the specificity of hybridization techniques such as uct. This process of transferring the energy from one Southern blotting or oligonucleotide hybridization. fluorescent dye, to a second fluorescent dye is called Amplification and hybridization techniques are Fluorescence Resonance Energy Transfer (FRET; R E usually performed separately. However, with the Figure 1). Hybridization probes use FRET to provide L C recent development of LightCycler™ technology, PCR a homogeneous real-time measure of amplification Y C amplification and hybridization probe detection occur product formation. T H G simultaneously in homogeneous solution. That is, I both amplification and hybridization analysis can pro- L ceed in the same reaction. Because the LightCycler Instrument uses rapid cycling techniques (12), the entire process is finished in 15–30 min. Sequence detection with adjacent oligonucleotides and fluorescence was suggested as early as 1985 (6). However, it was not until 1997 that fluorescent Figure 1: Hybridization probes produce fluorescence when both are annealed to hybridization analysis was demonstrated during PCR a single strand of amplification product. The transfer of resonance energy from the (11). All reagents for both amplification and detec- donor fluorophore (3'-fluorescein) to the acceptor fluorophore (5'-LC Red 640) is a process tion are added before temperature cycling is begun. known as fluorescence resonance energy transfer. Sequence-specific probe hybridization occurs during amplification, allowing real-time product Three advantages to using hybridization probes are: identification, quantification, and mutation detection ■ Fluorescence is the direct result of the hybridiza- (1, 2, 8, 11, 13, 14). The LightCycler is the only instru- tion of two independent probes. As expected, this ment currently available for real-time fluorescent results in very high specificity. hybridization probe analysis. Although other fluores- ■ Fluorescence from hybridization probes does not cent probes and dyes (including SYBR® Green I Dye depend on an irreversible cleavage of the probe by and TaqMan® Probes) can be used on the Light- polymerase exonuclease activity. Because the Cycler Instrument (11), this article will focus on the fluorescence is reversible, the strand status and unique characteristics and advantages of fluores- melting temperature of the probes can be followed. cent hybridization probes. The probe melting temperature is sequence depen- dent, providing a simple and elegant method to genotype mutations, including single base muta- Hybridization probe basics tions (2, 8), and multiplex mutation analysis (1). The hybridization probe system consists of two fluo- ■ Hybridization probes are easy to design, synthe- rescently labeled oligonucleotides. A donor probe size, and optimize. labeled with fluorescein at the 3' end absorbs light from the blue LED of the LightCycler Instrument. An adjacent acceptor probe absorbs resonance energy ROCHE MOLECULAR BIOCHEMICALS BIOCHEMICA · No. 1 n 1999 5 CONTENTS HHybridization probe design Mutation detection with hybridization probes The design of hybridization probes is straight- Hybridization probes provide a simple and elegant forward. Following a few general principles will system for real-time detection of mutations, includ- ensure success: ing single-base mutations (1, 2, 8). Only one reaction ■ Hybridization probes should anneal adjacent to each and one set of probes are required for genotyping other on the same strand of product (Figure 1). with the LightCycler System. A melting curve of ■ The spacing between adjacent hybridization hybridization probe fluorescense produces a high- probes is optimally one base. However, excellent resolution “dynamic dot blot” that can easily fluorescence transfer occurs with as many as five discriminate even the most stable single base bases between the two probes, and some observ- mismatches (2). Unlike a standard dot blot where able fluorescence can occur with up to 20 inter- hybridization occurs at a single temperature, melting vening bases or more. curve analysis on the LightCycler Instrument simpli- ■ One probe is best labeled at the 3' end with fluo- fies the optimization of probe hybridization with rescein, and the other at the 5' end with LC Red continuous monitoring of probe hybridization status 640. To prevent extension of the LC Red 640 as the temperature changes. A single base mismatch labeled probe, the 3' end must be blocked by under the probe decreases the melting temperature phosphorylation. by as little as 3°C for G::T mismatches, to as great as ■ Probe Tm should be approximately 5–10°C higher 7–10°C for A::C mismatches. Typically the probe R than the Tm of the primers. Usually, the probes are should be designed to produce the greatest temper- E L 23–35 bases in length with a G+C content ranging ature change between the wild type and mutant C Y from 38–60%. melting curves. Figure 3 demonstrates a typical C T ■ Probe sequences that cause secondary structures derivate melting curve for single base genotyping. H G I must be avoided, as for normal PCR primers. L 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. Quantification with hybridization probes Q Figure 2: Quantification of initial template copy number with Real-time or kinetic PCR is a powerful method for hybridization probes. Samples of ten-fold serial dilutions of template estimating the initial template copy number (7, 11, (human genomic DNA) were amplified using primers and hybridization 14). Fluorescence is acquired once each cycle and probes, specific for the human b-globin gene. Template copy numbers are the fluorescence is plotted against the cycle number. 10 (30 pg), 102 (300 pg), 103 (3 ng), 104 (30 ng), and 105 (300 ng). The A typical titration experiment on the LightCycler cycling conditions were 95°C for 0, 55°C for 10 sec. and 72°C for 5 s. Instrument with hybridization probes is shown in Temperature transition rates were programmed at 20°C/s. The 45 cycle Figure 2. PCR was completed in 20 min. In addition to hybridization probes, the double stranded DNA binding SYBR® Green I dye can also HHybridization probe synthesis be used for analysis of PCR products (11, 14), even Hybridization probes with a single label are easier to for quantification of low-copy transcripts (9). The synthesize and characterize than dual-labeled oligo- Light Cycler System is also compatible with dual- nucleotides such as exonuclease probes (TaqMan), labeled TaqMan® Probes that are commonly labeled hairpin probes (Molecular Beacons™), or hairpin with fluorescein (FAM) and rhodamine (TAMRA). primers (Sunrise™ Primers). The single fluorescent label can be added during or after automated oligo- nucleotide synthesis. 6 BIOCHEMICA · No. 1 n 1999 ROCHE MOLECULAR BIOCHEMICALS CONTENTS HHybridization probe characterization Probe purity can be assessed by HPLC, PAGE gels, and/or the concentration ratio of dye to oligonucleo- tide. This ratio can be calculated from two experi- mental absorbance values: 1. The absorbance at 260 nm (A260). 2. The absorbance at the absorbance maximum of the dye (Adye). Figure 3: Derivative melting curve (-dF/dT) showing single The predicted absorbance of the unlabeled oligonu- base genotyping. Samples are wild type (black) with a perfect cleotide at 260 nm (nmol/A260) is calculated from match to the hybridization probe and a melting temperature of nearest neighbor values [3] or conveniently from 60°C, the mutation (red) with a C::A mismatch to the hybridiza- commercial software such as Oligo 4.0 (National tion probe and a melting temperature of 54°C, and a heterozygous (yellow) sample with both wild type and mutant alleles. Biosciences). Finally, [dye] = Adye/ edye 6 [oligo] = [A260– (Adye x e260(dye)/edye)]/[10 /(nmol/A260)] For fluorescein labeling, it is easiest to start with a flu- The ratio [dye]/[oligo] should be about 1.0, indicat- orescein-coupled CPG-support. Such supports are ing that on average, one dye molecule is present for R E prelabeled with fluorescein and the oligonucleotide is each oligonucleotide. L C extended in the 5'-direction during synthesis. After Y C cleavage and deprotection, the result is a 3'-fluores- T Absorbance Emission H G cein-labeled oligonucleotide. LightCycler Red 640 I Dye Maximum e e Maximum L (LC Red 640) is a special dye, optimized specifically dye 260 (dye) (nm) (M–1cm–1) (M–1cm–1) (nm) for use as a fluorescence acceptor for hybridization probes. It is currently available for addition to amino- Fluorescein 494 68,000 12,000 524 derivatized oligonucleotides. The N-hydroxysuccin- LC Red 640 622 110,000 31,000 638 imide 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.
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