Designing Real-Time Assays on the SmartCycler II System

Transcription

1 Designing eal-time Assays on the SmartCycler II System Cepheid Technical Support Overview This document provides general guidelines for the design of real-time experiments on the Cepheid SmartCycler II System. The basic principles involved in designing PC experiments for simple or complex targets are similar; however additional care must be taken when designing primers and probes for complex targets and organisms. Genomes of higher organisms are not only large in size, but they are also riddled with repeat elements (LINEs, SINEs, alu), pseudogenes, and large duplications. s and probes should be designed to avoid these more complex regions. General steps in designing real-time experiments: 1. Selecting a target sequence. 2. unning a homology search. 3. Selecting a real-time chemistry. 4. Designing a specific probe. 5. Designing primers. Here we discuss tips and guidelines to facilitate your experimental design. More detailed information on optimizing your PC reaction performance is covered in SmartNote 6.2, Optimizing & Analyzing eal-time Assays on the SmartCycler II System. unning a Homology Search The first step in designing a real-time experiment is selecting a unique target sequence. Homology searches are a necessary step in identifying and avoiding complex sequence regions and other homologous sequences in the genome. With the sequencing efforts of the human, mouse, rat and other genomes nearly complete, homology searching has been greatly simplified. Figure 1: Gene on interest (cdna sequence) on chromosome X The following public domain sites provide sequence analysis programs: For example, to design an T-PC assay for a gene on chromosome X that has a 1600-bp mna, a BLAST search was run using the mna sequence. Figure 1 shows the homology between the mna of interest and homologous sequences in other parts of the genome. The sequence between bp exhibits homology to other sequences. For specificity, it is advisable to not select primers in this region of the gene. defining on-demand molecular diagnostics.

2 Designing Probes The following section gives a general overview of chemistries that are frequently used with the SmartCycler II System and describes the general considerations to keep in mind while designing probes for each. TaqMan Probes: TaqMan probes are short oligonucleotide probes labeled with a fluorescent reporter dye at the 5 end and a quencher at the 3 end (Figure 2). The probe chemistry utilizes the 5-3 exonuclease activity of Taq DNA polymerase for fluorescence detection and is sometimes referred to as a hydrolysis probe. The probes should be designed to anneal to the target sequence downstream from one of the primers. As the Taq polymerase extends the primers, the exonuclease activity cleaves the hybridized probe. This cleavage serves to separate the reporter from the quencher, thus increasing the fluorescence signal proportional to the number of amplicons generated. Figure 2: TaqMan probe hydrolysis during the amplification process A. Polymerization B. Probe Displacement C. Probe Cleavage D. Signal increases with cycles proportionately with cycles DNA = eporter = eporter = uencher = uencher Probe When designing TaqMan probes, use the following Probe melting temperature (T m ): The probe T m should be 5-10 C higher than the T m of the primer pair. The higher probe T m allows the probe to anneal to its complement before binding of the PC primers, thus ensuring maximum exonuclease activity during the primer extension phase. Without efficient probe cleavage, PC amplification can occur but the amplicon will not be detectable. Sequence length: The probe sequence should be bases long with a reporter dye on the 5 end, and a quencher dye on the 3 end, or at an internal nucleotide position. For selecting reporter dyes and quenchers, see SmartNote 6.3, Dye-uencher Considerations for the SmartCycler II System. GC content: Limit the GC content to 30 60% of the total sequence. Terminal G residues: Avoid placing a G residue on the 5 end of the probe. Guanine has a quenching effect, especially on fluorescein derivatives. It is recommended that the dye be attached to a C or T residue. Molecular Beacon Probes: Molecular Beacons, often referred to as conformational probes, are hairpin-shaped oligonucleotide probes containing a fluorophore covalently attached to the 5 end, and a non-fluorescent quencher fused to the 3 end (figure 3). The probe chemistry requires a complementary target for fluorescence detection. The probe itself has two self-complementary sequences at the 5 and 3 ends, allowing the probe to form a stem-and-loop structure. In the absence of a complementary target, the probe prefers to exist in a hairpin configuration. The proximity of the quencher to the reporter serves to quench the fluorescence emission of the reporter. When the probe hybridizes to a complementary target, the reporter dye is separated from the quencher dye, allowing the reporter to fluoresce. For the probe to anneal and remain in the hybridized conformation to the target sequence, it must form a hybrid that is even more stable than the hairpin, a property often exploited for allelic discrimination. During primer extension, the probe is displaced from the target and returns to the stem-and-loop formation. 2

3 Figure 3: Molecular Beacons during the amplification process A. A. Polymerization DNA = eporter = uencher eporter = uencher Probe This probe chemistry requires annealing to a complementary target for fluorescence detection. In the absence of a target, the quencher absorbs the fluorescence emitted by the reporter dye via contact quenching. After one cycle of PC extension, the newly synthesized target region is attached to the same strand as the probe. As amplification continues, the probe sequence contained within the loop portion of the probe anneals to the newly synthesized template strand, separating the reporter dye from the quencher and producing fluorescence. When designing Molecular Beacons probes, use the following Probe melting temperature (T m ): The probe T m should be 8-10 C higher than the primer T m. The higher probe T m ensures that the probe remains annealed to its complementary target prior to primer extension. Stem sequence: The stem sequence should be GC-rich (>80% of the total sequence) to form a stable duplex that is 5-8 bases long. The stem sequence should not be complementary to the target sequence. Loop sequence: The loop sequence should be bases long. The length of the loop determines specificity of the probe. Therefore, smaller loop sequences are less likely to hybridize to mismatched base pairs. This qpc chemistry is often exploited for developing SNP assays. Secondary structures: Use available software such as Zucker (reference) to determine the potential of the probe to form secondary structures. Higher order structures other than the hairpin configuration can impede quenching of the reporter dye and cause high background, poor signal, lower sensitivity, and specificity. Each Scorpions probe can be tagged with different fluorophores to produce different emission wavelengths to facilitate multiplexing. Because the probe is physically coupled with the primer, the reaction generates signal via unimolecular kinetics. The blocker is present on the Scorpion probe to limit non-specific amplification. Cycling is performed at a temperature optimal for DNA polymerase activity, making it an excellent choice for allelic discrimination. Figure 4: Scorpion probes during the amplification process Target-specific region A. Scorpion = eporter = uencher B. Scorpions primer extended on target DNA C. Extended primer is heat-denatured Blocker Target DNA C. Extended primer is heat-denatured Terminal G residues: Avoid placing a G residue on the 5 end because G has a quenching effect on fluorescein derivatives. Scorpions -Probes: Scorpions probes are bifunctional molecules that have a primer covalently linked to the probe (figure 4). Scorpions probes contain a reporter dye attached to the 5 end, followed by a complementary stem-and-loop structure (the loop contains specific probe sequence), a quencher dye, a DNA polymerase blocker, and a PC primer on the 3 end. D. Cooling causes scorpion to rearrange and flouresce in a target-specific manner 3

4 When designing Scorpions probes, use the following Figure 5: LUX primer-probes during the amplification process Probe melting temperature (T m ): The stem sequence T m should be 5-10 C higher than the probe T m. Amplicon length: Design the primer-probe to produce an amplicon of approximately bp. Secondary structures: Test the designed primers for hairpins and secondary structures. The primers should have few secondary structures. Lux eporter Dye uenched Fluorphore DNA Length criteria: The probe sequence should be bases long. The probe target should be 11 bases or fewer from the 3 end. The probe sequence should be designed so that it complements the target. Stem sequence: The stem sequence is 6-7 bases long, consisting of C and G residues. Terminal G residues: Avoid placing a G residue on the 5 end because guanine has a quenching effect on fluorescein derivatives. sequence: Check for primer-and-probe hybridization. If the primer hybridizes to the probe, the probe will remain linear during amplification, causing target-independent fluorescence that will contribute to background fluorescence. LUX -Probes: The Light Upon Extension (LUX ) primer-probes are oligonucleotides labeled with a single reporter dye, custom-synthesized according to the DNA or NA of interest. Typically bases long, the primer-probes are designed with a fluorescein-based reporter dye close to the 3 end in a hairpin structure (Figure 5). This configuration, a variation from the Sunrise primer-probe, renders fluorescence quenching capability so that a separate quenching moiety is not needed. When the primer is incorporated into a double-stranded PC product, the reporter dye is dequenched, resulting in a significant increase in fluorescence signal. To use the LUX platform, design one fluorogenic primer labeled with a single reporter dye and one corresponding unlabelled primer. The fluorogenic primer can be either the forward or reverse primer. For detailed information about LUX primer-probes, contact Invitrogen Corporation. When designing the LUX primer-probes, use the following Probe melting temperature (T m ): The stem sequence T m should be 5-10 C higher than the probe T m. Amplicon length: Design the primer-probe to produce an amplicon of approximately bp. Secondary structures: Test the designed primers for hairpins and secondary structures. The primers should have few secondary structures. Length criteria: The probe sequence should be bases long. The probe target should be 11 bases or fewer from the 3 end. The probe sequence should be designed so that it complements the target. Stem sequence: The stem sequence is 6-7 bases long, consisting of C and G residues. Terminal G residues: Avoid placing a G residue on the 5 end because guanine has a quenching effect on fluorescein derivatives. sequence: Check for primer-and-probe hybridization. If the primer hybridizes to the probe, the probe will remain linear during amplification, causing target-independent fluorescence that will contribute to background fluorescence. Sunrise -Probes: The Sunrise primer-probes are bifunctional molecules similar to Scorpions primer-probes (figure 6). The Sunrise primer-probes have reporter dyes attached to 3 end of the stem and quenchers attached to the 5 end of the stem. 4

5 The probe chemistry requires a complementary target for fluorescence detection. In the absence of a complementary target, the primer-probe retains the hairpin structure, and the proximity of the quencher to the reporter quenches fluorescence emission of the reporter. During the first few cycles of amplification, the probe hybridizes to the target, holding the reporter dye away from the quencher, thus producing fluorescence. The Sunrise primer-probes are not suitable for allelic discrimination. For detailed information about the Sunrise primer-probes, contact Oncor. Figure 6: Sunrise primer-probes during the amplification process = eporter = uencher DNA sequence: Check for primer-and-probe hybridization. If the primer hybridizes to the probe, the probe will remain linear during amplification, causing target-independent fluorescence that will contribute to background fluorescence. MGB Eclipse Probes: MGB Eclipse probes contain a Minor Groove Binding (MGB) peptide moiety in a three-dimensional conformation (figure 7). The MGB moiety and quencher molecule are attached to the 5 end of the probe. The reporter dye is attached to the 3 end of the probe. The probe chemistry requires a complementary target for fluorescence detection. In the absence of a complementary target, the probe remains in its three-dimensional conformation, suppressing flourescence. In the presence of a target, the probe anneals to the target, separating the reporter dye from the quencher to produce fluorescence. For detailed information about the MGB Eclipse probes, contact Epoch Biosciences. Figure 7: Eclipse probes during the amplification process When designing the Sunrise primer-probes, use the following A. A. Denaturation (95 C) (95 C) D MGB moiety Eclipse probe = eporter D = Dark uencher Probe melting temperature (T m ): The stem sequence T m should be 5-10 C higher than the probe T m. Amplicon length: Design the primer-probe to produce an amplicon of approximately bp. B. Hybridization B. Hybridization (56 C) (56 C) TA D Secondary structures: Test the designed primers for hairpins and secondary structures. The primers should have few secondary structures. C. Extension C. Extension (76 C) (76 C) D Length criteria: The probe sequence should be bases long. The probe target should be 11 bases or fewer from the 3 end. The probe sequence should be designed so that it complements the target. Stem sequence: The stem sequence is 6-7 bases long, consisting of C and G residues. Terminal G residues: Avoid placing a G residue on the 5 end because guanine has a quenching effect on fluorescein derivatives. When designing the MGB Eclipse probes, use the following Probe melting temperature (T m ): The probe T m should be 5-10 C higher than the primer T m. The higher probe T m ensures that the probe remains annealed to its complementary target before primer extension begins. Note that the moiety stabilizes probe-target binding and 5

6 increases the probe melting temperature, allowing the use of shorter probe sequences and providing greater allelic discrimination. Amplicon length: Design the probe to produce an amplicon of approximately bp. Length criteria: The probe sequence should be bases long. Terminal G residues: Avoid placing a G residue on the 5 end because guanine has a quenching effect on fluorescein derivatives. General Guidelines for Designing Probes: For optimal results, the probe should be designed before the PC primer pair. In addition, make sure the probe s complement is located within the amplicon sequence, but does not overlap the primer binding sequences. For allelic discrimination assays, the probe should be designed such that the SNP location is within the middle-third of the probe. For example, if the probe length is 21 nucleotides, place the SNP between positions 8 and 13. The following are general recommendations for working with dual-labeled probes: Purification: Make sure real-time PC probes are highly purified to minimize background signal and to ensure optimal results. Purification is especially critical for quantitative assays, multiplex assays, and any dye that requires an ester link. The following is an overview of available purification methods: - Desalting: emoves cleaved protecting groups but does not remove truncated products. Desalting is routinely performed by most oligo probe vendors. - P-HPLC (reverse-phase HPLC): Separates oligos by charge and results in a high level of purity and yield. - Dual HPLC: Anion Exchange HPLC followed by PHPLC. - PAGE (polyacrylamide gel electrophoresis): Separates oligos based on molecular weight. This method removes most truncated products and has a tendency to result in lower overall yields. - HPLC + PAGE: HPLC followed by PAGE for double purification. This purification method will generally result in the highest level of purity but the lowest yield. Storage: Always resuspend and store probes according to the manufacturer s recommendations. esuspend lyophilized probes to a 100 µm stock solution in Tris ph 8.0 or nuclease-free water. Prepare a working stock at a final concentration between 10 and 25 µm in Tris, ph 8.0 in µl aliquots. Store all probes at -20 C protected from light to protect against photo-bleaching and degradation. Avoid repeated freeze/thaw cycles to reduce degradation and maintain probe integrity. Working concentration: Empirically determine the optimal concentration of every new probe. The optimal assay concentration will depend on the amount of background signal, probe/complement binding efficiency, PC amplification efficiency and primer concentration. In general, the probe concentration can range from µm. When optimizing a real time PC assay with a newly synthesized probe, 0.2 µm is an acceptable working concentration at which to begin. To minimize background fluorescence, always titrate to the least amount of probe necessary to obtain adequate results without compromising assay sensitivity. Fluorescence background readings in a SmartCycler II assay should be below 500 fluorescence units. Background levels higher than 500 fluorescence units might indicate a poorly purified probe, the use of too much probe in the master mix, or degradation. Designing s The basic concepts of PC primer design are the same regardless of the complexity of the starting template. Choose primers so that they have a unique sequence within the template DNA that is to be amplified. The sensitivity of the PC amplification is dependent on the specificity and uniqueness of the primers. If the primers are not specific, they will bind to other nucleic acid sequences within the mix and amplify non-specific sequences, possibly sacrificing assay sensitivity. Ideally, primers should be designed after the probe and they should flank, but not overlap, the probe sequence. When designing PC amplification primers, use the following Length of Amplicons: The amplicon sequence should be bp in length to maintain a high degree of PC amplification efficiency in qpc assays. 6

7 GC Content of Amplicon: The GC content of the target sequence should be 40-55% GC avoiding palindromes or homo polymers in the amplicon sequence. Length of the s: The primer length should be approximately nucleotides long. If the primer composition is GC-rich, the length can be shortened to maintain a similar T m to AT-rich primers. Note that there is no rule for primer length. Forward and reverse primers can be of different lengths, but the T m of the forward and reverse primers should be within 0.5 C of each other for optimal performance. most applications will use between 0.2µM to 0.5µM. Use the least amount of primer possible to avoid primerdimer formation and to balance the primer and probe concentration. Forward and reverse primer concentrations might need to be titrated independently to give the best results. Melting temperature (T m ): The primer T m should be 5-10 C lower than the probe T m. Cepheid recommends a lower limit of no less than 55 C. The optimal T m for PC primers depends on a combination of the Mg+ 2 concentration and the annealing temperature of the PC assay. Use an oligonucleotide analysis program to analyze Mg+ 2 concentration for calculating the primer T m. Composition of the s: s with secondary or hairpin structures should be avoided. Additionally, a primer that folds back on itself will result in an overall decreased signal. It is best to design primers that are balanced in AT: GC composition. GC% should not exceed more than 60% and primers should contain no more than a three-base homo-polymer. To avoid primer-dimer formation, primers should also not contain complementary sequences that would allow self-annealing or annealing to the other primer (especially in multiplexed assays). When using intercalating dyes as a detection chemistry, one must consider more carefully the length and GC composition of the PC primers. Longer primers have a tendency to form higher order structures that can be bound by these non-specific dsdna binding dyes, thereby background fluorescence in the assay. Several oligonucleotide and nucleic acid analysis programs are available which can be used to determine secondary structure and primer-dimer potential. If possible, design multiple forward and reverse primers that flank the probe region and test various combinations or permutations of forward and reverse primers with the probe and choose the primer set that gives the lowest Ct value and highest end-point fluorescence. After the primer and probe have been designed, the optimal annealing temperature, primer concentration, and probe concentration must be determined. The primer concentration can range from 0.1µM to 1.0µM. However, 7

8 eferences Amersham Biosciences Product Specification Fluorolink Cy3-d-CTP PA etrieved July 18, 2003, from Annovis, Manual for the design and use of oligonucleotide specialty fluorescent probes. Behlke, M., Kelly, G., Dark uenchers. Integrated DNA Technologies Technical Bulletins. etrieved July 18, 2003, from bulletin/dark_uenchers. Behlke, M., Tao, W., Oligonucleotide Purification. Integrated DNA Technologies Technical Bulletins. etrieved July 18, 2003, from bulletin/oligonucleotide_purification. Behlke, M., esuspending Dry Oligos. Integrated DNA Technologies Technical Bulletins. etrieved July 18, 2003, from bulletin/esuspending_lyophilized_oligos. Bonnett, G., et al., Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Nat. Acad. Sci.96, pp Clegg,.M Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 211: Innis, M., Gelfand, D., Sninsky, J., PC Applications: Protocols For Functional Genomics. Academic Press, San Diego, CA. Perkin Elmer, TaqMan Universal PC Master Mix Protocol, ev A. Wong, Marisa L. and Medrano, Juan F. (July 2005) eal-time PC for mna quantitation. BioTechniques, 39(1): SmartCycler and I-COE are registered trademarks of Cepheid. Alexa Fluor, Texas ed, and SY are registered trademarks of Molecular Probes, Inc. Black Hole uencher and BH are trademarks of Biosearch Technologies. Eclipse is a trademark of Epoch Biosciences, Inc. Scorpions is a trademark of DxS Ltd. Sunrise is a trademark of Oncor. TaqMan is a registered trademark of oche Molecular Systems. Cy is a trademark of Amersham Biosciences. All other trademarks are the property of their respective owners. COPOATE HEADUATES 904 Caribbean Drive Sunnyvale, CA USA toll free: phone: fax: EUOPEAN HEADUATES Vira Solelh Maurens-Scopont France phone: fax: