Probe designing in Real Time PCR

Parameter for designing Taqman probes 1. Tm criteria: The primer melting temperature (Tm) should be around 58-60°C, and TaqMan® probe Tm should be 10°C higher than the Primer Tm. The Tm of both the primers should be equal 2. Length criteria: Primers should be 15-30 bases in length 3. GC content: The G+C content should ideally be 30-80%. If a higher G+C content is unavoidable, the use of high annealing and melting temperature co-solvents such as glycerol would be deemed essential

Parameter for designing Taqman probes 4. GC clamp: The total number of Gs and Cs in the last five nucleotides at the 3' end of the primer should not exceed two. This helps to introduce relative instability to the 3' end of primers to reduce non-specific priming 5. Amplicon length: Maximum amplicon size should not exceed 400 bp (ideally 50-150 bases) 6. Runs and repeats: The probes should not have runs of identical nucleotides (especially four or more consecutive Gs), G+C content should be 30-80%, there should be more Cs than Gs, and not a G at the 5' end 7. Genomic DNA avoidance: False-positive results are obtained due to amplification of contaminating

Taqman probes applications 1. Quantitative real time PCR. 2. DNA copy number measurements. 3. Bacterial identification assays. 4. SNP genotyping. 5. Verification of microarray results

Parameter for designing Molecular Beacon to detect the synthesis of products during polymerase chain reactions, any region within the amplicon that is outside the primer binding sites can be selected In order to discriminate between amplicons that differ from one another by as little as a single nucleotide substitution, the length of the probe sequence should be such that it dissociates from its target at temperatures 7-10 ˚C higher than the annealing temperature of the PCR If single-nucleotide allele discrimination is not desired, longer and more stable probes can be chosen Loop: This is the 15-30 base pair region of the molecular beacon which is complementary to the target sequence

Parameter for designing Molecular Beacon ensured by choosing a stem that melts 7-10˚C higher than the annealing temperature of the PCR 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 have a very high GC content (75 to 100%) DNA folding program, such as the Zuker DNA folding program available on the internet at http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form1.cgi, is utilized to estimate the melting temperature of the stem

Parameter for designing Molecular Beacon In general, 5 basepair-long GC-rich stems will melt between 55 and 60˚C, 6 basepair-long GC-rich stems will melt between 60 and 65˚C, and 7-basepair long GC-rich stems will melt between 65 and 70˚C Do not use G residues near the end to which the fluorophore (5’) is attached (instead, use them at the end where the quencher (3’) is attached), G residues tend to quench the fluorophore Small stems within the probe's hairpin loop that are 2- to 3-nucleotides long do not adversely effect the performance of molecular beacons.

Parameter for designing Molecular Beacon no regions of substantial complementarity that may cause the molecular beacon to bind to one of the primers amplicons should be less than 150-basepairs long Molecular beacons are internal probes that must compete with the other strand of the amplicon for binding to the strand that contains their target sequence Having a shorter amplicon allows the molecular beacons to compete more efficiently, and therefore produces stronger fluorescence signals during real-time PCR smaller amplicons result in more efficient amplification

Parameter for designing Molecular Beacon the magnitude of the molecular beacon signal can be increased by performing asymmetric PCR, in which the primer that makes the strand that is complementary to the molecular beacon is present at a slightly higher concentration than the other prime Molecular beacons can also be designed with the help of a dedicated software package called 'Beacon Designer,' which is available from Premier Biosoft International (www.premierbiosoft.com) A detailed description on the design, synthesis and application of molecular beacons used for genotyping single nucleotide polymorphisms appeared in Marras SAE, Kramer FR, and Tyagi S (2003) Genotyping single nucleotide polymorphisms with molecular beacons. In Kwok, P. Y. (ed.), Single nucleotide polymorphisms: methods and protocols. The Humana Press Inc., Totowa, NJ, Vol. 212, pp. 111-128 Vet, J.A.M. and Marras, S.A.E. (2004) Design and optimization of molecular beacon real-time polymerase chain reaction assays. In Herdewijn, P. (ed.), Oligonucleotide synthesis: Methods and Applications. Humana Press, Totowa, NJ, Vol. 288, pp. 273-290

Molecular Beacon applications 1 . SNP detection 2 . Real-time nucleic acid detection 3 . Real-time PCR quantification 4 . Allelic discrimination and identification 5 . Multiplex PCR assays 6 . Diagnostic clinical assays

Parameter for designing Scorpion probes Amplicon length: The primer pair should be designed to give an amplicon of approximately 100-200 bp Secondary structures: The designed primers should be tested for hairpins and secondary structures. Ideally the primers should have as little secondary structure as possible Tm criteria: The Tm's of the two primers should be similar. Also, the stem Tm should be 5-10ºC higher than the probe Tm Complementary probe: The scorpion® should be written as the reverse complement of the target

Parameter for designing Scorpion probes Length criteria: Probe sequences should ideally be about 17-27 bases Also, the probe target should be 11 bases or less from the 3' end of the scorpion® Probe stem: The stem sequence can be of 6 to 7 bases, mostly Cs and Gs, avoiding motifs. The 5' stem sequence should begin with a C as G may quench the FAM Primer: Probe hybridization: There is always the possibility of the primer hybridizing to the probe element, this will lead to leniarization of the probe in an amplification-independent manner causing significant, target-independent fluorescence

Scorpion probes applications 1. SNP analysis 2. Real-time PCR 3. Allelic discrimination 4. Single tube genotyping assay

Parameter for designing SYBR Green primer Careful primer design is critical If primer dimers or other non-specific PCR products form, they will incorporate SYBR Green dye This may lead to inaccurate quantification, especially when detecting sequences of low abundance Amplicon length: The primer pair should be designed to give an amplicon of approximately 250-400 bp SYBR Green dye fluorescence depends on the presence of double-stranded DNA, so products shouldn't be too short G/C content should be 20-80%

Parameter for designing SYBR Green primer Template regions with obvious secondary structures or long runs of the same nucleotide should be avoided when designing primers as this hinders the primers from annealing and extension Avoid a 3' terminal T on primers if possible. Thymidine tends to misprime more readily than other bases Tm criteria: The Tm of 60-65 °C will be sufficient BLAST your primer sequences. Run a similarity search of primers against whole organism/non-redundant databases to determine if primers might anneal to other (unwanted) targets Run positive and negative controls with conventional endpoint PCR

Choosing a Flourophore & Quencher Fluorophores: Quenchers: FAM TET, VIC HEX JOE CY3 TAMRA CY3.5, Redmond Red Texas Red, ROX CY5 LC640 CY5.5 LC705 BHQ-3 BHQ-2 BHQ-1 QSY-7 TAMRA Eclipse DABCYL BHQ3 Currently not available. Unstable. They are working on the chemistry. DABCYL Good quencher for beacons not TaqMan probes. Requires CLOSE proximity for quenching. QSY7 Good quencher but difficult to make. Eclipse Dark quencher from Synthetic Genetics. Similar to DABCYL, may provide better results for Tex Red and ROX. Vendors:IDT is the most reliable in terms of quality of probes. However, turn around time is often 1 week. NOTE: HEX/TAMRA as reporters: Choose BHQ1 or 2. HEX and TAMRA can be difficult to purify but IDT does it well. CY5: The best quencher would be BHQ3 but currently unavailable due to the instability of the chemistry. So R&D is using BHQ2 now. Redmond Red is from Synthetic Genetics. The next slide shows which Fluorophore/Quencher combinations we recommend.

Choosing a Flourophore & Quencher BHQ3 Currently not available. Unstable. They are working on the chemistry DABCYL Good quencher for beacons not TaqMan probes. Requires CLOSE proximity for quenching QSY7 Good quencher but difficult to make Eclipse Dark quencher from Synthetic Genetics. Similar to DABCYL, may provide better results for Tex Red and ROX Vendors:IDT is the most reliable in terms of quality of probes. However, turn around time is often 1 week NOTE: HEX/TAMRA as reporters: Choose BHQ1 or 2. HEX and TAMRA can be difficult to purify but IDT does it well. CY5: The best quencher would be BHQ3 but currently unavailable due to the instability of the chemistry. So R&D is using BHQ2 now Redmond Red is from Synthetic Genetics

Choosing a Flourophore & Quencher List of dye and quencher TaqMan Probe – FAM and TAMRA (higher background) FAM and BHQ1 or BHQ2 (heat transfer, not light, low backgroud, suitable for multiplexing) Molecular Beacons – FAM and DABCYL FAM and BHQ1 Scorpion – same as Molecular Beacons

Choosing a Flourophore & Quencher Multiplexing – choose dye that are spectrally distinct from each other as possible or have little spectral overlap. Add in one reference dye Duplex reaction – FAM and HEX (JOE/VIC)(ROX as reference dye) Triplex reaction – FAM, HEX (JOE/VIC) and CY5 (ROX as reference dye) Quadriplex reaction – FAM, HEX (JOE/VIC), CY5 and Texas Red Quencher should be dark quencher, even thought TAMRA is an effective quencher, but emission spectra for TAMRA do have some overlap with other dyes like HEX

Real Time primer/probe Use desalted primer with probe-based detection chemistry If the primer are to be used with the SYBR Green I detection, it need to be HPLC purify Sequence specific probe should be double HPLC purified or PAGE purified Aliquot the primer or probe before storing at -20°C Multiple freeze/thaw cycles can damage oligos, probes especially fluorescent tags Primer – not more than 20 times, for probe – not more than 5 times

Real Time primer/probe Simple test can be performed to ensure that the probe is quencher properly Read the fluorescence from an aliquot of a probe Digest 100nM of probe with 10U DNase or S1 nuclease in 25ul 1X buffer Incubate at room temperature for 30 min Take a second fluorescence reading The reading should be increase >5000 counts in raw fluorescence signal (digestion free the fluorophore)

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