Introduction

Platinum® qPCR SuperMix for SNP Genotyping is a ready-to-use reaction mix for the amplification and identification of single-nucleotide polymorphisms (SNPs) in genomic DNA using PCR-based SNP genotyping technologies such as fluorescent primers or probes. The SuperMix has been specifically formulated for discrimination of alleles by real-time qPCR or end-point PCR followed by allelic-discrimination analysis on a real-time instrument or fluorescent microplate reader. It provides enhanced fluorescent signals for better discrimination of alleles and good separation with minimal scattering between replicate samples. The SuperMix format and integrated UDG carryover prevention make this reagent well suited for high-throughput applications.
 
This SuperMix has been developed using Applied Biosystems’ TaqMan®-based SNP genotyping products.
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Materials

Platinum® qPCR SuperMix for SNP Genotyping is supplied at 2X concentration and contains Platinum® Taq DNA polymerase, Tris-HCl (pH 8.4), 6 mM MgCl2, 400 µM dGTP, 400 µM dATP, 400 µM dCTP, 800 µM dUTP, uracil DNA glycosylase (UDG), and PCR enhancer and proprietary stabilizers.

  • Platinum® Taq DNA polymerase is recombinant Taq DNA polymerase complexed with specific monoclonal antibodies that inhibit polymerase activity at room temperature. Full polymerase activity is restored after the denaturation step in PCR cycling, providing an automatic “hot start” in PCR and significantly increasing amplification efficiency, sensitivity, and yield.
  • UDG and dUTP are included in the SuperMix to prevent the reamplification of carryover PCR products between reactions. dUTP ensures that any amplified DNA will contain uracil. UDG, or uracil-N-glycosylase, removes uracil residues from single- or double-stranded DNA, preventing dU-containing DNA from serving as template in future PCRs. Incubation of subsequent PCRs with UDG before cycling destroys any contaminating dU-containing PCR product from previous reactions. After this decontamination step, UDG is inactivated by the high temperatures during normal PCR cycling, thereby allowing the amplification of genuine target sequences.

Platinum® qPCR SuperMix for SNP Genotyping is supplied at 2X concentration to allow for the addition of template, primers, and probes. ROX Reference Dye is provided as a separate tube to normalize the fluorescent signal on instruments that are compatible with this option.
 
Reagents are provided for 250 or 1250 amplification reactions of 20 µl each.
 
Component                                                                   250-rxn Kit                        1250-rxn Kit
Platinum® qPCR SuperMix for
SNP Genotyping                                                              2 × 1.25 ml                             12.5 ml
ROX Reference Dye                                                           500 µl                                   500 µl
 
Storage
 
Store components at –20ºC for long-term storage. After initial use, Platinum® qPCR SuperMix for SNP Genotyping may be stored at 4ºC for up to one month to minimize freeze-thawing. For periodic use over several months, we recommend preparing aliquots of SuperMix to minimize freeze-thawing.
 
Store ROX Reference Dye in the dark.
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Ordering Information

Sku Name Size Price Qty
CS11040 ChargeSwitch® gDNA Serum Kit, 0.2-1 mL 50 preps USD 334.00
CS11000 ChargeSwitch® gDNA Blood Kit, 50-100 µL 50 preps USD 198.00
CS11203 ChargeSwitch® gDNA Micro Tissue Kit 50 preps USD 228.00
CS11204 ChargeSwitch® gDNA Mini Tissue Kit 25 preps USD 80.00
CS11021 ChargeSwitch® gDNA Buccal Cell Kit 50 preps USD 211.00
CS11020 ChargeSwitch® gDNA Normalized Buccal Cell Kit 50 preps USD 211.00

General Protocol

The following protocol uses TaqMan® probes in a SNP genotyping assay on ABI real-time instruments or a standard thermal cycler. Note the separate cycling conditions for the ABI 7500 in Fast Mode, and the lower amount of ROX Reference Dye required for the ABI 7500 and 7500 Fast systems. This generic protocol may also be suitable with some modifications for other real-time instruments.

For additional information about specific instruments click here . Since PCR is a powerful technique capable of amplifying trace amounts of DNA, all appropriate precautions should be taken to avoid cross-contamination.

  1. Program the real-time instrument or standard thermal cycler to perform a brief UDG incubation immediately followed by PCR amplification, as shown below. Optimal cycling temperatures and times may vary for different target sequences and primer/probe sets

  2. Standard Cycling Program Fast Cycling Program (for the ABI 7500 in Fast Mode)
    50°C for 2 minutes hold (UDG incubation) 95°C for 2 minutes hold 40 cycles of: 95°C, 15 seconds 65°C, 30 seconds (60 seconds for theSelect Fast Mode on the Thermal Profile tab 50°C for 2 minutes hold (UDG incubation) 95°C for 2 minutes hold 40 cycles of: 95°C, 3 seconds 65°C, 30 seconds



  3. Prepare each reaction in a microcentrifuge tube or PCR plate wellas specified below.For multiple reactions, prepare a master mix of common components, add the appropriate volume to each tube or plate well, and then add the unique reaction components (e.g., template).

    Note:   Preparation of a master mix is essential in quantitative applications to reduce pipetting errors.

    Component Single reaction
    Platinum® qPCR SuperMix for SNP Genotyping10 µl
    Forward primer, 10 µM0.4 µl
    Reverse primer, 10 µM 0.4 µl
    Probe for SNP allele 1, 10 µM0.2 µl
    Probe for SNP allele 2, 10 µM0.2 µl
    Template (100 pg to 1 µg of genomic DNA) 1 µl
    ROX Reference Dye, 25 µM (optional) 0.4 µl/0.04 µl *
    DEPC-treated waterto 20 µl

    *See Support Protocol 3 - ROX Reference Dye or the amount/concentration of ROX to use for your specific instrument.

  4. Cap or seal the reaction tube/PCR plate, and gently mix. Make sure that all components are at the bottom of the tube/plate; centrifuge briefly if needed.

  5. Place reactions in the thermal cycler programmed as described above and run the program.

  6. For real-time instruments, perform real-time analysis and/or an allelic-discrimination end-point reading at the end of the run. For standard thermal cyclers, transfer the PCR product to a fluorescent microplate reader for SNP genotyping analysis.
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Support Protocol 1: Template

The target template for SNP genotyping is purified genomic DNA. For a 20-µl reaction, use 10 ng to 1 µg of purified genomic DNA in a 1µl volume. To purify genomic DNA, we recommend the PureLink™ or ChargeSwitch® genomic DNA purification kits from Invitrogen.
 
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Support Protocol 2: Magnesium Concentration

Platinum®qPCR SuperMix for SNP Genotyping includes magnesium chloride at a final concentration of 3 mM, which is optimal for SNP genotyping experiments.
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Support Protocol 3: ROX Reference Dye

ROX Reference Dye is included in each kit to normalize the fluorescent reporter signal for instruments that are compatible with this option. ROX is supplied at a 25 µM concentration, and is composed of a glycine conjugate of 5-carboxy-X-rhodamine, succinimidyl ester in 20 mM Tris-HCl (pH 8.4), 0.1 mM EDTA, and 0.01% Tween® 20. Use the following table to determine the amount of ROX to use with a particular real-time instrument per 20-µl reaction:

Instrument ROX per 20-µl reaction* Final Concentration
ABI 7000, 7300 7700, 7900HT, and 7900HT Fast0.4 µl500 nM
ABI 7500; Stratagene Mx3000™, Mx3005P™, and Mx4000™0.04 µl50 nM

*To enable accurate pipetting, you can dilute the appropriate amount of ROX in DEPC-treated water immediately before use. For example, to add 0.4 µl per reaction, dilute 1:2.5 immediately before use and add 1 µl of the dilution to each reaction.
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Support Protocol 4: Instrument Settings

Platinum® qPCR SuperMix for SNP Genotyping can be used with real-time qPCR instruments that can detect three colors (one for each SNP, plus one for ROX Reference Dye). Supported real-time instruments include the ABI PRISM® 7000, 7700, and 7900HT; the ABI 7300 and 7500 Real-Time PCR Systems; the Stratagene Mx3000P®, Mx3005P™, and Mx4000® the Corbett Research Rotor-Gene™ and the MJ Research DNA Engine Opticon® and Opticon® 2.
 
You can also perform end-point PCR on a standard thermal cycler and analyze the results using a fluorescent microplate reader capable of detecting in three channels.
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Support Protocol 5: Detection Methods

SNP genotyping using fluorescent dual-labeled probe technology such as TaqMan® probes requires two PCR primers as well as two allele-specific probes that hybridize to the SNP portion of the amplicon. Predeveloped TaqMan® SNP genotyping assays are available from Applied Biosystems Assays-on-Demand, and include a mix of PCR primers and two allele-specific TaqMan® MGB Probes, labeled with FAM and VIC. Custom TaqMan® SNP genotyping probes labeled with FAM and VIC may be designed using Applied Biosystems’ Primer Express 2.0 software.
 
When designing custom probes, note that the probe sequences should be free of secondary structure and should not hybridize to each other or to primer 3´-ends. The optimal concentration of the probes may vary between 50 and 800 nM, with a recommended starting concentration of 100 nM each.
 
PCR primers used with custom probes should be designed according to standard PCR guidelines. They should be specific for the target sequence and free of internal secondary structure, and should avoid complementation at 3´-ends within each primer, with each other, or with the dual-labeled probes. Optimal results may require a titration of primer concentrations between 100 and 500 nM. A final concentration of 200 nM per primer is effective for most reactions.
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Troubleshooting


Problem
Possible Cause
Solution
No PCR product is evident in the qPCR graph or allelic-discrimination plot, or on a gel
The protocol was not followed correctly
Verify that all steps have been followed and the correct reagents, dilutions, volumes, and cycling parameters have been used.
Template contains inhibitors, nucleases, or proteases, or has otherwise been degraded.
Purify or re-purify your template.
 
Primer and/or probe design is suboptimal
Verify your primer and probe selections. We recommend using validated pre-designed primers or designing primers or primer/probe combinations using dedicated software programs or primer databases.
PCR product is evident in the gel, but not on the qPCR graph
qPCR instrument settings are incorrect
Confirm that you are using the correct instrument settings (dye selection, reference dye, filters, acquisition points, etc.) for your application.
 
Problems with your specific qPCR instrument
For instrument-specific information about probes, consult your instrument documentation and/or your probe technology documentation. For additional instrument-specific tips and troubleshooting, visit this page.

References

  1. Tyagi, S. and Kramer, F.R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology 14, 303.
  2. Tyagi, S., Bratu, D.P., and Kramer, F.R. (1998) Multicolor molecular beacons for allele discrimination. Nature Biotechnology 16, 49.
  3. Kostrikis, L.G., Tyagi, S., Mhlanga, M.M., Ho, D.D., and Kramer, F.R. (1998) Spectral genotyping of human alleles. Science 279, 1228.
  4. Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5'3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 88, 7276.
  5. Gibson, U. E. M., Heid, C. A., and Williams, P.M. (1996) A novel method for real-time quantitative RT-PCR. Genome Res. 6, 99
  6. Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Real-time quantitative PCR. Genome Res. 6, 986.
  7. Chou, Q., Russel, M., Birch, D., Raymond, J., and Bloch, W. (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucl. Acids Res. 20, 1717.
  8. Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., and Daiss, J.L. (1994) Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction. BioTechnology 12, 506.
  9. Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Focus® 19.2, 46.
  10. Longo, M., Berninger, M., and Hartley, J. (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93, 125.
  11. Lindahl, T., Ljungquist, S., Siegert, W., Nyberg, B., and Sperens, B. (1977) DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli. J. Biol. Chem. 252, 3286.
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