TaqMan Plant Transgenic Screening NPTII Detection Kit ...tools.thermofisher.com/content/sfs/manuals/cms_041826.pdf · selectable marker in many plant transformations, particularly

TaqMan® Plant Transgenic Screening NPTII Detection Kit

Protocol

© Copyright 2007, 2010 Applied Biosystems

For Research Use Only. Not for use in diagnostic procedures.

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Use of the TaqMan® Plant Transgenic Screening NPTII Detection Kit is covered by US patent claims and corresponding patent claims outside theUS. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only thisamount of product solely in [environmental testing, quality control/quality assurance testing, and food testing] applications including reporting resultsof purchaser’s activities for a fee or other commercial consideration, and also for the purchaser's own internal research. No right under any otherpatent claim (such as apparatus or system claims for real-time PCR) is conveyed expressly, by implication, or by estoppel. Further information onpurchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404,USA.

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Part Number 904150 Rev. D

06/2010

November 2007 TaqMan Plant Transgenic Screening NPTII Detection iii

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ContentsIntroduction 1NPTII Detection Kit Components 4Technical Support 5

To Reach Us on the Web 5Hours for Telephone Technical Support 5To Reach Us by Telephone or Fax in North America 5Documents on Demand 7To Reach Us by E-Mail 7Regional Offices Sales and Service 8

Storage and Stability 10Materials Required But Not Supplied 10Model LS-50B TaqMan System 11

Upgrading to the LS-50B TaqMan System 11Reagent Preparation 12Information Regarding Protocols 13

Sources of Contamination 13Storage Conditions Prior to Analysis 13AmpErase UNG for Prevention of PCR Product Carryover 14Fluorescence Analysis 15

Reaction Protocol 16Temperature Cycling for the NPTII System 17Analysis by LS-50B Luminescence Spectrometer 18

PCR Cycle Optimization 19Annealing Temperature 19Two-temperature Cycles 19Extension Time 19High G+C Content DNA 20

Interpretation of Results 21Determination of Threshold ∆RQ 21Establishing Confidence Levels for Probe Performance 22

Performance Characteristics 23Appendix A Troubleshooting 24Appendix B Preparing Plant Genomic DNA 26Appendix C References 27

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iv TaqMan Plant Transgenic Screening NPTII Detection November 2007

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IntroductionThe NPTII gene encodes resistance to the antibiotic kanamycin and is used as a selectable marker in many plant transformations, particularly those methods involving Agrobacterium tumefasciens Ti plasmid. The Plant Transgenic Screening Kit for NPTII detection is designed to detect the NPTII gene with a 5' nuclease assay that uses TaqMan® fluorogenic probes. The kit includes the core TaqMan PCR reagents, a DNA positive control kit, and a set of primers with a TaqMan probe specific for amplification of a 193-bp region of the NPTII gene.

A 5' nuclease assay that uses a TaqMan fluorogenic probe provides a simple, reliable procedure for determining the presence or absence of a specific sequence. Direct detection of PCR product, with no downstream processing, is accomplished within minutes of PCR completion by monitoring the increase of fluorescence of a dye-labeled DNA probe. The ease and simplicity of this method, coupled with the plate reader capability of the Perkin-Elmer LS-50B Luminescence Spectrometer, permit the analysis of thousands of samples per day with high sample-to-sample reproducibility.

The NPTII Detection Kit employs a probe technology that exploits the fork-like-structure-dependent, polymerization-associated, 5'–3' nuclease activity of AmpliTaq® DNA Polymerase (Lawyer et al., 1989; Holland et al., 1991; Lyamichev et al., 1993) to allow direct detection of the PCR product by the release of a fluorescent reporter during PCR (Lee et al., 1993). The probe consists of an oligonucleotide with a 5'-reporter dye and a quencher dye. The fluorescent reporter dye, FAM, is covalently linked to the 5' end of the oligonucleotide. We have also used TET and HEX as reporter dyes. Each of the reporters is quenched by TAMRA, typically located at the 3' end.

When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence, primarily by Förster-type energy transfer (Förster, 1948; Lakowicz, 1983). During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5'–3' nucleolytic activity of the AmpliTaq DNA Polymerase cleaves the probe only if it hybridizes to the target. AmpliTaq DNA Polymerase does not digest free probe. The probe is then displaced from target and polymerization of the strand continues (Figure 1).

This process occurs in every cycle and does not interfere with the exponential accumulation of product. The separation of the reporter dye from the quencher dye results in increased fluorescence of the reporter. This increase in fluorescence is measured and is a direct consequence of target amplification during PCR (Figure 2).

November 2007 Introduction 1

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The specificity of this method results from the requirement of the enzyme for primer and probe complementarity to target in order for amplification and cleavage to occur. Thus, the fluorescence signal is generated only if the target sequence for the probe is amplified by the PCR. Due to these requirements, any non-specific amplification is not detected.

An Applied Biosystems LS-50B Luminescence Spectrometer with a plate reader accessory is used to monitor the increase of the reporter fluorescence following PCR. This increase in fluorescence is compared to the fluorescence of a No Template Control PCR. The results are calculated as outlined in “Interpretation of Results” on page 21 and defined as ∆RQ, the difference between the ratio of reporter fluorescence to quencher fluorescence in the Sample and No Template Control tubes.

Figure 1. Stepwise representation of the fork-like-structure-dependent, polymerization-associated, 5'–3' nuclease activity of AmpliTaq DNA Polymerase during one extension phase of PCR (Lyamichev et al., 1993).

2 TaqMan Plant Transgenic Screening NPTII Detection November 2007

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Figure 2. Comparison of post-PCR emission scans: Sample and No Template Control

The NPTII Reagent Kit contains enough PCR reagents for up to 200 (50 µL) reactions, and includes enough primers, probe, and template for 50 control reactions. The control reactions detect the amplification of a 193-bp region of the NPTII gene in plasmid pBI121, a 13-kb plant transformation vector derived from the Ti plasmid. During amplification, the positive control generates a reporter fluorescence signal that is significantly greater than that of the No Template Control.

Figure 3. NPTII amplicon, with positions of primers and probe sequences with respect to the gene coding sequence (Beck et al., 1982). See list of kit components for sequences of primers and probes.

Reporter (FAM) λem

Quencher (TAMRA) λem

Wavelength (nm)

Sample

No Template Control

Em

issi

on In

tens

ity

November 2007 Introduction 3

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NPTII Detection Kit ComponentsThe TaqMan Plant Transgenic Screening Kit for NPTII detection (P/N 402188) comprises two boxes: TaqMan PCR Core Reagents (P/N N808-0229) and TaqMan NPTII Detection Reagents (P/N 402158).

Table 1. TaqMan PCR Core Reagents (P/N 808-0229): 200 reactions

Table 2. TaqMan NPTII Detection Reagents (P/N 402158)

Reagent Volume Description

AmpErase™ UNG (P/N N808-0096 alone, P/N N808-0068 with dUTP)

100µL 1 tube of 1 U/µL uracil-N-glycosylase

dUTP 320 µL 1 tube of 20 mM deoxyuridine triphosphate dissolved in autoclaved, deionized, ultrafiltered water; titrated with NaOH to pH 7.0

dATP 320 µL 1 tube each of 3.2 µmol of dATP, dCTP, or dGTP at 10 mM concentration in autoclaved, deionized, ultrafiltered water; titrated with NaOH to pH 7.0.

dCTP 320 µL

dGTP 320 µL

AmpliTaq DNA Polymerase (P/N N801-0060)

50 µL 1 tube containing 5 U/µL AmpliTaq DNA Polymerase

10X PCR Buffer II 1.5 mL 1 tube of 500 mM KCl, 100 mM Tris-HCl, pH 8.3, room temperature (solution has been autoclaved).

25 mM MgCl2 Solution 3.0 mL 2 tubes of 25 mM MgCl2 solution, 1.5 mL each

Reagent Volume Description

NPTII Forward Primer 600 µL 1 tube of 5 µM primer in TE Buffer. Sequence: 5'-CACGACGGGCGTTCCTTGC-3' (Position: 213-231)

NPTII Reverse Primer 600 µL 1 tube of 5 µM primer in TE Buffer. Sequence: 5'-GGTGGTCGAATGGGCAGGTAGC-3' (Position: 406-385)

NPTII Probe 200 µL 1 tube of 2 µM of fluorescent probe in TE Buffer. Sequence: 5'-(FAM)-ACTGAAGCGGGAAGGGACTGGCT- (TAMRA)-p-3', where p indicates phosphorylation. (Position: 253-275)

Control DNA 50 µL 1 tube containing 100 fg/µL of plasmid pBI121 (Clontech) formulated in TE Buffer.


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Storage and StabilityUpon receipt, store the NPTII Detection Kit or its components at –15 to –25 °C in a constant-temperature freezer.

Materials Required But Not SuppliedIn addition to the reagents supplied in this kit, the following items are required. This list does not include equipment or reagents required for the synthesis of primers and probes or for DNA extraction. Many of the items listed are available from major laboratory suppliers unless otherwise noted. Equivalent sources may be acceptable where noted.

Reagents

Equipment

Deionized distilled water (DI H2O) major laboratory suppliers (MLS)

Methanol, HPLC grade MLS

Tris-EDTA (TE) Buffer Sigma (T9285)

GeneAmp PCR Instrument System Applied Biosystems, Norwalk, CT

Microcentrifuge Costar, Greenwich, CT (Cat. No. 8450) for 1.5-mL and 500-µL tubes, or equivalent

Microplates, 96-well, opaque white Applied Biosystems (P/N L225-1692)

Mineral Oil (used with the DNA Thermal Cycler 480)

Applied Biosystems (P/N 0186-2302)

PCR Reaction Tubes (see Table 4 on page 18) GeneAmp PCR Reaction Tubes ...(0.5 mL, polypropylene) GeneAmp Thin-Walled Reaction Tubes ...(0.5 mL, polypropylene) MicroAmp™ Reaction Tubes ...(0.2 mL polypropylene

Applied Biosystems (P/N N801-0180, N801-0280) Applied Biosystems (P/N N801-0737) Applied Biosystems (P/N N801-0533, N801-0540, N801-0553, N801-0580)

Pipettes, positive-displacement, and tips MLS

Pipettes and tips MLS

Vortexer MLS


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Model LS-50B TaqMan SystemThe LS-50B TaqMan System consists of the following:

• Model LS-50B Luminescence Spectrometer

• Plate Reader

• 485DF22 Interference Filter and Holder

• FLDM or WPR software

The LS-50B Luminescence Spectrometer is an optical unit that incorporates a pulsed xenon source. The excitation monochromator covers the wavelength range 200–800 nm.

The emission monochromator covers the wavelength range 200–650 nm, with the standard photomultiplier. Zero order can be selected and the emission slits are variable between 2.5 and 20 nm in 0.1 nm increments. An emission filter wheel is fitted and has the following positions, which can be selected through the software:

• open

• blank

• 1% attenuator

• cut-off filters (290, 350, 390, 430, and 515 nm)

The 515 nm cut-off filter is used in this protocol.

The system includes either Fluorescent Data Management (FLDM) or Well Plate Reader (WPR) software and Microsoft Excel-compatible macros and corresponding software manuals to facilitate data analysis.

The Plate Reader is used for measuring samples in 96-well microplates. Controlled by the software, the accessory can measure a chosen number of wells in either the X or Y direction.

Upgrading to the LS-50B TaqMan System

If you currently have an LS-50B Luminescence Spectrometer, call your Applied Biosystems Service Representative for information about upgrading to perform TaqMan PCR. See back cover for Applied Biosystems sales offices.

November 2007 Model LS-50B TaqMan System 11

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Reagent PreparationPreparation of Reagents Not SuppliedUse reagent grade chemicals unless otherwise noted. Prepare all solutions using deionized distilled water. Wear gloves and follow safety recommendations provided by manufacturer for handling chemicals. Comply with any and all laws, regulations, or orders with respect to the disposal of any hazardous or toxic chemical, material, substance or waste. Store all reagents at room temperature (15–25 °C), unless otherwise noted.

Preparation of Reagents SuppliedPrior to use, allow the kit components to thaw on ice. When the reagents are thawed, mix each tube component by vortexing gently.

Using a microcentrifuge, collect the tube contents at the bottom of the tube. Store the tube on ice until ready for use. When finished with the kit, return it to the –15 to –25 °C constant-temperature freezer. If the kit is to be used more than once every two days, it may be stored at 2–6 °C between uses.


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Information Regarding ProtocolsThe PCR formulations and thermal cycling conditions described in this protocol have been optimized for the controls found in the NPTII Kit. To avoid risk of PCR product carryover, use dUTP during optimization. Verify that the target DNA is being amplified by performing agarose gel electrophoresis and ethidium bromide staining of the PCR product.

We strongly recommend using a Master Mix cocktail, to reduce tube-to-tube variability.

Sources of Contamination

Because the interpretation of results depends upon the comparison of a Sample (containing DNA) to a No Template Control (without DNA), it is extremely important that all sources of contamination be anticipated and controlled.

All reaction mixes should be set up in an area isolated from PCR product analysis and sample preparation. The use of dedicated or disposable vessels, solutions, and pipettors (preferably positive displacement pipettors with disposable tips) for DNA preparation, reaction assembly, and sample analysis will minimize PCR product carryover and sample cross-contamination (Kwok, 1990; Orrego, 1990). See “Materials Required But Not Supplied” on page 10.

Since sample protein and fluorescent contaminants may interfere with this assay and give false positive results, it may be necessary to include a No Enzyme Control tube that contains target, but no enzyme. If, after PCR, the fluorescence of the No Enzyme Control is greater than that of the No Template Control, fluorescent contamination may be present in the sample.

Storage Conditions Prior to Analysis

Since AmpErase UNG has no effect on the fluorescence signal generated during TaqMan PCR, its use as a Hot Start method and in prevention of PCR product carryover is ideal. (Loewy et al., 1994; PCR Technical Information, 1995). AmpErase UNG is active below 55 °C, so the annealing temperature must be kept at or above this temperature. To prevent UNG digestion of amplicons before gel electrophoresis, hold the reaction at 72 °C until analyzed, or rapidly cool the reaction and store at –15 to –25 °C.

November 2007 Information Regarding Protocols 13

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AmpErase UNG for Prevention of PCR Product Carryover

The capability for single DNA molecule amplification provided by the PCR process (Saiki et al., 1985; Mullis and Faloona, 1987; Saiki et al., 1988) has required the adoption of special laboratory practices to avoid false positive amplifications (Higuchi and Kwok, 1989). The AmpErase UNG provided in this product (either the kit with dUTP or sold separately as P/N N808-0096) is a pure, nuclease-free, 26 kDa enzyme encoded by the Escherichia coli uracil N-glycosylase gene that has been inserted into an E. coli host to direct the expression of the native form of the enzyme (Varshney et al., 1988).

When dUTP replaces dTTP as a dNTP substrate in PCR and the method described below is used, AmpErase UNG treatment can prevent the reamplification of carry-over PCR products in subsequent experiments (Sninsky and Gelfand, 1995; Longo et al., 1990).

Because of the enormous amplification possible with GeneAmp PCR, small levels of DNA carry-over from samples with high DNA levels, positive control templates, or from previous PCR amplifications can result in product even in the absence of purposefully added template DNA. Although the protocol and reagents described here are capable of degrading or eliminating carried-over PCR products, we encourage users to continue using the specific devices and suggestions described in this protocol to minimize cross-contamination from non-dU-containing PCR products or other samples (Kwok, 1990; Higuchi and Kwok, 1989).

AmpErase UNG InactivationTen-minute incubation at 95 °C is necessary to cleave the dU-containing PCR product generated in the low temperature (18–50 °C) incubation, to reduce AmpErase UNG activity, and to denature the native DNA in the experimental sample. Because UNG is not completely deactivated during the 95 °C incubation, it is important to keep the reaction temperatures greater than 55 °C to prevent amplicon degradation. A ten-minute incubation at 95 °C does not affect significantly the activity of AmpliTaq DNA Polymerase, which has a half-life of approximately 35 minutes at 95 °C.

Preventing PCR Product Carryover Primers used should contain dA nucleotides near the 3' ends so that any primer dimer generated is efficiently degraded by AmpErase UNG at least as well as any dU-containing PCR products. The further a dA nucleotide is from the 3' end the more likely that partially degraded primer dimer molecules may serve as templates for a subsequent PCR amplification. Production of primer dimer could lower the amplification yield of the desired target region. If primers cannot be selected with dA nucleotides near the ends, the use of primers with 3' terminal dU nucleotides should be considered. Single-stranded DNAs with terminal dU nucleotides are not substrates for AmpErase UNG (Delort et al., 1985) and thus the primers will not be degraded. Biotin-dUMP derivatives are not substrates for AmpErase UNG.


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The concentration of AmpErase UNG and the time of the incubation step necessary to prevent amplification of contaminating dU-containing PCR product depends on the PCR conditions necessary to amplify your particular DNA sequence and the level of contamination expected. In most cases the general recommendation of using AmpErase UNG at 1 U/100 µL reaction and incubation at 50 °C for two minutes will suffice. If primer-dimer amplification dominates the PCR, reduce the length of the initial incubation with or without increasing the UNG concentration to 2 U/100 µL reaction.

Do not attempt to use AmpErase UNG in amplification of dU-containing PCR template, as in nested-PCR protocols. The UNG will degrade the dU-containing PCR product, preventing further amplification.

Since AmpliTaq DNA Polymerase has some activity at low temperatures before cycling begins, the polymerase can extend mispriming to single stranded regions common in isolated DNA or even produce artifacts from the primers. AmpErase UNG is most active near 50 °C, so it can eliminate many side-products produced during the preparation of Master Mixes up to the point where restriction temperatures are reached. Thus, it provides an alternative to AmpliWax™ PCR Gem 100 or 50 for producing the high specificity of Hot Starts when amplifying from approximately 1000 or less copies (Kwok et al., 1982; Chou et al., 1992).

Fluorescence Analysis

Erroneous results may arise from inadequately cleaned 96-well microplates or cuvettes. It is recommended that microplates be cleaned immediately after use to prevent PCR product and probes from drying onto the plate. The plate should be washed thoroughly with detergent and water, followed by thorough rinsing with DI water, followed by rinsing with HPLC-grade methanol.

To verify plate cleanliness, use the reporter and quencher emission wavelengths to monitor the empty plate for the presence of fluorescent residue prior to sample introduction. The well-to-well fluorescence of the cleaned 96-well microplate should be consistent and should not exceed 10 FU.

November 2007 Information Regarding Protocols 15

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Reaction ProtocolThe procedure, which has been optimized for the NPTII control, involves a PCR amplification of the target DNA followed by fluorescence analysis. Analysis requires that at least one buffer blank and three No Template Controls be run concurrently with the Test Samples. The No Template Control is the complete PCR formulation without the target DNA. Changes in any of the components other than the target DNA in the PCR formulation require a separate No Template Control.

We strongly recommend preparation of a Master Mix, shown in Table 3, containing all the kit components except the Control (target) DNA.

Table 3. Master Mix Preparation for 12 Reactions

Note The nucleotides are supplied in four separate vials. Combine equal amounts of each nucleotide in a separate tube before preparing the Master Mix.

To set up and run 12 NPTII reactions:

1. Thaw the reagents and store them on ice while preparing the Master Mix.

2. Prepare the Master Mix, allowing seven volumes of Master Mix for each set of six Samples and four volumes of Master Mix for each set of three No Template Controls.

3. Aliquot 343 µL (seven volumes) of Master Mix into one microcentrifuge tube labeled “Sample.”

Component Volume (µL) Final Concentration

DI H2O 327 —

10X PCR II Buffer 60 1X

25 mM MgCl2 60 2.5

dATP 12 200 µM

dCTP 12 200 µM

dGTP 12 200 µM

dUTP 12 400 µM

NPTII Forward Primer 36 300 nM

NPTII Reverse Primer 36 300 nM

NPTII probe 12 40 nM

AmpliTaq DNA Polymerase (5 U/µL) 3 0.025 U/µL

AmpErase UNG (1 U/µL) 6 0.01 U/µL

Mix Total 588


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4. Add 7 µL pBI121 Control DNA to the “Sample” tube. Cap it immediately and vortex gently to mix.

5. Aliquot 196 µL (four volumes) of Master Mix into a second microcentrifuge tube labeled “No Template Control.”

6. Add 4 µL of 1X TE Buffer, diluted according to the manufacturer’s procedure, to the “No Template Control” tube. Cap it immediately to prevent DNA contamination and vortex gently to mix.

7. Aliquot 50 µL from the “Sample” Master Mix tube into each of the six Sample reaction tubes.

8. Aliquot 50 µL from the “No Template Control” Master Mix tube into each of the three No Template Control reaction tubes.

Note If using the DNA Thermal Cycler 480, add 50–75 µL mineral oil to each tube as a vapor barrier.

9. Perform amplification by PCR as soon as all of the tubes have been prepared.

10. To read fluorescence, transfer 40 µL from each tube to individual wells of the microplate. Avoid transferring mineral oil to the microplate.

11. Run the control DNA template for 35 cycles.

Temperature Cycling for the NPTII System

The NPTII Detection Kit is optimized and quality-control tested for performance on the Applied Biosystems GeneAmp PCR System 9600. Table 4 on page 18 shows typical profile times for this kit with various Applied Biosystems thermal cyclers. Refer to the user’s manual for your particular instrument for details on operation.

November 2007 Reaction Protocol 17

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Table 4. Thermal Cycler Times and Temperatures for use with NPTII Control

Analysis by LS-50B Luminescence Spectrometer

Use a Applied Biosystems LS-50B Luminescence Spectrometer with a plate-reader attachment for fluorescence detection. For each plate, a well containing 1X PCR buffer must be included and used to autozero for each wavelength analyzed. Refer to the TaqMan PCR Software for Windows User’s Manual (P/N 903831) for details. In conjunction with the NPTII probe, we recommend the following configuration on the LS-50B shown in Table 5.

Table 5. LS-50B Configuration

Thermal Cycler

Tube Examples of Times and Temperatures

Tube TypeVolume in µL/Tube

(vapor barrier)

Each of 35 CyclesFinal Step

Initial Step Melt Anneal/Extend

DNA Thermal Cycler 480

GeneAmp PCR

Reaction

orGeneAmp

Thin-WalledReaction

STEP CYCLE STEP CYCLES SOAK

10–150(plus 50–75 µLvapor barrier)

2 min. 50 °C 10 min. 95 °C

1 cycle

1 min.92 °C

1 min. 60 °C

4 °C forever

GeneAmp PCR System 9600

MicroAmpReaction

5–100(no vapor barrier

needed)

HOLD CYCLE HOLD

2 min. 50 °C 10 min. 95 °C

30 sec.94 °C

45 sec60 °C

4 °C forever

GeneAmp PCR System 2400

MicroAmpReaction

5-100(no vapor barrier

needed)

HOLD CYCLE HOLD

2 min. 50 °C 10 min. 95 °C

30 sec.94 °C

45 sec.60 °C

4 °C forever

Slit widths

excitation 10 nm

emission 10 nm

Wavelengths

excitation 488 nm

emission reporter dye: 518 nm

quencher dye: 582 nm

Emission filters 515 nm long-pass cutoff

485DF22 interference

Read time 0.2 sec


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PCR Cycle OptimizationA typical PCR cycle consists of the following:

• a melting step (94–96 °C) to separate the complementary strands of DNA

• a primer annealing step (55–70°C, depending on the primers) to allow hybridization of the primers to the single stranded DNA and initiation of polymerization

• a primer extension step (72 °C) to complete the copy initiated during annealing

Begin with a template melting step (at least one minute at 94–96 °C), then start cycling. The final extension step allows polymerization to complete all strands. If using AmpErase UNG, all reaction temperatures must be greater than 55 °C.

Annealing Temperature

Higher annealing temperatures (>55 °C) generally result in much more specific product (Saiki et al., 1988; Rychlik et al., 1990). The optimal annealing temperature can be determined empirically by testing at 5 °C (or smaller) increments until the maximum in specificity is reached. At these temperatures, AmpliTaq DNA Polymerase has significant activity so extension of primed templates is occurring.

Two-temperature Cycles

Two-temperature rather than three-temperature cycles are recommended for the NPTII application. Since extension is completed at the anneal temperature, two-temperature cycles reduce the time needed to complete the amplification. For custom fluorescent probes with lower melting temperatures (Tm<70°C, under PCR conditions) two-temperature PCR cycles may increase target-specific fluorescent signal. Higher concentrations of MgCl2 promote the use of higher temperatures during the combined anneal-extension step.

Extension Time

The extension temperature and length of the target sequence affect the required extension time. Typically, AmpliTaq DNA Polymerase has an extension rate of 2,000 to 4,000 bases per minute at 70–80 °C (Gelfand, 1995). Polymerization rates are significant even below 55 °C, and with some templates, up to 85 °C (Innis et al., 1988; Jeffreys et al., 1988). As the amount of DNA increases in later cycles, the number of AmpliTaq DNA Polymerase molecules may become limiting for the extension time allotted. Increasing the extend times in later cycles may be needed to maintain efficiency of amplification. Use the autosegment extension feature, for the DNA Thermal Cycler or DNA Thermal Cycler 480, the AutoX feature on the GeneAmp PCR System 2400, or the AUTO program for the GeneAmp PCR System 9600.

November 2007 PCR Cycle Optimization 19

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High G+C Content DNA

High G+C content DNA needs very high annealing (>65 °C) and melting temperatures or cosolvents or the use of 7-deaza-2'-deoxy-GTP mixed with dGTP to overcome secondary structure (McConlogue et al., 1988; Sarkar et al., 1990; Smith et al., 1990). The half life of AmpliTaq DNA Polymerase is less than 35 minutes at temperatures above 95 °C (Gelfand and White, 1990), suggesting 95–96 °C as the maximum practical melting temperature. It is very important in the early cycles to be sure to melt the template DNA completely. When using genomic DNA as the starting template, melting at 97 °C for the first few cycles can help ensure single stranded template for the PCR. The melting temperature should be reduced for the later cycles because the smaller PCR product usually melts completely at a lower temperature (unless the PCR product is excessively G+C rich) than the starting genomic DNA.


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Interpretation of ResultsIn the NPTII Kit, the signal results from changes in the fluorescence emission intensity of the reporter dye following the cleavage of the probe. Interfering fluorescence fluctuations are normalized by applying two calculations.

The first calculation employs the quencher dye as a passive internal standard. This is accomplished by dividing the emission intensity of the reporter dye by the emission intensity of the quencher dye to give a ratio which we define as the RQ (reporter:quencher) value for a given reaction tube. Non-specific effects such as concentration change due to volume fluctuations are normalized by this ratio.

Any other fluctuation that is not due to PCR-related nuclease digestion is normalized by taking the RQ value for a tube that contains all components including target (defined as RQ+) and subtracting from this the RQ value of the No Template Control tube, which contains all of the same components except template (defined as RQ-). This final value, the ∆RQ, reliably indicates the magnitude of the signal generated by the given set of PCR conditions.

The following equation expresses the relationship of these terms:

∆RQ = (RQ+) – (RQ-)

where

For the system demonstrated by the NPTII Detection Kit, the reporter intensity is measured at 518 nm and the quencher intensity is measured at 582 nm. One excitation wavelength (488 nm) is used.

Determination of Threshold ∆RQ

To assure a statistically high confidence level in the results of the NPTII Kit, run the protocol with at least three No Template Controls per microplate. Based on t-distribution values, any ∆RQ value above the threshold ∆RQ has a 99% confidence level of being a positive result (Beyer, 1984).

You may use the TaqMan PCR software with Microsoft Excel for data analysis. Excel is distributed with the LS-50B TaqMan System. You can also use the following procedure.

To determine threshold ∆RQ:

1. On one microplate, measure both Reporter and Quencher fluorescence and determine the Reporter to Quencher ratio for each No Template Control tube (RQ-).

RQ+ = Emission Intensity of Reporter

Emission Intensity of ReporterRQ- = Emission Intensity of Quencher

Emission Intensity of QuencherPCR with Target

PCR without Target

November 2007 Interpretation of Results 21

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2. Determine the mean and standard deviation of RQ-.

3. Multiply the RQ- standard deviation by 6.965 to determine the threshold ∆RQ for the system.

For example, if the mean RQ- for a system is 0.15 with a standard deviation of 0.01, the threshold ∆RQ is 6.965 × 0.01 or 0.07. Any ∆RQ>0.07 is a positive result and indicates the sample contains target.

When more than three No Template Controls are run, the multiplier for the standard deviation (6.965) decreases. Refer to a table of t-distribution values for the appropriate multiplier (Beyer, 1984).

Establishing Confidence Levels for Probe Performance

Interpretation of results for the NPTII Detection Kit is based on a statistical analysis of the ratios of the Reporter and Quencher fluorescence signals. These fluorescence signals are dependent on a variety of system conditions including thermal cycler optimization, luminescence spectrometer configuration, pipetting techniques, DNA template, and probe.

Greater confidence can be obtained when the reactions are carried out in duplicate or triplicate and a Master Mix is common to all reactions. Use of positive displacement pipettes also improves precision.

To evaluate reproducibility, calculate the coefficient of variation (CV) on replicate samples. Inconsistent results (CVs exceed 10%) may be caused by pipetting errors and incomplete mixing of DNA solutions. To achieve smaller CVs, make Master Mixes for replicates and, if necessary, dilute the template with TE buffer or DI water and gently vortex the diluted template. When diluting the template, decrease the volume of DI water in the Master Mix appropriately.

Note To validate confidence levels, a full microplate of three No Template Controls, one buffer blank, and 92 Samples should be run at least once.


Applied Biosystems

Performance CharacteristicsThis NPTII Detection Kit is designed to give either a positive or a negative result, when analyzed on the TaqMan LS-50B System. A positive result is defined as any value greater than the threshold ∆RQ determined by analysis of three or more No Template Controls for a defined set of system conditions.

Table 6 shows typical raw and calculated values for control reactions developed using the TaqMan NPTII detection kit. These values represent the range of acceptable values using a TaqMan LS-50B System with both excitation and emission slit widths set to 10 nm.

Table 6. Typical Raw and Calculated Values for Control Reactions with the NPTII Kit

Adjust slit widths for emission and excitation filters so that values for No Template Control fall within the range given. In general, these values for FAM and TAMRA should be >50 to ensure accurate calculation of ýRQ. Within a run, mean RQ values should have a coefficient of variation (CV) less than 10%. If CV exceeds 10%, see Appendix A on page 24.

PCR inhibitors can co-purify with genomic DNA. While they have not yet been well-characterized, it is known that their presence is influenced by the plant species, the type of plant tissue, and the protocol used for DNA purification. Use the control DNA supplied in this NPTII Kit to determine whether PCR inhibition occurs.

To perform an inhibition assay, mix 1 µL (100 fg) of control DNA with genomic DNA prepared from a non-transformed plant. If the ∆RQ of this DNA mixture is less than the ∆RQ of the control DNA alone, PCR inhibition is occurring. Increasing the number of cycles during PCR may help overcome this problem. The best way to overcome PCR inhibition is to modify the DNA purification method. References for purifying genomic DNA from plants are provided in Appendix B on page 26.

Sample FAM TAMRA R/Q ∆RQ

No Template Control 80–100 80–110 0.9–1.0 0

Positive Control 350–500 86–96 3.5–5.4 2.5–4.4

November 2007 Performance Characteristics 23

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Appendix A TroubleshootingUse the TaqMan PCR Reagent Kit (P/N N808-0230) periodically to check the performance of the TaqMan LS-50B System.

Note Confirm the size and specificity of the PCR product by gel electrophoresis.

Table 7. Troubleshooting for the NPTII Detection Kit

Observation Possible Cause Recommended Action

∆RQ ð threshold ∆RQ and no PCR product generated in the reaction

PCR component omitted, incorrect primer(s) or template used

Troubleshoot PCR optimization, including all reagents. Then repeat NPTII Kit reaction with NPTII control

Inhibitor present in PCR Spike DNA from non-transformed plants into control reaction to check for inhibition

∆RQ ð threshold ∆RQ and both RQ+ and RQ- reactions show PCR product

Negative Reaction (RQ-) contaminated with DNA

Check technique and equipment to confine contamination

Avoid using dTTP; use dUTP only

Incorporate UNG for control of carryover contamination

∆RQ ð threshold ∆RQ and both RQ+ and RQ- reactions show PCR product

Failure of carryover contamination method

Troubleshoot the carryover contamination method

∆RQ ð threshold ∆RQ with PCR product in RQ+ reaction but not in RQ- reactio

Probe degradation (independent of AmpliTaq DNA Polymerase), incorrect mixing or probe accidentally omitted

Recheck probe concentration and rerun PCR. Use a different batch of probe

Post-PCR fluorescence at or near background fluorescence

Probe omitted Rerun system with probe

∆RQ Šthreshold ∆RQ in No Enzyme control

Nuclease or fluorescence contamination

Review template preparation protocol for possible sources of contamination

∆RQ of a known transformation positive less than ∆RQ of control DNA

Amount of DNA in sample less than amount of DNA in control

Add more cycles to PCR

Sample contains PCR inhibitors

Improve purity of sample DNA. (See Appendix B on page 26 for references)


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Emission >1000 FU or values out of range

Detector saturated Dilute samples. Refer to LS-50B User’s Manual to adjust excitation and emission slits

Replicate sample CV>10% Template pipetting error Use positive displacement pipettes. Dilute DNA with water or TE buffer, vortex, and adjust volume of Master Mix accordingly

Replicate sample CV>10% Dirty microplate See “Fluorescence Analysis” on page 15 for plate cleaning suggestions

Too little signal Increase probe concentration in reactions. Check that all parameters (emission filter, emission and excitation λ, slit widths, integration time) are set correctly

Sudden decrease in FAM emission with previously analyzed probe system

Lamp in LS-50B dimming Replace LS-50B lamp. (Refer to LS-50B User’s Manual)

Directional trends in well-to-well fluorescent emissions across microplate

Instrument alignment errors Check alignment of reader optics and microplate holder. (Refer to LS-50B User’s Manual)

Observation Possible Cause Recommended Action

November 2007 Appendix A Troubleshooting 25

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Appendix B Preparing Plant Genomic DNAThis appendix supplies references for extraction and quantification of genomic plant DNA.

DNA Extraction TechniquesAny particular plant species presents unique extraction problems, so it is up to researchers to optimize a DNA extraction technique for their system. Our own scientists and many in other laboratories have had excellent results using the various CTAB purification schemes (Doyle and Doyle, 1990).

For individual systems, journals such as Biotechniques contain numerous reports detailing modifications that improve the quality and or quantity of purified DNA in various species (Baker et al., 1990, and references therein; Doyle and Doyle, 1990).

Quantitating DNARefer to molecular biology manuals such as Current Protocols in Molecular Biology (Ausubel et al., 1987) for information on the following:

• Quantitation of DNA, restriction digest procedures

• Pouring and loading of agarose gels

• Running and interpretation of gels

Another good source of general information is Molecular Cloning: A Laboratory Manual (Sambrook et al., 1980).


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Appendix C ReferencesAusubel, F. M., Brent, R., Kingstin, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds., 1987. Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York.

Baker, S. B., Rugh, C. L., and Kamalay, J. C., 1990. “RNA and DNA isolation from recalcitrant plant tissue.” Biotechniques 9: 268–272.

Beck, E. et al., 1982. Derived from GeneBank Accession number U0004 19385. Gene 19: 327–336.

Beyer, W. H., ed., 1984. CRC Standard Mathematical Tables. 27th ed. Boca Raton, FL: CRC Press.

Chou, Q., Russell, M., Birch, D. E., Raymond, J., and Bloch, W., 1992. “Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.” Nucleic Acids Research 20: 1717–1723.

Delort, A.-M., Duplaa, A.-M., Molko, D., and Teoule, R., 1985. “Excision of uracil residues in DNA: Mechanism of action of Escherichia coli and Micrococcus luteus uracil DNA glycosylases.” Nucleic Acids Research 13: 319–335.

Doyle, J. and Doyle, J., 1990. “Isolation of plant DNA from fresh tissue.” Focus 12: 13–15.

Förster, V. Th., 1948. “Zwischenmolekulare Energiewanderung und Fluoreszenz.” Annals of Physics (Leipzig) 2: 55–75.

Gelfand, D. H., and White, T. S., 1990. “Thermostable DNA Polymerases.” In: Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds. PCR Protocols. A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, pp. 129–141.

Gelfand, D. H., 1995. Roche Molecular Systems, Inc., personal communication with PE Applied Biosystems staff scientists.

Higuchi, R. and Kwok, S., 1989. “Avoiding false positives with PCR.” Nature 339: 237–238.

Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H., 1991. “Detection of specific polymerase chain reaction product by utilizing the 5’ to 3’ exonuclease activity of Thermus aquaticus DNA polymerase.” Proceedings of the National Academy of Sciences, USA 88: 7276–7280.

Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. D., 1988. “DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.” Proceedings of the National Academy of Sciences, USA 85: 9436–9440.

November 2007 Appendix C References 27

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Jeffreys, A. J., Wilson, V., Neumann, R., and Keyte, J., 1988. “Amplification of human minisatellites by the polymerase chain reaction: Towards DNA fingerprinting of single cells.” Nucleic Acids Research 16: 10953–10971.

Kwok, S., Kinard, S., Spadoro, J., and Sninsky, J. J., 1982. “Enhancement of PCR specificity by uracil N-glycosylase.” VII International Conference on AIDS/III STD World Congress. Abstract.

Kwok, S., 1990. “Procedures to minimize PCR-product carry-over.” In: PCR Protocols. A Guide to Methods and Applications, Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds. Academic Press, Inc., San Diego, CA.

Lakowicz, J. R., 1983. “Energy Transfer.” In: Principles of Fluorescent Spectroscopy, Plenum Press, N.Y., pp. 303–339.

Lawyer, F. C., Stoffel, S., Saiki R. K., Myambo, K. B., Drummond R., and Gelfand, D. H., 1989. “Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from the extreme thermophile, Thermus aquaticus.” Journal of Biological Chemistry 264: 6427–6437.

Lee, L. G., Connell, C. R., and Bloch, W., 1993. “Allelic discrimination by nick-translation PCR with fluorogenic probes.” Nucleic Acids Research 21: 3761–3766.

Loewy, Z. G., Mecca, J., and Diaco, R., 1994. “Enhancement of Borrelia burgdorferi PCR by uracil N-glycosylase.” Journal of Clinical Microbiology 32: 135–138.

Longo, N., Berninger, N. S., and Hartley, J. L., 1990. “Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions.” Gene 93: 125–128.

Lyamichev, V., Brow, M. A. D., and Dahlberg, J. E., 1993. “Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases.” Science 260: 778–783.

McConlogue, L., Brow, M. A. D., and Innis, M. A., 1988. “Structure-dependent DNA amplification by PCR using 7-deaza-2-deoxyguanosine.” Nucleic Acids Research 16: 9869.

Mullis, K. B. and Faloona, F. A., 1987. “Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.” In: Wu, R., ed. Methods in Enzymology, Vol. 155, Academic Press, Inc., San Diego, CA, pp. 335–350.

Orrego, C., 1990. “Organizing a laboratory for PCR work.” In: PCR Protocols. A Guide to Methods and Applications, Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds. Academic Press, Inc., San Diego, CA.

PCR Technical Information, 1995. In: Perkin-Elmer Systems, Reagents & Consumables, 1995-1996, Foster City, CA, p. 96.


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Rychlik, W., Spencer, W. J., and Rhoads, R. E., 1990. “Optimization of the annealing temperature for DNA amplification in vitro.” Nucleic Acids Research 18: 6409–6412.

Saiki, R. K., Scharf, S. J., Faloona, F. A., Mullis, K. B., Horn, G. T., Erlich, H. A., and Amheim, N., 1985. “Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.” Science 230: 1350–1354.

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A., 1988. “Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.” Science 239: 487–491.

Sambrook, J., Fritsch, E. F., and Maniatis, T., 1980. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press.

Sarkar, G., Kapeiner, S., and Sommer, S. S., 1990. “Formamide can dramatically improve the specificity of PCR.” Nucleic Acids Research 18: 7465.

Smith, K. T., Long, C. M., Bowman, B., and Manos, M. M., 1990. “Using cosolvents to enhance PCR amplification.” Amplifications 5: 16–17.

Sninsky, J. J. and Gelfand, D. H., 1995. Roche Molecular Systems, Inc., personal communication with PE Applied Biosystems staff scientists.

Varshney, U., Hutcheon, T., and van de Sande, J. H., 1988. “Sequence analysis, expression, and conservation of Escherichia coli uracil DNA glycosylase and its gene (ung).” Journal of Biological Chemistry 263: 7776–7784.

November 2007 Appendix C References 29

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