RealMODTM Real-time PCR...

RealMODTM

Real-time PCR KitManual Ver. 1.1

Manual for

RealMODTM Real-time PCR Kit

(Labeled probe or Interchelating dye)

INT-25331 RealMODTM Real-time PCR Core Kit 200 T INT-25341/25342 RealMODTM Real-time PCR Master mix Kit(2X) 1 ml / 5 ml

INT-25332 RealMODTM Green Real-time PCR Core Kit 200 T INT-25343/25344 RealMODTM Green Real-time PCR Master mix Kit(2x) 1 ml / 5 ml

L i S ta rF i sh S . r . l . V ia Cavour , 35 - 20063 Cernusco S /N (MI ) , I t a l y Te l . +39-02-92150794 - Fax . +39-02-92157285 in fo@l is ta r f i sh . i t -www. l i s ta r f i sh . i t

General

Information

Introduction

TaqManⓇ Probe Method

Interchelating Dye Method

… 2

Product Information … 5

Notice Before Use … 6

Storage … 8

Precautions and Safety Information … 8

Product Warranty and Satisfaction Guarantee … 8

Reagents and Instruments Required … 8

Manual

Contents

1. Description… 9

2. Protocol for Real-time PCR… 11

3. Experimental Data… 17

4. Troubleshooting Guide… 20

5. Guideline for Primer Design… 22

6. Related Products… 22

Contents

Introduction

1) Principle of TaqManⓇ Probe Method

The RealMODTM Real-time PCR Core Kit and Master Mix provides a system 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. This method permits the analysis of thousands of samples per day with high sample-to-sample

reproducibility.

This method can be incorporated into an established PCR system by designing a fluorescent DNA probe

specific to the PCR target sequence flanked by PCR primers.

TaqManⓇ(TaqMan® is a trademark of Roche Molecular Systems Inc.) assays, also referred to as 5’-nucleaseassays, exploit the 5' to 3‘ exonuclease activity of Taq DNA polymerase. Each reaction contains agene specific primer and a fluorescently labeled TaqManⓇ probe. The probe contains a 5’ reporterdye and a 3’ quencher dye. The 3’-end is also blocked to prevent extension during PCR. The probeis designed to anneal the target sequence between the forward and reverse PCR primers. Whilethe probe is intact, the quencher suppresses the fluorescence of the reporter dye. Duringamplification, Taq DNA polymerase cleaves the probe and displaces it from the target, allowingextension to continue. Cleavage of the probe separates the reporter dye from the quencher dye,resulting in an increase in fluorescence. The increased fluorescence only occurs if the targetsequence is amplified and is complimentary to the probe, thus preventing detection of non-specificamplification. For any given cycle within the exponential phase, the amount of product, and hencefluorescence signal, is directly proportional to the initial copy number. Thus, higher copy numbertemplates will cross a fluorescence detection threshold before lower copy templates.

Polymerization : A fluorescent reporter(R) dye and a quencher(Q) are attached to the 5’ and 3’

ends of a TaqManⓇ probe respectively. Strand displacement : When the probe is intact the reporter dye emission is quenched.Cleavage : During each extension cycle the DNA polymerase cleaves the reporter dye from the

probe.Polymerization completed : Once separated from the quencher, the reporter dye emits its

characteristic fluorescence.

(Text adapted from Applied Biosystems)

R Q

R

Q

2) Principle of Interchelating dye Method

Interchelating dye provides the simplest and most economical format for detecting and quantitatingPCR products in real-time reactions. Interchelating dye binds double-stranded DNA, and uponexcitation emits light. Thus, as a PCR product accumulates, fluorescence increases. Theadvantages of interchelating dye are that it is inexpensive, easy to use, and sensitive. Thedisadvantage is that interchelating dye will bind to any double-stranded DNA in the reaction,including primer-dimers and other non-specific reaction products, which results in anoverestimation of the target concentration. For single PCR product reactions with well designedprimers, interchelating dye can work extremely well, with spurious non-specific background onlyshowing up in very late cycles.Interchelating dye is the most economical choice for real-time PCR product detection. Since thedye binds to double-stranded DNA, there is no need to design a probe for any particular targetbeing analyzed. However, detection by interchelating dye requires extensive optimization. Sincethe dye cannot distinguish between specific and non-specific product accumulated during PCR,follow up assays are needed to validate results.

(Text adapted from Ambion(Applied Biosystems))

High Resolution Melt(HRM) Dye

High Resolution Melting(HRM) is a novel, homogeneous, close-tube, post-PCR method, enablinggenomic researchers to analyze genetic variations (SNPs, mutations, methylations) in PCRamplicons. It goes beyond the power of classical melting curve analysis by allowing to study thethermal denaturation of a double-stranded DNA in much more detail and with much higherinformation yield than ever before. HRM characterizes nucleic acid samples based on theirdisassociation (melting) behavior. Samples can be discriminated according to their sequence,length, GC content or strand complementarity. Even single base changes such as SNPs (singlenucleotide polymorphisms) can be readily identified. The most important High Resolution Meltingapplication is gene scanning - the search for the presence of unknown variations in PCR ampliconsprior to or as an alternative to sequencing. Mutations in PCR products are detectable by HighResolution Melting because they change the shape of DNA melting curves. A combination of new-generation DNA dyes, high-end instrumentation and sophisticated analysis software allows todetect these changes and to derive information about the underlying sequence constellation.In order to improve the signal intensity and the resolution of melting curve analysis, the Real-MOD®

Green Real-Time Series were adapted HRM dye for Interchelating method.

• Optimize the primer concentration—start at 300 nM• Optimize the MgCl2 concentration—start between 1.0–3.0 mM• Test different primer pairs to determine which one is optimal• Include replicates for significance—triplicates are recommended• Use calibrated pipettes to ensure accurate pipetting• Set up reactions on ice to minimize sample degradation and nonspecific reactivity• Keep reaction volumes at 20 μL or above, to minimize pipetting and mixing errors• Centrifuge before placing in the real-time instrument• Run samples within 2 hr of preparing

Tip : Optimizing PCR Reagents for Successful HRM Analysis

Benefit of RealMODTM System

Scan for mutations with greater confidence

Avoid spending time optimizing your Experiments

Scan gDNA for mutations without the complexity and expense of other methods

High resolution—sharp, clean melt curves for unambiguous discrimination between wild-type and variant sequences

All-inclusive formulation—

HRM Master Mix eliminates need to optimize Mg2+, dye, or other components for each target

One instrument—perform PCR and variant identification using your real-time PCR instrument; no need for HPLC or gels

Accurate results—very low false negative rate

Reproducible results—minimal lot-to-lot, run-to run, and well-to-well variation

Flexible formulation—

RealMODTM Green Real time

Core Kit / Master mix Kit

provides individual components for rapid optimization of HRM mix when more flexibility is needed

Low reagent consumption and waste—one mix used for PCR and HRM; no need for additional reagents, solvents, or gels

High specificity—minimal primer-dimer andnonspecific amplification—

tested across awide range of targets, sequence contexts, and PCR primer pairs

Comprehensive coverage—

reagents have been tested across a wide range of variant types including all categories of SNP mutations, insertions, deletions, etc.

Product Information

Product Cat. No. Size Kit Contents

RealMODTM Real-time PCR Core Kit 25331 200

Tests

RealMODTM DNA Polymerase(5U/μl) 50 μl

10x Real-time PCR Buffer 1 ml 10x Mg2+ free Buffer 1 ml 10mM dNTP Mix(2.5mM each) 1 ml 25mM MgCl2 1 ml

RealMODTM Green Real-time PCR Core Kit 25332 200

Tests

RealMODTM Green DNA Polymerase(5U/μl) 500 μl

10x Green PCR Buffer 1 ml 10x Mg2+ free Buffer 1 ml 10 mM dNTP Mix(2.5mM each) 1 ml 25 mM MgCl2 1 ml HRM Dye 250 μl

Core Kit

Product Cat. No. Size Kit Contents

RealMODTM Real-time PCR Master mix Kit(2x) 25341 / 25342 1 ml / 5 ml 2x RealMODTM PCR Solution 1 ml / 5 ml

RealMODTM Green Real-time PCR Master mix Kit(2x)

25343 1 ml 2x RealMODTM Green PCR Solution 1 ml HRM Dye 50 μl

25344 5 ml 2x RealMODTM Green PCR Solution 5 ml HRM Dye 250 μl

2x Master Mix Kit

RealMODTM Real-time PCR Core Kit

Cat. No. 25331

RealMODTM Green Real-time PCR Core Kit

Cat. No. 25332

RealMODTM Real-time PCR Master mix Kit(2x) Cat. No. 25341 / 25342 Size. 1 ml / 1 ml x 5

RealMODTM Green Real-time PCR Master mix Kit(2x)

Cat. No. 25343 / 25344 Size. 1 ml / 1 ml x 5

Notice Before Use

1) DNA Polymerase

• i-StarTaq™ DNA Polymerase(Ultra-grade) is a modified version of recombinant i-Taq™ DNAPolymerase. It is provided in an inactive state and activated through a pre-PCR step of 5 ~ 10minutes at 95 ºC . Once activated, i-StarTaq™ DNA Polymerase(Ultra-grade) has the same enzymeunit activity, thermal stability, and half-life at 95 ºC as i-Taq™ DNA Polymerase. Because theenzyme is supplied in an inactive state, complete reaction premixes can be made in advance andpipetted easily into reaction tubes or 96-well microplates with a multi-channel pipettor. This is a veryimportant point for high-throughput applications. Since the enzyme does not become active until atemperature well above primer.

• i-TaqTM Plus DNA Polymerase(Ultra-grade) to perform an automated, convenient, and efficient HotStart. i-TaqTM Plus DNA Polymerase(Ultra-grade) is a chemically modified form of i-TaqTM DNAPolymerase. The modification renders the enzyme inactive. Upon thermal activation, the modifier isreleased, resulting in active enzyme. The high-temperature incubation step required for activationensures that active enzyme is generated only at temperatures where the DNA is fully denatured.When i-TaqTM Plus Polymerase is added to the reaction mixture at room temperature, the inactiveenzyme is not capable of primer extension. Any low-stringency mis-priming events that may haveoccurred will not be enzymatically extended and subsequently amplified.

2) Hot Start

• The initial inactive state of i-StarTaq™ DNA Polymerase(Ultra-grade) and i-TaqTM plus DNAPolymerase(Ultra-grade) allow a Hot Start PCR to be performed without the cumbersome, time-consuming, and expensive methods of conventional Hot Start techniques. A Hot Start PCRprevents primer dimer formation and other non-specific amplifications. Hot Start PCR results inhigher specificity, higher sensitivity, increased yield and decreased background.

3) Contamination

• Because the interpretation of results depends upon the comparison of a sample(containing DNA) toa No Template Control (without DNA), it is extremely important that all sources of contamination beanticipated and controlled. All reaction mixes should be set up in an area isolated from PCR productanalysis and sample preparation. Use dedicated or disposable vessels, solutions, and pipettors(preferably positive displacement pipettors with disposable tips) for DNA preparation, reactionassembly, and sample analysis to minimize PCR product carryover and sample cross-contamination. Since sample protein and fluorescent contaminants may interfere with this assayand give false positive results, it may be necessary to include a No Enzyme Control tube thatcontains target, but no enzyme. If the fluorescence of the no enzyme control is greater than that ofthe no template control after PCR, fluorescent contamination may be present in the sample.

4) Rn and ΔRn Values

• Normalization is accomplished by dividing the emission intensity of the reporter dye by the emissionintensity of the Passive Reference to obtain a ratio defined as the Rn (normalized reporter) for agiven reaction tube. Rn+ is the Rn value of a reaction containing all components including thetemplate. Rn– is the Rn value of an unreacted sample. This value may be obtained from the earlycycles of a real-time run, those cycles prior to a detectable increase in fluorescence. This value mayalso be obtained from a reaction not containing template. ΔRn is the difference between the Rn+value and the Rn– value. It reliably indicates the magnitude of the signal generated by the given setof PCR conditions.

5) Threshold Cycle

• The threshold cycle or CT value is the cycle at which a statistically significant increase in ΔRn isfirst detected. Threshold is defined as the average standard deviation of Rn for the early cycles,multiplied by an adjustable factor. On the graph of Rn versus cycle number shown below, thethreshold cycle occurs when the Sequence Detection System begins to detect the increase in signalassociated with an exponential growth of PCR product.

6) CAUTIONS

• RUO : For research purpose only. Not for use in diagnostic procedures for clinical purposes. For invitro use Only.

• Do not use any reagent after the expiration date.• Do not use together with reagents of other products.• Follow the instructions

Reagents and Instruments Required (Not Supplied)1) Thermal Cycler for Real time PCR(authorized instruments)2) Reaction tube or plate for real time PCR3) PCR primers4) TaqMan® probe* for detection (licensed probes)5) dH2O6) Micropippets and Micropippet tips (autoclaved prior to use)

* The TaqMan® probe consists of an oligonucleotide with a 5’-reporter dye and a 3’-quencher dye. A fluorescent reporterdye,such as FAM dye, is covalently link to the 5’ end of the oligonucleotide. TET dye and VIC dye have also been used as reporter dyes. Each of the reporters is quenched by TAMRA dye, or non-fluorescent quencher attached via a linker arm that is usually located at the 3’ end.

Storage• RealMODTM Real-time PCR Core Kit & RealMODTM Green Real-time PCR Core Kit

Upon receipt, store at –15 to –25 ºC in a constant temperature freezer. If stored under therecommended conditions, the product will maintain performance through the control date printed onthe label.

• RealMODTM Real-time PCR Master mix Kit & RealMODTM Green Real-time PCR Master mix Kit

Upon receipt, store at 4 ºC in a constant temperature freezer. If stored under the recommendedconditions, the product will maintain performance through the control date printed on the label.

• HRM Dye(amber vial) of RealMODTM Green Real-time PCR Series should be protected from light.

Precautions and Safety InformationCHEMICAL HAZARD. Some of the chemicals are potentially hazardous and can cause injury, illness,or death.• Read and Understand the material safety data sheets(MSDSs) provided by the chemical

manufacturer before you store, handle, or work with any chemicals or hazardous materials.• Minimize contact with the chemicals. Wear appropriate personal protective equipment when

handling chemicals(e.g. safety goggles, gloves, or protective clothing).• Minimize the inhalation of chemicals. Do not leave chemical containers open, use only with

adequate ventilation(e.g. fume hood).

1. Description

RealMODTM Real-time PCR Core Kit & RealMODTM Real-time PCR Master mix Kit(2X) contain thecomponents needed to perform PCR, specially designed for real time PCR by using TaqMan® probe.This product combines the high performance and Ultra pure grade of i-StarTaqTM DNA Polymerase,which is an enzyme for hot start PCR utilizing chemical modification, with a newly developed bufferwhich provides superior specificity, increased amplification efficiency for real time PCR. Accordingly, asuccessful real time PCR is promised with high sensitivity, wide dynamic range and accuratequantification.

RealMODTM Real-time PCR Core Kit provides individual components needed to perform real-time PCR.RealMODTM Real-time PCR Master mix Kit is a 2x concentration of premix to perform real-time PCR.

1.1. RealMODTM Real-time PCR Core Kit

RealMODTM Real-time PCR Master mix Kit(2X)

• Flexible To Use : RealMODTM Real-time PCR Core Kit

- Separate components provide flexibility in assay set-up• Optimized To Use : RealMODTM Real-time PCR Master mix Kit(2X)

- Used with rapid assay development guidelines to minimize optimization time.- Enhanced performance to accommodate difficult G/C-rich sequences.- Optimized buffer components are proprietary buffer enhancements that add to the performan-

-ce and reliability of the 5' nuclease assay• Hot Start PCR

- Contains ultra pure grade of i-StarTaqTM DNA Polymerase for a robust 5' nuclease assay• High Sensitivity

- One of advantages of Real-time PCR is higher sensitivity than conventional PCR whichobserve end point’ by monitoring the beginning of amplification. When real time PCR isanalyzed by RealMODTM Real-time PCR Series, high signal is shown even under pg. This resultis caused by highly purified enzyme and the combination of optimized PCR buffer.

• Wide dynamic range- Real-time PCR analysis widely applies to qualitative analysis and also quantitative analysis.

In this case, the most important factor is dynamic range. Technically, when the amount ofstarting template is halved, Ct value of one cycle is increased. In the qualitative analysis case,the most important factor is to keep stable pattern irrespective of the concentration oftemplate. In this point, RealMODTM Real-time PCR Series is applicable to wide dynamic range.In addition, it can be applicable to DNA analysis of high concentration and also lowconcentration.

• High reproducibility and convenience- High reproducibility is necessary for accurate quantitative analysis. RealMODTM Real-time PCR

Series provides high reproducibility and guarantees constant results from repeated analysis.In addition, Master mix and also Core Kit for customer’s needs by each research areprovided.

Characteristics of RealMODTM Real-time PCR Series

At the real-time PCR analysis, TaqManⓇ probe is ideal method because it is mixed with a idea ofblotting. But it has drawbacks that it highly costs and cannot apply to genotyping research thru themelting curve analysis with PCR products. In this case, interchelating dye is generally used such asSYBR Green, LC Green to implicate real time PCR analysis. This method has a merit that it has a wideapplication but on the other hand, it is negative because this could be affected by non-specificamplification and primer dimer.RealMODTM Green Real-time PCR Series which can restrict non-specific amplification and primer dimer iscalled as a competitive product in the entire amplicons.RealMODTM Green Real-time PCR Core Kit and RealMODTM Green Real-time PCR Master mix Kit(2X)

provide components needed to perform PCR, except primers, template and water necessary toperform real-time PCR using High Resolution Melt (HRM) dye. Direct detection of Polymerase ChainReaction(PCR) product is monitored by measuring the increase in fluorescence caused by the bindingof HRM dye to double-stranded(ds) DNA. The HRM dyes are specifically designed for high-resolutionmelting curve analysis to detect DNA sequence variants. The addition of HRM dyes increase themelting temperature of DNA by 1-3 ℃ and may require adjustment of cycling parameters. The HRMdyes of RealMODTM Green Real-time PCR Core Kit and RealMODTM Green Real-time PCR Master mix Kit(2X)

are tailored specifically for High Resolution Melting and have the unique ability to detect hetero-duplexes during melting analysis after PCR. Just add the dye to your sample before PCR. The HRMdyes of RealMODTM Green Real-time PCR Core Kit are extremely stable, do not inhibit PCR, and are"saturation" dyes that can detect multiple PCR products in a mixture during melting analysis.RealMODTM Green Real-time PCR Core Kit and RealMODTM Green Real-time PCR Master mix Kit(2X)

combine the high performance of i-TaqTM Plus DNA Polymerase(Ultra pure grade), which is anenzyme for hot start PCR, with a newly developed buffer which provides superior specificity, increasedamplification efficiency and high aptitude for high-speed real time PCR. Accordingly, a successful realtime PCR is promised with high sensitivity, wide dynamic range and accurate quantification.

RealMODTM Real-time PCR Core Kit provides individual components needed to perform real-time PCR.RealMODTM Real-time PCR Master mix Kit is a 2x concentration of premix to perform real-time PCR.

• High resolution melt(HRM) dye- The LC Green plus* dye was adapted to interchelating dye of RealMODTM Green Real-time PCR

Series Kits

• High specificity- RealMODTM Green Real-time PCR series is designed to have a powerful amplification efficiency

and minimize a noise peak by using high purity DNA polymerase with hot starter PCR functionto restrict the non-specific amplification.

• High sensitivity and accuracy- HRM(High resolution melt) dye is applied to be able to have more sensitivity and more

accurate analysis. It results in the better Signal sensitivity because the intensity of dye isstronger than that of SYBR Green.

• Optimal to the melting curve analysis- RealMODTM Green Real-time PCR series make it possible to analyze melting curve with high

resolution because HRM dye is more sensitive at Tm value than that of SYBR Green. This factmakes it easy to distinguish non-specific PCR products and also have an advantage to applyto the downstream application such as SNP research etc.

1.2. RealMODTM Green Real-time PCR Core Kit

RealMODTM Green Real-time PCR Master mix Kit(2X)

Characteristics of RealMODTM Green Real-time PCR Series

2. Protocol for Real-time PCR

2.1. RealMODTM Real-time PCR Series

1) Prepare a reaction mixture according to Table 1 or Table 2.

Table 1. RealMODTM Real-time PCR Core Kit

Table 2. RealMODTM Real-time PCR Master mix Kit(2X)

[Reagents]

RealMODTM DNA Polymerase(5U/μl)

10X Real-time PCR Buffer

dNTP mixture(2.5 mM each)

PCR Forward Primer

PCR Reverse Primer

TaqMan® probe

Template

DW

16 Test /20 μl scale

1.6 μl

32 μl

32 μl

Variable

Variable

Variable

0.5 ~ 2 μl each

Up to 20 μl each


4 μl

80 μl

80 μl

Variable

Variable

Variable

1 ~ 5 μl each

Up to 50 μl each

Final conc.

0.0125 unit/μl

1X

0.25mM each

0.2 μM *A

0.2 μM *A

*B

*C

-


0.25 μl

5 μl

5 μl

Variable

Variable

Variable

1 ~ 5 μl each

Up to 50 μl

[Reagents]

2x RealMODTM Real-time PCR Master mix Solution

PCR Forward Primer

PCR Reverse Primer

TaqMan® probe

Template

DW

1 Test /50 μl scale*D

25 μl

Variable

Variable

Variable

1 ~ 5 μl each

Up to 50 μl

1 Test /20 μl scale*E

10 μl

Variable

Variable

Variable

0.5 ~ 2 μl each

Up to 20 μl

Final conc.

1x

0.2 μM *A

0.2 μM *A

*B

*C

-

*A : The final concentration of primers can be 0.2 μM in most reactions. When it does not work, determine the optimalconcentrations within the range 0.1-1.0 μM.

*B : Probe concentration differs depending on kind of real time PCR instrument, and kind of fluorescence labelingmaterial. The adding amount should be determined by referring to the operation manual of instrument and aproduct insert supplied with probe.

*C : Final template concentration varies depending on the copy number of target present in the template solution.Optimal amount should be determined by preparing the dilution series. DNA template should be used in less than100 ng for 20 μl reaction. When the RT reactant(cDNA) is used as a template, it should be added in less 10 %volume of PCR reaction mixture.

*D : Real-time reaction setup volume for ABI PRISM 7000/7700/7900HT, Applied Biosystems 7300/7500/7500 FastReal-time PCR System and other Real-time PCR instrument.

*E : Real-time reaction setup volume for LightCycler Real-time PCR instrument.

Shuttle PCR standard protocol is recommended. Try this protocol firstly, and optimize thereaction condition if needed(see the next page : example for the cycling parameters for variousReal-time PCR instrument) .

Note : This product combines the high performance of i-StarTaqTM DNA Polymerase(ultra pure grade), which is an enzyme

for hot start PCR. Initial denaturation step prior to PCR should be at 95°C for 5 ~ 10 min as the initial denaturation,

required for chemically modified Taq polymerase.

2) Mix the upper reaction mixture thoroughly and aliquot appropriate volumes into the Real-time PCR tube or plates

3) Program your Real-time PCR instrument according to the program “Suggested CyclingParameters(Table 3)”.

Table 3. Suggested Cycling Parameters for RealMODTM Real-time PCR Series

[ Stage ]

Stage 1. Initial Denaturation

Stage 2. PCR

Temperature

95oC

95oC 50oC ~ 60oC

Time

5 min

5 sec. 30 sec.

Number of Cycle

1

35 ~ 45

4) Place the Real-time PCR tube or plate in the Real-time PCR instrument and start thecycling program.

5) After the reaction is completed, verify the amplification curve. Establish the standardcurve when quantification is done.Refer to the operation manual of an used Real-time PCR instrument.

1) ABI PRISM 7000/7700/7900HT, Applied Biosystems 7300/7500 Real-Time PCR System

[ Example. Suggested Cycling Parameters for various Real-time PCR instrument ]

PCR Standard Protocol

*1 Fluorescence detection step should be 30 seconds with ABIPRISM 7700 and 7900HT, 31 seconds with ABI PRISM 7000 and7300 Real-Time PCR System, and 34 seconds with 7500 Real-timePCR System.

Stage 1: Initial denaturation

Reps: 1, 95°C 5 min.

Stage 2: PCR

Reps: 40 95°C 5 sec.50 ~ 60°C 30 sec. (31sec, 34sec)*1

2) Applied Biosystems 7500 Fast Real-Time PCR System



Reps: 1, 95°C 5 min.

Stage 2: PCR

Reps: 40 95°C 3 sec.60°C 25 sec.

3) LightCycler Real-Time PCR System



Reps: 1, 95°C 5 min. 20°C / sec

Stage 2: PCR

Reps: 40 95°C 5 sec. 20°C / sec60°C 20 sec. 20°C / sec

2.2. RealMODTM Green Real-time PCR Series

1) Prepare a reaction mixture according to Table 4 or Table 5.

Table 4. RealMODTM Green Real-time PCR Core Kit

Table 5. RealMODTM Green Real-time PCR Master mix Kit(2X)

[Reagents]

RealMODTM Green DNA Polymerase(5U/μl)

10X Green PCR Buffer

dNTP mixture(2.5 mM each)

PCR Forward Primer

PCR Reverse Primer

HRM Dye

Template

DW


16 μl

32 μl

32 μl

Variable

Variable

8 μl

0.5 ~ 2 μl each

Up to 20 μl each


40 μl

80 μl

80 μl

Variable

Variable

20 μl

1 ~ 5 μl each

Up to 50 μl each

Final conc.

0.0125 unit/μl

1x

0.25 mM each

0.2 μM *A

0.2 μM *A

-

*B

-


2.5 μl

5 μl

5 μl

Variable

Variable

1.25 μl

1 ~ 5 μl each

Up to 50 μl

[Reagents]

2x RealMODTM Green Real-time PCR Master mix Solution

PCR Forward Primer

PCR Reverse Primer

HRM Dye

Template

DW

1 Test /50 μl scale*C

25 μl

Variable

Variable

1.25 μl

1 ~ 5 μl each

Up to 50 μl

1 Test /20 μl scale*D

10 μl

Variable

Variable

0.5 μl

0.5 ~ 2 μl each

Up to 20 μl

Final conc.

1x

0.2 μM *A

0.2 μM *A

-

*B

-

*A : The final concentration of primers can be 0.2 μM in most reactions. When it does not work, determine the optimalconcentrations within the range 0.1-1.0 μM.

*B : Final template concentration varies depending on the copy number of target present in the template solution.Optimal amount should be determined by preparing the dilution series. DNA template should be used in less than100 ng for 20 μl reaction. When the RT reactant(cDNA) is used as a template, it should be added in less 10 %volume of PCR reaction mixture.

*C : Real-time reaction setup volume for ABI PRISM 7000/7700/7900HT, Applied Biosystems 7300/7500/7500 FastReal-time PCR System and other Real-time PCR instrument.

*D : Real-time reaction setup volume for LightCycler Real-time PCR instrument.

Shuttle PCR standard protocol is recommended. Try this protocol firstly, and optimize thereaction condition if needed(see the next page : example for the cycling parameters for variousReal-time PCR instrument) .

2) Mix the upper reaction mixture thoroughly and aliquot appropriate volumes into the Real-time PCR tube or plates

3) Program your Real-time PCR instrument according to the program “Suggested CyclingParameters(Table 6)”.

Table 6. Suggested Cycling Parameters for RealMODTM Green Real-time PCR Series

[ Stage ]

Stage 1. Initial Denaturation

Stage 2. PCR

Stage 3. Dissociation step(Melting Curve Analysis)

Temperature

95oC

95oC 50oC ~ 60oC

95oC 50oC ~ 60oC(Beginning)

95oC(Final)

Time

30 sec.

5 sec. 30 sec.

15 sec.1 min

Number of Cycle

1

35 ~ 45

1

4) Place the Real-time PCR tube or plate in the Real-time PCR instrument and start thecycling program.

5) After the reaction is completed, verify the amplification curve. Establish the standardcurve when quantification is done.Refer to the operation manual of an used Real-time PCR instrument.

1) ABI PRISM 7000/7700/7900HT, Applied Biosystems 7300/7500 Real-Time PCR System

[ Example. Suggested Cycling Parameters for various Real-time PCR instrument ]

2) Applied Biosystems 7500 Fast Real-Time PCR System

3) LightCycler Real-Time PCR System


*1 Fluorescence detection step should be 30 seconds with ABI PRISM 7700 and 7900HT, 31 seconds withABI PRISM 7000 and 7300 Real-Time PCR System, and 34 seconds with 7500 Real-time PCR System.


Reps: 1, 95°C 30 sec.

Stage 2: PCR

Reps: 40 95°C 5 sec.60°C 30 sec. (31sec, 34sec)*1

Stage 3: Dissociation Stage



Reps: 1, 95°C 30 sec.

Stage 2: PCR

Reps: 40 95°C 5 sec.60°C 25 sec.

Stage 3: Dissociation Stage



Reps: 1, 95°C 10 sec. 20°C/sec.Stage 2: PCR

Reps: 40, 95°C 5 sec. 20°C/sec.60°C 20 sec. 20°C/sec.

Stage 3: Melting curve analysis

3. Experimental Data

Figure 1. Real-time PCR analysis using the Real-MODTM Real-time Core Kit with various target genes.Synthetic RNA template(SIV NA gene, JEV, Inf M gene and SIV NP gene) and several kinds of genomic DNA(Q-fever, beta-actin) was serially diluted (10X) resulting in 106~103 copies per sample. All reactions were performedin duplicate.Panel A, Q-fever (bacterial gDNA); panel B, beta-actin(human gDNA); panel C, M gene of Influenza A(cDNA);panel D, NA gene of Swine influenza virus(cDNA); panel E, Japanese Encephalitis Virus(JEV, cDNA); panel F,NP gene of Swine influenza virus(cDNA)

RealMODTM Real-time PCR Series was tested to various target gene amplification studies.

Real-MODTM Real-time PCR Series exhibits exceptional sensitivity and an extremely wide dynamicrange in qPCR assays.Real-time PCR was implemented with various samples. PCR results showed very high performanceand could prove that RealMODTM Real-time PCR Series can meet various field conditions.

A B C

E FD

A B C D

E F G H

Figure 2. Real-time PCR analysis using the Real-MODTM Real-time Master mix Kit with various target genes.Synthetic RNA template(SIV NA gene, HA gene, Inf M gene and SIV NP gene) and several kinds of genomicDNA(van A, van B, van C1 gene, RNase P gene) was serially diluted (10X) resulting in 106~103 copies persample. All reactions were performed in duplicate.Panel A, van A gene(bacterial gDNA); panel B, van B gene(bacterial gDNA); panel C, RNase P gene(humangDNA); panel D, M gene of influenza A(cDNA); panel E, van C1 gene(bacterial gDNA); panel F, NA gene ofInfluenza A(cDNA); panel G, HA gene of Inf A H1(cDNA); panel H, NP gene of Swine influenza virus(cDNA)

Figure 3. Melting peak analysis with M. penumoniae and M. fermentans

Melting curve analysis of Mycoplasma isolates was performed after quantification step. The two meltingtemperatures are marked with arrows. It could be distinguished M. pneumoniae from M. fermentans. All sampleswere run with concentration of 104 copies/μl.

RealMODTM Green Real-time PCR Series shows high resolution of melting curve analysis

RealMODTM Green Real-time PCR Series are very useful when different samples are analyzed thru meltin-g curve. To prove this, real time PCR was implemented with targeting 16S genes of two different myco-plasmas. Competitor’s product could not distinguish two different mycoplasma but RealMODTM Green

Real-time PCR Series exactly distinguished two different mycoplasma with showing high resolution.

Comparison of HRM Dye with SYBR I Dye

Comparison test was implemented. Competitors SYBR Green products could not hold the proportionalpatterns at the low density. But RealMODTM Green Real-time PCR Series can hold the proportionallysame amplification curves and could get the superior result to the sensitivity(Fig. 4.).RealMODTM Green Real-time PCR Series has an excellent resolution at the melting curve analysis andthis follows more accurate analysis. By following the comparison test with competitor’s products, therewas a remarkable difference at peak sharpness. In case that the experiment is repeated with differenttemplate density, competitor’s products showed collapse of signal at low density template. RealMODTM

Green Real-time PCR Series showed proportionally same peak at even low density template (Fig. 5)

M. pneumoniae

M. fermentans

A-1

A-2 B-2

B-1

< SYBR Green I Dye of Competitor A > < HRM Dye of RealMODTM Green PCR Series >

Figure 4. Comparison of RealMODTM Green Real-time PCR Core Kit and Competitor AThe GAPDH DNA fragment was amplified using RealMODTM Green Real-time PCR Core Kit with SYBR Green I andHRM dye. The amplication curves were clearly observed with template gradient when HRM dye was used. WithSYBR Green I, the peaks were not proportioned amount of templates.

A-3A-4 B-3

B-4

< SYBR Green I Dye of Competitor A > < HRM Dye of RealMODTM Green PCR Series >

Figure 5. High resolution of melting curve-analysisPanel A-3, Amplification curve(100 ng, 10 ng, 1 ng, 100 pg) with SYBR Green I; panel A-4, Melting curve withSYBR Green I; panel B-3, Amplification curve(100 ng, 10 ng, 1 ng, 100 pg) with HRM dye of RealMODTM Green

Real-time PCR Series; panel B-4, Melting curve with HRM dye of RealMODTM Green Real-time PCR Series

4. Troubleshooting Guide

This troubleshooting guide may be helpful in solving problems that may frequently arise. The scientistsare always happy to answer any questions you may have about the information or protocolin this manual or other molecular biology applications.

Problems Recommendations

No product, or weak product signal in in PCR

1) Annealing step or annealing/extension step too short(labeled probes)

Always use the annealing time or annealing/extension timespecified in the protocol. In some cases, increasing the time insteps of 10 s can improve results, especially with theLightCycler.

2) Annealing/extension time too short(labeled probes)

Always use the annealing/extension time specified in theprotocol. In some cases, increasing the time in steps of 10 s canimprove results, especially with the.

3) Pipetting error or missing reagent Check the concentrations and storage conditions of thereagents, including primers, probe, and template nucleic acid.Repeat the PCR.

4) Wrong detection step(labeled probes)

Ensure that fluorescence detection takes place during theannealing step or during the combined annealing/extension stepwhen using labeled probes.

5) No detection activated Check that fluorescence detection was activated in the cyclingprogram.

6) PCR product too long For optimal results, PCR products should be between 100 and 150 bp. PCR products should not be outside the range of 60–

300 bp.

7) Primer design not optimal(labeledprobes)

Check for PCR products by gel electrophoresis. If no specificPCR products are detected, prepare the primer design .Check for temperature of the labeled probe, which of Tm valueis higher than temperature of primer at least 5 ~ 10 ℃.

8) Primer design not optimal(HRM) Check for PCR products by melting curve analysis or gelelectrophoresis. If no specific PCR products are detected,recheck the primer design.

9) Mg2+ concentration not optimal Always start with the Mg2+ concentration provided in RealMODTM

Real time PCR series(2 mM final concentration). For a few targets,an increase up to 6 mM Mg2+ may be helpful. Perform thetitration in 0.5 mM steps.

10) Problems with starting template Check the concentration, storage conditions, and quality of thestarting template. If necessary, make new serial dilutions oftemplate nucleic acid from the stock solutions. Repeat the PCRusing the new dilutions.

11) Insufficient amount of starting template

Increase the amount of template, if possible. Ensure thatsufficient copies of the target nucleic acids are present in yoursample.

Problems Recommendations

No product, or weak product signal in in PCR

12) Insufficient number of cycles Increase the number of cycles.

13) Probe design not optimalIncorrect nucleotide concentration or nucleotide degradation(labeledprobes)

If the amplification reaction was successful, there may be aproblem with the probe. Design the probe again.

14) Annealing temperature too high Decrease annealing temperature in steps of 2°C.

15) Annealing temperature too low Increase annealing temperature in steps of 2°C.

16) Probe synthesis not optimal Check the quality of dual-labeled probes or Molecular Beacons by incubation with DNase I. A correctly synthesized probe, containing both fluorophore and quencher, will show a significant increase in fluorescence after DNase I incubation.

Poor dynamic range of CT value

1) Template amount too high Do not exceed maximum recommended amounts of template.LightCycler : Do not use more than 1 μg template.All other cyclers : Do not use more than 500 ng template.

2) Template amount too low Increase template amount, if possible

High fluorescence in “No Template” control

1) Contamination of reagents Discard all the components of the assay (e.g., master mix,primers, and probes). Repeat the assay using new components.

2) Contamination during reaction setup, Take appropriate precautions during such as using aerosol-barrier pipet tips.

Primer–dimers and/or nonspecific PCR products (HRM)

1) Mg2+ concentration not optimal Always start with the Mg2+ concentration provided in RealMODTM

Real time PCR series(2 mM final concentration). For a few targets,an increase up to 6 mM Mg2+ may be helpful. Perform thetitration in 0.5 mM steps.

2) Annealing too low temperature Increase annealing temperature in increments of2°C.

3) PCR product too long For optimal results, PCR products should be between 100 and150 bp. PCR products should not exceed 500 bp

4) To much amount of primer Decrease the amount of primer

5) Template amount too low Increase template amount, if possible

7. Related Products

Product Cat. No. Size Product Cat. No. Size

iNNOPLEXTM JEV/WNV Real-time RT-PCR Typing Kit

IP10131 50 TestsMaximeTM PCR PreMixKit(i-StarTaq)

25165 96 Tubes

25167 480 Tubes

iNNOPLEXTM YFV/DENV Real-time RT-PCR Typing Kit

IP10132 50 TestsMaximeTM PCR PreMixKit(i-StarMAX II)

25281 96 Tubes

iNNOPLEXTM SIV Real-time RT-PCR Kit(NP) D40253 50 Tests i-D MaximeTM PCR

PreMix Kit25178 96 Tubes

6. Guideline for Primer Design

It is essential to design primers which allow good reactivity for a successful real time PCR reaction. Please

follow the guideline stated as below to design primers which offer high amplification efficiency and minimizes

non-specific reaction.

- Amplification product

- Primer

Amplified size 80 ～ 150 bp is most recommended. (Possible to amplify a target up of 300 bp.)

Length 17 ～ 25 mer

GC content 40 ～ 60％ (45 ～ 55％ is recommended.)

Tm Tm values of Forward primer and Reverse primer must not largely differ. Tm value is calculated with the software for calucation of Tm value.

Sequence The sequence should not be partially rich in each base in the whole sequence. Avoid including parts which has high GC or AT content, (especially 3'-end).

- Not include polypyrimidine (serial T/C sequence).- Not include polypurine (serial A/G sequence).

Sequence of 3' end

The termini part of 3' should not have high content of GC or AT.It is recommended to be G or C at 3' end.It is not recommended to be T at 3' end.

Complementarity

Complementary sequence of more than 3 bases should not exist within a primer or even between primer pairs. Primer pair should not have a complementary sequence of more than 2 bases at the 3' end each.

Specificity Specificity of primers should be confirmed through BLAST search.

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