mutareal® factor ii

MutaREAL Factor II

Version 2.0 / November 2018

KF291632

KF291696

32

96

Only for in vitro diagnostics

Real-Time PCR Kit

Real-Time PCR kit for the analysis of mutation G20210Ain the FII gene based on the FRET technology

on LightCycler 1.5, 2.0 and 480.

®

Immundiagnostik AG, Stubenwald-Allee 8a, 64625 Bensheim, Germany www.immundiagnostik.com Tel.: +49 (0)6251/ [email protected] Fax: +49 (0)6251/ 849430

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© 2018 Immundiagnostik AG / Version 2.0

MutaREAL® Factor II

Table of Contents

1 Intended Use 3

2 Introduction 3

3 Concept of the Assay 3

4 Kit Components 4

5 Required Materials 4

6 Storage and Handling 4

7 Considerations and Precautions 4

8 Test Procedure 5

8.1 PCR Preparation 5

8.2 PCR Protocol 6

9 Evaluation 6

10 Troubleshooting 7

11 Test Limitations 7

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1 Intended Use

The Factor II Real-Time PCR Kit is a FRET-based test for the analysis of the mutationG20210A in the FII gene.

2 Introduction

Prothrombin (Factor II) is the precursor of the active coagulation enzyme Thrombin(Factor IIa), which has a key position in the haemostasis and thrombosis. It convertsfibrinogen to fibrin for the blood coagulation, stimulates the aggregation of blood plateletsand activates the coagulation factors V, VII and XIII. The base substitution G20210A isassociated with an increased level of prothrombin and thus with an increasedsusceptibility to venous thrombosis.

References:De Stefano et al., The New England Journal of Medicine, 1999, 341:801-6Gerhardt et al., The New England Journal of Medicine, 2000, 342:374-80Franco et al., British Journal of Haematology, 1999, 104:50-4

3 Concept of the Assay

This sequence specific detection assay is based on fluorescence resonance energytransfer (FRET). The assay contains two specific primers flanking the target region aswell as two hybridization probes. One of the hybridization probes binds to a specific targetregion known to carry the gene mutation of interest. The second hybridization probe bindsto a sequence in close proximity to the first probe, not covering the mutation. In order to achieve FRET, one of the hybridization probes is labeled with a donor-fluorophor, the other one is labeled with an acceptor-fluorophor. If both hybridizationprobes are in immediate proximity, the donor-fluorophor transfers energy to the acceptorfluorophor following excitation. Due to this energy transfer, the acceptor-fluorophor emitslight of a longer wavelength. Since energy transfer can only occur if both hybridizationprobes are bound to the target sequence, the amount of hybridized probe pairs, andthereby the fluorescence signal proportionally increases to the amount of amplified PCRproduct. Following amplification, genotyping is then performed via melting curve analysis.For this purpose, the temperature is slowly increased after a short denaturation step andthe dissociation behavior of the probe is monitored by constantly measuring the emittedfluorescence signal. If the targeted mutation is not present, the fluorescence signaldecreases only at high temperatures since the perfectly matched hybridization probedissociates late due to its high binding affinity to the target region. However, in thepresence of the mutation, the fluorescence signal decreases earlier with increasingtemperatures since the mismatched and therefore less stable probe dissociates earlierfrom the target region.

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4 Kit Components

Factor II Real-Time PCR-Kit Volume

Reagents 32-rxn 96-rxn

Enzyme Mix (blue) 438 µL 3 x 438 µL

Detection Mix (yellow) 368 µL 3 x 368 µL

Positive Control (red) 15 µL 45 µL

Negative Control (green) 50 µL 150 µL

5 Required Materials

Required Materials - not provided:Roche LightCycler® 1.5, 2.0 or 480 Real-Time PCR-Systemo The CE conformity is only given when one of the above mentioned

components is used.LightCycler® capillaries, Roche or 96-well plate/stripes (white)

LightCycler® cooling block, Roche or cryo container for PCR reaction tubes

Pipettes (0.5 – 200 µL)o 0.5 - 10 µLo 10 - 200 µL

1.5 mL reaction tubes

6 Storage and Handling

All reagents should be stored at -20 °C.

Avoid several freeze / thaw cycles of the reagents (if necessary prepare aliquots).

The detection mixes have to be protected from exposure to light.

7 Considerations and Precautions

The regulations and principles for working in a biomolecular laboratory have to be strictlyfollowed.

All steps have to be performed in an uninterrupted manner

All PCR reagents have to be cooled while in use

The DNA purity (A260/A280 ratio) should be between 1.8 and 2.0

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8 Test Procedure

8.1 PCR Preparation

Gently thaw all components on ice. Mix them gently (do not vortex) and spin them downbefore use. Keep in mind to protect the detection mixes from exposure to light. During thePCR setup all reagents have to be kept cool.

For the amplification one PCR reaction tube (type dependent on the instrument used) isneeded per sample plus two additional tubes for the negative and positive controls. Thefollowing table shows the volume of each reagent per sample. For the analysis amastermix should be prepared for the number of samples (incl. negative and positivecontrol) (N) plus 10 % to compensate inaccuracies. The mastermix should be prepared inthe same order as indicated in the table.

Reagent Volume per 25 µLReaction

Master Mix Volume

Detection Mix (yellow) 10.5 µL 10.5 µL * (N + (N * 0.1))

Enzyme Mix (blue) 12.5 µL 12.5 µL * (N + (N * 0.1))

Thoroughly mix the mastermix by pipetting up and down or inverting (do not vortex)and shortly spin down. Aliquot 23 µL into each PCR tube. For the negative control add 2 µL of the provided negative control (green).

For the positive control add 2 µL of the provided positive control DNA (red).

Add 2 µL of each sample DNA to the corresponding PCR tube.

LightCycler® 1.5 and 2.0: Close the capillaries with the lid, transfer them into theLightCycler® carousel and centrifuge in the LightCycler® centrifuge (if a table centrifuge isused, centrifuge the capillaries in the adapters of the cooling block for 15 s at 3000 rpm).Subsequently transfer the carousel into the LightCycler® and use the PCR protocoldescribed in 8.2.

LightCycler® 480: Close the wells with the sealing-foil and centrifuge the plate for 15 s at2000 rpm. Subsequently transfer the plate into the LightCycler® 480 and use the protocoldescribed in 8.2.

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8.2 PCR Protocol

Step Temperature[°C]

Time [s] Ramp Rate[°C/s]

Cycles Acquisition

Initial Denaturation 94 120 max. 1 x None

Denaturation 94 10 max.

45 x

None

Primer Annealing 55 25 max. Single

Elongation 72 25 max. None

Melting Curve

94 30 max. 1 x None

40 30 max. 1 x None

80 0 0,1 - 0,2* 1 x Constant

Cooling 40 30 max. 1 x ---

*depending on the number of selected filters for the LightCycler® 480, it can be necessary to adjustthe acquisitions per °C.

9 Evaluation

For the evaluation of the melting curves add an analysis of the type "genotyping". Therebythe derivation of the fluorescence curve is generated. The detection wavelength is640 nm.

Temperature mutated allele: 53,0 °C (+/-2 °C) Temperature wildtype allele: 60,0 °C (+/-2 °C)

The following graphic shows the typical results for the possible genotypes: pink curve -negative control, violet and blue curve - homozygous wildtype, green Kurve -heterozygous mutation, black curve - homozygous mutation.

The provided positive control contains a template heterozygous for the point mutationG20210A.

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10 Troubleshooting

Problem Solution

No or low fluorescence peakwith positive control orsamples

Check PCR-program of the real time PCR instrument inuse and repeat with corrected protocol.

The Detection Mix was thawn / frozen more than twice orstored longer than four days at 2-8 °C. Repeat analysiswith a fresh aliquot or new Detection Mix.

Quality of DNA template is not sufficient. Use freshlyextracted DNA and measure the concentration/puritybefore use.

The Detection Mixes were not protected from light. Repeatanalysis with a fresh aliquot or new PCR reagents.

11 Test Limitations

The accuracy of genetic testing is never 100%. However, an accuracy of more than 98%was determined based on the validation data. The attending physician is free to use thetest results as a guidance in the decision making process in terms of diagnosis andtherapy. However, these recommendations are based on genetic test outcomes and needto be interpreted in the context of medical history and known familiar risks of eachindividual patient. The attending physician is fully responsible for the final diagnosis andtreatment.

mutareal® factor ii

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