User Manual
Contents
1. TUBE T DESCRIPTION LABEL OR LID COLOUR Mastermix 2X _ ey 4x340uL 2x340yL 1x 340 uL I Oligomix Primer and probe Mix MTHFR White 4x27uL 2x27uL 1x24yL C677T Passive Reference ROX 2 x 30 uL 1 x 30 uL 1x 15 HL STORE AT 2 8 C TUBE T DESCRIPTION LABEL OR LID COLOUR DNA containing a part of the target sequences Wig ups C677T Blue 1x20gL 1x10gL 1x10yL homozygote for Positive MTHFR C677T control HET Mix of HOMO WT and HOMO MTHFR MUT positive controls for C677T Green 1x20 pL 1x10 HL 1x10 uL MTHFR C677T Positive control DNA containing a part of the url Hugevssquenea inulta C677T Red 1x20gL 1x10uL 1x10pL homozygote for Positive MTHFR C677T control MANUAL RS MTHFR677 eR070109 anauitica ADVANCED BIOMEDICINE www abanalitica it 3 STORAGE AND STABILITY OF THE REAGENTS Each component of the kit should be stored according to the directions indicated on the label of the single boxes In particular Box F store at 30 C 20 C BAG store at 2 8 C When stored at the recommended temperature all test reagents are stable until their expiration date The 2X AD Real Time Mix Oligomix and the positive control reagents are sensitive to the physical state variations it is recommended not to let the reagents undergo more than two freeze thaw cycles If the single test runs are limited to a small number of2. ANALITICA ADVANCED BIOMEDICINE www abanalitica it User Manual REALQUALITY RS MTHFR C677T code RQ S29 48A code RQ S29 96A Kit for the identification and genotyping of C677T mutation in the human gene encoding the Methylene tetrahydrofolate Reductase C MANUAL RS MTHFR677 eR070109 This product was developed using a technology licenced by DxS Ltd Manchester UK 1 PRODUCT INFORMATION 1 1 Intended Use 2 KIT CONTENT 3 STORAGE AND STABILITY OF THE REAGENTS 4 PRECAUTIONS FOR USE 5 SAFETY RULES 5 1 General safety rules 5 2 Safety rules about the kit 6 MATERIALS REQUIRED BUT NOT PROVIDED 6 1 Reagents 6 2 Instruments 63 Materials 7 INTRODUCTION 8 TEST PRINCIPLE 9 PRODUCT DESCRIPTION 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES 10 1 Peripheral blood 11 PROTOCOL 11 1 DNA Extraction 11 2 DNA Amplification 11 2 1 Instrument programming 11 2 2 Amplification Mix preparation 11 2 3 Pre Read run 11 2 4 Absolute Quantification run 11 2 5 Post Read run 11 3 Data analysis 12 TROUBLESHOOTING N co O 0 O 11 12 13 13 13 13 14 14 17 18 18 19 19 23 ANALITICA 1 MANUAL_RS MTHFR677_eR070109 www abanalitica it 13 DEVICE LIMITATIONS 14 DEVICE PERFORMANCES 14 1 Analytical specificity 14 2 Diagnostic sensitivity and specificity 14 3 Analytical sensitivity 14 4 Accuracy 15 REFERENCES 16 RELATED PRODUCTS MANUAL RS MTHFR677 eR070109 2 2
3. do not vortex then centrifuge briefly Prepare the reaction mix rapidly at the room temperature or work on ice or on the cooling block 5 SAFETY RULES 5 1 General safety rules e Wear disposable gloves to handle the reagents and the clinical samples and wash the hands at the end of work e Since no known diagnostic method can assure the absence of infective agents it is a good rule to consider every clinical sample as potentially infectious and handle it as such e All the devices that get directly in touch with clinical samples should be considered as contaminated and disposed as such In case of accidental spilling of the samples clean up with 1096 Sodium Hypochloride The materials used to clean up should be disposed in special containers for contaminated products MANUAL RS MTHFR677 eR070109 6 ANALITICA www abanalitica it e Clinical samples materials and contaminated products should be disposed after decontamination by immersion in a solution of 5 Sodium Hypochloride 1 volume of 5 Sodium Hypochloride solution every 10 volumes of contaminated fluid for 30 minutes OR autoclaving at 121 C at least for 2 hours NOTE do not autoclave solutions containing Sodium Hypochloride 5 2 Safety rules about the kit The risks for the use of this kit are related to the single components Dangerous components none The Material Safety Data Sheet MSDS of the device is avaialble upon request ANALITICA 7 MANUAL_
4. 2 2 The kit was not stored properly or it was used beyond the expiry date e Order a new product and make sure to follow the instructions in paragraph 3 and on the labels The amplification reaction was inhibited e Repeat the analysis using a suitably extracted DNA paragraph 11 1 e Ifan extraction system employing Ethanol wash steps was used make sure no ethanol residue remains in the DNA sample 2 Low fluorescence intensity The fluorophores may have decayed e Store the oligomix as indicated in the instructions in paragraph 3 do not expose to direct light also during the thawing of the reagents An extremely low amount of DNA was amplified e Do not amplify less than 2 5 ng of DNA The optimum amount of DNA to be used for this amplification is 20 50 ng reaction ANALITICA 23 MANUAL_RS MTHFR677_eR070109 www abanalitica it The 2X AD Real Time Mix and Oligomix reagents were thawed for more than two times e Check if the reagents were thawed for more than two times and repeat the experiment with another aliquot of these reagents 3 Variable fluorescence intensity from sample to sample The reaction mix was not mixed well prior to aliquoting e Once prepared mix the amplification mix thoroughly by inverting the tube in which it was prepared several times Some wells contained air bubbles at the bottom e Once you aliquot the mix check the bottom of the plate to make sure no air bubbles are present at the bottom of the wells and or
5. samples it is recommended to aliquot the reagents Oligomix and ROX contain fluorescent molecules it is recommended to store these reagents far from any light source 4 PRECAUTIONS FOR USE e The kit must be used only as an IVD and handled by qualified investigators who are educated and trained in molecular biology techniques applied to diagnostics e Before starting the kit procedure read carefully and completely the instruction manual e Keep the product away from heating sources and the direct light e Do not use any part of the kit if over the expiration date In case of any doubt about the storage conditions box integrity or method application contact AB ANALITICA technical support at laboratorio abanalitica it ANALITICA 5 MANUAL_RS MTHFR677_eR070109 ADVANCED BIOMEDICINE www abanalitica it In the amplification of nucleic acids the investigator has to take the following special precautions Use filter tips Store the biological samples the extracted DNA positive control included in the kit and all the amplification products in different places from where amplification reagents are stored Organise the work space in different pre and post PCR units do not share consumables pipettes tips tubes etc between them Change the gloves frequently e Wash the bench surfaces with 5 sodium hypochloride Thaw the reagents prior to use once thawed mix the solutions well by inverting the tubes several times
6. 5 25 25 25 25 26 27 28 ADVANCED BIOMEDICINE 1 PRODUCT INFORMATION 1 1 Intended Use The RS MTHFR C677T kit is an IVD for identification and genotyping of C677T mutation in gene encoding the human Methylene tetrahydrofolate Reductase by means of Real time PCR amplification of genomic DNA extracted from human peripheral blood It is an in vitro diagnostic test for detection and genotyping of MTHFR mutation and it represents an auxiliary instrument for diagnosis and evaluation of putative thrombophylic patients As such it is recommended to use this kit as indicated in the instructions herein The present manual refers to the following product RS MTHFR C677T Kit for identification and genotyping of C677T mutation in gene encoding the human Methylene tetrahydrofolate Reductase by Real time PCR amplification Contains all the reagents needed for the Real time amplification This product is in accordance with 98 79 CE Directive regarding the In Vitro medical diagnostic devices CE mark Code Product PKG RQ S29 48A RS MTHFR C677T 48 reactions RQ S29 96A RS MTHFR C677T 96 reactions NOTE The product was validated on ABI 7300 Applied Biosystems The compatibility with the ABI7000 Applied Biosystems and SmartCycler Cepheid Celbio instruments was confirmed experimentally ANALITICA 3 MANUAL_RS MTHFR677_eR070109 ADVANCED BIOMEDICINE www abanalitica it 2 KIT CONTENT STORE AT 30 20 C
7. AM1 Legend Allele x Allele Y A Both m Nrc Undetermined By selecting the Report menu the data shall be displayed as a table ANALITICA ADVANCED BIOMEDICINE www abanalitica it 21 MANUAL_RS MTHFR677_eR070109 Seldom it may occur that the software can not assign the genotypes to all or some of analyzed samples automatically These samples are reported in the graph as X undetermined In this case it is possible to assign the genotypes manually as indicated below by referring to positions of the samples in the graph and or by analyzing the curves in the amplification file Absolute Quantification 1 Activate the button ES 2 Use it to identify the undetermined sample or the group of undetermined samples and assign the correct genotype one by one by selecting it from the Call menu MANUAL RS MTHFR677 eR070109 22 ANALITICA ADVANCED BIOMEDICINE www abanalitica it 12 TROUBLESHOOTING 1 Absence of FAM and JOE signal in the samples and positive controls The instrument was not programmed correctly e Repeat the amplification taking care of the instrument programming pay particular attention to the thermal profile the selected fluorophores and the correspondence between the plate protocol and the plate itself The amplification mix was not prepared correctly e Prepare a new amplification mix making sure to follow the instructions given in the paragraph 11
8. ANUAL_RS MTHFR677_eR070109 16 ANALITICA www abanalitica it At this point the instrument will connect to the pc and a plate scheme will appear on the screen Setup level 4 Click rapidly two times anywhere on the plate the Well Inspector window will appear 5 Activate the Add detector button select the two detector created previously and click Add To Plate Document 6 Create the same ayout as in the Pre Read plate 7 Make sure that the Passive reference option is set to ROX 8 Go from the Setup level to the Instrument level 9 Set the following thermal profile Cycle Repeats Step Time C Taq activation 1 1 1 03 00 95 0 Amplification 2 50 4 00 10 95 0 cycles Fluorescence collection step 10 Set the Sample Volume to 25 uL 11 Save the file 11 2 2 Amplification Mix preparation Once thawed mix the reagents by inverting the tubes several times do not vortex then centrifuge briefly Prepare the reaction mix rapidly at room temperature or work on ice or on the cooling block Take care to work shaded from the direct light as much as possible Prepare a master mix of an appropriate volume that must be sufficient for all the samples to be processed for the positive controls and the negative control when calculating the volume consider an excess of at least one reaction volume as follows NB ROX is an inert dye whose fluorescence does not change during the amplification reaction on instrument
9. RS MTHFR677_eR070109 ADVANCED BIOMEDICINE www abanalitica it 6 MATERIALS REQUIRED BUT NOT PROVIDED 6 1 Reagents v DNA extraction reagents Y Dnase and Rnase free sterile water 6 2 Instruments Y Laminar flow cabinet use is recommended while preparing the amplification mix to avoid contamination it would be recommended to use another laminar flow cabinet to add the extracted DNA Y Micropipette range 0 5 10 uL 2 20 uL 10 100 uL 20 200 uL 100 1000 UL Y Microcentrifuge max 12 14 000 rpm v Plate centrifuge optional Y Real time amplification instrument The product was validated on ABI 7300 Applied Biosystems The compatibility with the ABI7000 Applied Biosystems e SmartCycler Cepheid Celbio instruments was confirmed experimentally 6 3 Materials v Talc free disposable gloves Y Disposable sterile filter tips range 0 5 10 uL 2 20 uL 10 100 uL 20 200 uL 100 1000 uL v 96 well plates for optical measurements and the adhesive optical film MANUAL RS MTHFR677 eR070109 8 ANALITICA www abanalitica it 7 INTRODUCTION Venous thrombosis is the obstruction of the circulation by clots that have been formed locally in the veins or have been released from a thrombus elsewhere formed The usual sites of thrombus formation are the superficial and deep veins of the legs but it also may occur in veins in the brain retina liver and mesentery An important question is whether the risk for the development of
10. U IRiceannect ted I nina 3 Select Analysis gt Analysis settings 4 For automatic genotype assignment deselect Analyze post read data only by doing so the basal fluorescence acquired during the Pre Read phase will be subtracted from the fluorescence data acquired during the Post Read phase and Keep Manual Calls from previous Analysis option flag the Automatic Allele Calling instead 5 Click OK amp Reanalyze Analysis Settings Allelic Discrimination Assay x m Data Analysis Analyze post read data only Allele Calling Keep Manual Calls from previous Analysis Marker RENSES v Automatic Allele Calling Quality Value fo 9000 Two cluster Calling On OK amp Reanalyze ILo Cancel Apply MANUAL RS MTHFR677 eR070109 20 Qanaitica wom abanalien The software will group the samples in the following manner Allele X Homozygotes XX Downer right angle of the graph Allele Y YY Homozygotes Upper left angle of the graph Heterozygotes XY In the centre of the graph between the wt and mutant homozygotes Negative control Downer left angle of the NTC graph undetermined dispersed B File view Tools Instrument Analysis Window Help zexi D gal E gt Ha Its Marker SNPs1 gt car ateex y Ege E a a _ mit Graph Selection ic Discriminati Allele Y F
11. a 30 percent higher plasma prothrombin levels than noncarriers and have a risk of deep vein thrombosis that is 3 6 times higher than that in the general population The mutation is rare among nonwhites In whites the carrier rate ranges from 0 7 to 4 percent G20210A appears to be an important risk factor for cerebral vein thrombosis Furthermore the G20210A mutation seems to be synergistic with the use of oral contraceptives Factor V gene encodes the homonym protein which is present in the blood as inactive pro cofactor It can be activated by thrombin resulting in the formation of a two chain molecule factor Va that serves as a cofactor of factor Xa in the conversion of prothrombin into thrombin Inactivation of factor Va occurs through selective proteolytic cleavages in its heavy chain at Arg306 Arg506 and Arg679 by activated protein C APC The hypothesis is that thrombosis can result from a variety of genetic mutations affecting critical sites in the factor V protein A G A transition in exon 10 of the Factor V gene results in the replacement of arginine at position 506 by glutamine in the resulting protein This mutated form of factor V is known as the Factor V Leiden mutant The activated factor V Leiden is not cleaved by APC and is therefore designated APC resistant The population of carriers of factor V Leiden in the white population ranges from 2 to 15 percent Again the mutation is extremely rare in non whites Heterozygous carr
12. al specificity The specificity of the RS MTHFR C677T code RQ S29 kit is guaranteed by an accurate and specific selection of sequence specific primers and fluorogenic probes designed to amplify only the gene sequence of interest and also by the use of the stringent amplification conditions The alignment of primers and probes in the most important databanks shows the absence of non specific pairing 14 2 Diagnostic sensitivity and specificity A significant number of samples previously genotyped for the 677 position of the MTHFR gene with other CE IVDs were tested The RS MTHFR C677T device assigned the correct genotype to all of the analyzed samples 100 diagnostic sensitivity and specificity 14 3 Analytical sensitivity Serial dilutions of human genomic DNA WT homozygous mutant and heterozygous for MTHFR 677 mutation were amplified in order to determine the analytical sensitivity Even the smallest amount tested 2 ng of DNA was shown to be high enough for genotype determination by this device In order to define the maximum amplifiable amount of DNA experiments were performed amplifying up to 250 ng of DNA per reaction even in these conditions the assay was shown to be fully functioning ANALITICA 25 MANUAL_RS MTHFR677_eR070109 ADVANCED BIOMEDICINE www abanalitica it 14 4 Accuracy This value was calculated by the number of correct amplifications over the total number of executed amplifications The RS MTHFR C677T h
13. as an accuracy of 100 MANUAL RS MTHFR677 eR070109 26 ANALITICA ADVANCED BIOMEDICINE www abanalitica it 15 REFERENCES Bagley PJ Selhub J Proc Natl Acad Sci U S A 1998 95 13217 13220 Bertina RM Koeleman BPC Koster T Rosendaal RJ Dirven RJ de Ronde H van der Velden PA Reitsma PH Nature 1994 369 64 67 Malik NM Syrris P Schwartzman R Kaski JC Crossman DC Francis SE Carter ND Jeffery S Clin Sci 1998 95 311 315 Motti C Gnasso A Bernardini S Massoud R Pastore A Rampa P Federici G Cortese C Atherosclerosis 1998 139 377 383 Poort SR Rosendaal FR Reitsma PH Bertina RM Blood 1996 88 3698 3703 Saiki RK S Scharf F Faloona KB Mullis GT Horn HA Erlich and N Arnheim Science 230 1350 1354 1985 Williamson D Brown K Luddington R Baglin C Baglin T Blood 1998 91 1140 1144 ANALITICA 27 MANUAL_RS MTHFR677_eR070109 www abanalitica it 16 RELATED PRODUCTS REALQUALITY RS FATTORE Il G20210A Kit for the identification and genotyping of G20210A mutation in gene encoding the human coagulation Factor Il by Real time PCR amplification Contains all the reagents needed for the Real time amplification This product is in accordance with 98 79 CE Directive regarding the n Vitro medical diagnostic devices CE mark Code Product PKG RQ S27 48A RS FATTORE II G20210A 48 reactions RQ S27 96A RS FATTORE G20210A 96 reactions REALQUALITY RS FATTORE V Leiden Kit for identification and gen
14. ca it Click New Marker Name the marker in the New Marker Name field es MTHFR C677T Flag the two new detectors and click OK Select the newly created marker 6 Click Add then End At this point the instrument will connect to the pc and a plate scheme will appear on the screen Setup level 7 Click rapidly two times anywhere on the plate the Well Inspector window will appear 8 Put in Use the Marker and define the Task Unknown for the samples and the controls and NTC for the negative control and the Sample Name of every position to be loaded on the plate 9 Make sure that the Passive reference option is set to ROX 10 Go from the Setup level to the nstrument level 11 Modify the Sample Volume to 25 pL and check that the preset temperature is the default 60 C one 12 Save the file 11 2 1 2 Creation of the Absolute Quantification Document 1 Select File gt New 2 Select Absolute Quantification in the Assay scroll menu 3 Name the plate i e Amplification MTHFR677 yymmdd in the Plate Name field then click End New Document Wizard x Define Document Select the assay container and template for the document and enter the operator name and comments Assay Absolute Quantification Standard Curve Container 36 well Clear Template Blank Document x Browse Operator fieatimeuser Comments SDS v1 2 Pi NR amplification MTHFR77 aammad Fine Annulla M
15. centrifuge the plate at 4000 rpm for about 1 minute There is a great difference in concentration among the amplified DNA samples e Use the samples extracted with validated extraction methods and or determine the DNA concentration before amplification in order to render them more homogeneous 4 The automatic call of the genotypes does not allow the genotype assignment in all some samples also when considering the Pre Read data There was an error in Pre Read process e Try to use the Post Read data only for the automatic call by flagging the Post Read data only option in the Analysis Setting Allelic Discrimination Assay window e If the same result is obtained proceed with manual genotype assignment paragraph 11 3 or repeat the analysis of the undetermined samples For any further problems contact AB ANALITICA s technical support at laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 MANUAL RS MTHFR677 eR070109 24 ANALITICA ADVANCED BIOMEDICINE www abanalitica it 13 DEVICE LIMITATIONS The kit can have reduced performances if e The clinical sample is not suitable for this analysis improper storage after extraction use of heparin as anticoagulant e the histological sample is not suitable for this analysis use of alternative fixatives instead of neutral formalin buffer not correct sample storage e The kit was not stored at recommended temperature 14 DEVICE PERFORMANCES 14 1 Analytic
16. emselves with fluorescent molecules In the first case the Fluorescence Resonance Energy Transfer FRET among the two fluorophores or other mechanisms which lead to fluorescence emission and involve a fluorophore and a non fuorescent quencher molecular beacon scorpion primer etc are used The mechanism that determines the fluorescence emission is based on the presence of a quencher molecule located in proximity of a reporter molecule that blocks the fluorescence emission by the reporter When the quencher is separated from the reporter the latter emits fluorescence Main advantages of the Real time PCR technique compared to the conventional amplification techniques are for example the possibility to execute a semi automated analysis in which the time needed for the visualizzation of the amplicons is eliminated and the absence of the post amplification sample manipulation that reduces the possible contamination phenomena ANALITICA 11 MANUAL_RS MTHFR677_eR070109 www abanalitica it 9 PRODUCT DESCRIPTION The RS MTHFR C677T kit allows to define the genotype of a subject in the position 677 of the gene encoding the human N N methylenetetrahydrofolate reductase The wild type WT and mutant MUT alleles are distinguished due to the use of fluorogenic sequence specific probes Each allele specific primer is marked with a different fluorescent dye FAM for the WT allele and JOE for MUT allele this makes possible to discrimina
17. from the analysis by activating the option Omit Well in the Analysis menu before the analysis the selected samples will be displayed in the graph with an X symbol undetermined The positions occupied in the graph derive from the fluorescence data collected during the Post Read phase The three groups corresponding to three different genotypes should already be evident ANALITICA 19 MANUAL_RS MTHFR677_eR070109 ADVANCED Bi IOMEDICINE www abanalitica it B File view Tools Instrument Analysis Window Help la xl OSH S amp EL E E e 1 I Setup Plate Y Spectra Y Allelic Discrimination Y Report X Marker SNPs1 gt Calt Allele x AAJA _ Omit Graph Selection Allelic Discrimination 10 00 r Legend Allele x x Allele Y 8 00 Both RX e 6 00 F3 Undetermined L lt gt E x NX 4 00 2 00 m 0 00 0 00 0 20 0 40 0 60 0 80 1 00 1 20 1 40 Allele X JOE1 6 4 2 3 4 5 7 8 9 10 11 12 I oF i ueyy Fr y ppp gt yy mI CT m UDP JUD Qdm JUD dm 1 1 L cl on jon on AU JU du om DU 1D d p H0 0 d o Un dU JU d d n ef om om nm om um sm um J F fom fom fom Qo Jom om om om L1 sE M 0 0 A 0 A 0 1 D ee CE p o EE EE E EE 2 YI j
18. iers have a risk of deep venous thrombosis that is 7 times higher than that in the general population for homozygous carriers the risk is 80 times higher In 5 10 of patients affected by deep venous thromobosis with APC resistance without Factor V Leiden mutation the APC resistence might be due to other risk factors such as pregnancy or high levels of Factor VIII The presence of multiple mutations may have synergistic effects Therefore it is important to determine the genotype of each subject for a couple of different mutation MANUAL RS MTHFR677 eR070109 10 ANALITICA ADVANCED BIOMEDICINE www abanalitica it 8 TEST PRINCIPLE PCR method Polymerase Chain Reaction was the first method of DNA amplification described in literature Saiki RK et al 1985 It can be defined as an in vitro amplification reaction of a specific part of DNA target sequence by a thermostable DNA polymerase This technique was shown to be a valid and versatile molecular biology instrument its aplication contributed to a more efficient study of new genes and their expression and it brought to a revolution in the laboratory diagnostic and forensic medicine field The REAL TIME PCR technology represents an advancement of the basic PCR technique it allows to measure the number of DNA molecules amplified during the exponential amplification phase The amplicon monitoring is essentially based on the labeling of the primers and probes or of the amplicons th
19. otyping of G1691A Leiden mutation in gene encoding the human coagulation Factor V by Real time PCR amplification Contains all the reagents needed for the Real time amplification This product is in accordance with 98 79 CE Directive regarding the n Vitro medical diagnostic devices CE mark Code Product PKG RQ S25 48A RS FATTORE V Leiden 48 reactions RQ S25 96A RS FATTORE V Leiden 96 reactions REALQUALITY RS MTHFR A1298C Kit for the identification and genotyping of A1298C mutation in gene encoding the human Methylene tetrahydrofolate Reductase by Real time PCR amplification Contains all the reagents needed for the Real time amplification This product is in accordance with 98 79 CE Directive regarding the n Vitro medical diagnostic devices CE mark Code Product PKG RQ S31 48A RS MTHFR A1298C 48 reactions RQ S31 96A RS MTHFR A1298C 96 reactions MANUAL RS MTHFR677 eR070109 28 ANALITICA www abanalitica it Qanauitica 29 MANUAL RS MTHFR677 eR070109 www abanalitica it ANALITICA AB ANALITICA srl Via Svizzera 16 35127 PADOVA ITALY ADVANCED BIOMEDICINE Tel 39 049 761698 Fax 39 049 8709510 www abanalitica it e mail info abanalitica it
20. planations regarding the extraction method contact AB ANALITICA s technical support at laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 Please notice that the positive controls included in the kit are appropriate for amplification of 20 50 ng of DNA reaction However the assay was shown capable of identifying the correct genotype also in a much broader concentration range 2 250 ng DNA reaction ANALITICA 13 MANUAL_RS MTHFR677_eR070109 www abanalitica it 11 2 DNA Amplification Thaw all the reagents at room temperature prior to preparing the amplification mix DNA and positive controls also Assure that the 2X AD Real Time Mix Oligomix and positive controls do not undergo more than two freeze thaw cycles It is recommended to use the thaw time to program the instrument 11 2 1 Instrument programming The instructions provided herein refer to the AB 7300 SDS software version 1 2 3 For other details please consult the user manual of the instrument 1 Turn the pc on 2 Activate the instrument and the SDS software 11 2 1 1 Creation of the Pre Read Document 1 Select File gt New 2 Select Allelic Discrimination under the Assay scroll menu 3 Name the plate ie Pre Read MTHFR677 yymmdd in the Plate Name field 4 Click Next New Document Wizard X Define Document Select the assay container and template for the document and enter the operator name and comments Assay Allelic Discrimina
21. run Load the plate on the instrument making sure to position it correctly Open the Pre read document created previously Click Pre Read at the Instrument level At the end of the run a window indicating the end of the reaction will appear after a couple of minutes Click OK 6 Close the file AON C1 11 2 4 Absolute Quantification run 1 Open the Absolute Quantification document created previously MANUAL RS MTHFR677 eR070109 18 ANALITICA ADVANCED BIOMEDICINE www abanalitica it 2 3 4 5 Click Start at the nstrument level At the end of the run a window indicating the end of the reaction will appear after a couple of minutes Click OK Close the file It is recommended to proceed with the Post Read Run immediately in order to obtain correct reading of the end point fluorescence data obtained during the Amplification Run 11 2 5 Post Read run 1 2 3 At the end of the run a window indicating the end of the reaction will 11 3 1 2 Open the Post Read document Name and save the Post Read document es Post Read MTHFR677 yymmdd Click Post Read appear after a couple of minutes Click OK Data analysis In the Post Read document select the Results tab then select the Allelic Discrimination sublevel Select the wells on the plate for which the data will be analyzed if also another targets were amplified on the same plate the wells containing other targets must be excluded
22. s that allow its use Applied Biosystems Stratagene etc it allows to normalize the well to well differences due to artefacts such as pipetting errors or instrument limitations In case in which different instruments are to be used do not add ROX to the reaction mix and adjust the reaction volume appropriately with sterile water ANALITICA 17 MANUAL_RS MTHFR677_eR070109 www abanalitica it Reagent 1 Rx 2X AD Real Time Mix 12 5 uL Oligo Mix MTHFR C677T 1 uL ROX 0 5 uL H20 10 uL Total Volume 24 uL Mix by inverting the tube in which the mix was prepared several times Then centrifuge briefly Pipette 24 uL of the mix on the bottom of each well on the plate Add 1 uL of extracted DNA to each well or 1uL of each of the three positive control DNAs in the correct positions on the plate Please notice that the positive controls included in the kit are appropriate for amplification of 20 50 ng of DNA reaction However the assay was shown capable of identifying the correct genotype also in a much broader concentration range 2 250 ng DNA reaction Always amplify a negative control together with the samples to be analyzed add sterile water to the amplification mix instead of extracted DNA Hermetically seal the plate by using the optic adhesive film and the appropriate sealer Make sure there are no air bubbles in the bottom of the wells and or centrifuge the plate at 4000 rpm for about 1 minute 11 2 3 Pre Read
23. sults in an Ala gt Val substitution in the MTHFR protein Such substitution reduces the MTHFR enzymatic activity to 50 that is furtherly decreased to only 30 during heat exposure thermolabile variant The thermolabile variant causes high plasma homocysteine level especially after the oral methionine load The frequency of C677T mutation in the european population is 3 3 7 about 42 46 of general population are heterozygotes while the mutated homozygotes represent 12 13 of the general population in Europe Another MTHFR mutation A1298C associated with reduced enzymatic activity was identified recently it decreases the enzymatic activity to 60 when taken separately and to only 40 when associated with C677T mutation This mutation causes an increase in plasma homocysteine level in C677T mutant patients ANALITICA 9 MANUAL_RS MTHFR677_eR070109 ADVANCED BIOMEDICINE www abanalitica it Elevated homocysteine levels are considered to be a risk factor for vascular diseases arterial thrombosis Prothrombin or Factor Il is the inactive precursor of thrombin The gene comprises a 5 UTR 14 exons with 13 introns and a 3 UTR Recently a common genetic variation was found in the 3 UTR that is associated with elevated prothrombin levels and an increased risk of venous thrombosis The importance of this G A transition at nucleotide 20210 is not yet fully understood but several investigators have reported that heterozygous carriers have
24. te the patient genotype in a single reaction The kit also provides positive controls relative to each of the three possible genotypes WT HET MUT these controls contain DNA fragments that correspond to the genic region of interest and as such these controls are not dangerous for the user The correct amplification of the positive controls is a guarantee of the good amplification functioning MANUAL RS MTHFR677 eR070109 12 ANALITICA ADVANCED BIOMEDICINE www abanalitica it 10 COLLECTION MANIPULATION AND PRE TREATMENT OF THE SAMPLES 10 1 Peripheral blood Sample collection should follow all the usual sterility precautions Blood must be treated with EDTA Other anticoagulation agents as heparin are strong inhibitors of TAQ polymerase and so they could alter the efficiency of the amplification reaction Fresh blood can be stored at 2 8 C if processed in a short time If DNA extraction is not performed immediately the sample must be frozen 11 PROTOCOL 11 1 DNA Extraction For the DNA extraction from peripheral blood AB ANALITICA recommends the use of the QIAamp DNA Blood Mini Kit QIAGEN Hilden Germany For use follow the user manual of the manufacturer During validation of this kit DNA samples obtained with the following automatic semiautomatic extraction methods were used QIAGEN EZ 1 QIAGEN Roche MagNA Pure Roche Maxwell 16 System Promega and QuickGene Fuji Film For any further information or ex
25. tion e Container S5 weltem vj Template Blank Document e Browse Operator fieatimeuse Comments SDS v1 2 Default Plate Name Pre Read_MTHFR67 7_aammag m ma 5 Select the marker the marker is the set of the two detectors that MANUAL RS MTHFR677 eR070109 14 ANALITICA ADVANCED BIOMEDICINE discriminate different allelic variants of the same locus Y n case in which the marker is already present in the list select the marker of interest and click Aad New Document Wizard x Select Markers Select the markers you will be using in the document Find a Passive Reference Rox Marker Hame Detector 1 MTHFR C677T Fatt IAT Add gt gt i FY Leiden MUT MTHFR C677T MTHFR C677T VVT Remove New Detector New Marker lt Indietro Avanti gt Fine Annulla Y n case in which the marker is not on the list e Select New Detector and insert the name and the following characteristics for each detector MTHFR C677T WT FAM none Your choice MTHFR C677T MUT JOE none Your choice ICITTCEENNS S x Cyr W Name MTHFR C5677T wT Name MTHFR C677T MUT Description Description Reporter Dye FAM Y Reporter Dye JOE Quencher Dye none X Quencher Dye none Color m Color l Notes Notes Create Another Cancel Create Another Cancel panauitica 15 MANUAL_RS MTHFR677_eR070109 www abanaliti
26. venous thrombosis can be predicted Apart from the local activation of the coagulation system by e g trauma surgery immobilization pregnancy and use of oral contraceptives also the genetic background of an individual plays an important role An increased risk of venous thrombosis can last throughout life because of the presence of mutations in genes encoding proteins involved in the haemostatic or fibrinolytic processes At present several mutations that play an important role in the development of venous thrombosis have been identified in the following genes Factor II Factor V and MHTFR methylentetrahydrofolate reductase N N methylenetetrahydrofolate reductase MTHFR catalyses the reduction of N N methylenetetrahydrofolate to N methyltetrahydrofolate next N methyltetrahydrofolate acts as a methyl group donor for the remethylation of homocysteine to methionine by using vitamin B12 as a cofactor Rare mutations with autosomal recessive inheritance may cause severe MTHFR deficiency characterized by a decrease of enzyme activity to less than 20 the following homocysteinemia and homocystinuria occur at low plasma levels of folic acid The consequent clinical symptoms are severe and include grave effects on psychological and motor development and important thrombosis phenomena A common genetic polymorphism associated to severe MTHFR deficiency was identified a Cytosine to Thymine substitution at position 677 of the MTHFR re
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