MTHFR C677T Detection Kit v1 USER MANUAL
Contents
1. 3 Sibani S Christensen B O Ferrall E Saadi I Hiou Tim F Rosenblatt DS Rozen R 2000 Characterization of six novel mutations in the methylenetetrahydrofolate reductase MTHFR gene in patients with homocystinuria Hum Mutat 15 3 280 7 13 SYMBOLS od Use by Lot Batch Catalog number Temperature limitation Caution consult accompanying documents Manufacturer JESS Gf vD In Vitro Diagnostic Medical Device Code MB22v 1f Date April 2011 14 CONTACT INFORMATION Anatolia Tani ve Biyoteknoloji A Egitim Mh Kasap Ismail Sk No 10 23 Kadikoy 34722 ISTANBUL TURKEY Phone 90 216 330 04 55 Fax 490 216 330 00 42 E mail info anatoliageneworks com www anatoliageneworks com Registered Trademarks Anatolia Geneworks Montania Magnesia and Bosphore are registered trademarks of Anatolia Tani ve Biyoteknoloji A S Code MB22v1f Date April 2011 102. 66 082085 068405 F3 7 054724 P 041043 7 Fa y d yl 027362 013881 E 0 00000 013681 eb Fig 4 Amplification Curve of a Bosphore MTHFR Detection v1 test Analysis of the results should be performed by trained personnel who have received the required training for analysing Real Time PCR data We recommend that the test results must be evaluated by an expert clinician taking the patient s clinical findings and the results of other tests into consideration All analysis is done automatically in routine use However when the trained personnel who have received the required training from manufacturer consider it as necessary the system allows pulling down the threshold as much as possible in order to detect slight amplifications In this case attention should be paid to keep the threshold line above the background Amplification should be observed from both FAM and Cy5 filters of the heterozygous positive control during the test The amplification of both filters from the tested samples should be compared with that of the positive control Test results should not be reported unless the assay results meet the criteria stated above Please contact the manufacturer if impairment in the product s performance is observed See the last page for contact information The q
3. MTHFR C677T Detection Kit v1 USER MANUAL For in vitro Diagnostic Use Miia aires EE W o Document Code MB22v1f Approval Date April 2011 10 11 12 13 14 Product Description Content Storage Required Materials and Devices Important Notes and Safety Instructions Product Use Limitations Polymorphism Method Procedure 9 1 DNA Isolation 9 2 Kit Components 9 2 1 PCR Mix 9 2 2 Detection Mix 1 9 2 3 Positive Control 9 3 Preparing the PCR 9 4 Programming the Montania 483 Real Time PCR Instrument Analysis Troubleshooting References Symbols Contact Information Code MB22v1f Date April 2011 10 1 PRODUCT DESCRIPTION Bosphore MTHFR C677T Detection Kit v1 detects MTHFR mutation namely C677T a change of cytosine nucleotide with the thymine at position 677 in human biological samples Wild type MTHFR allele is amplified and fluorescence detection is accomplished using the Cy 5 filter Allele with MTHFR polymorphism is amplified and fluorescence detection is accomplished using the FAM filter 2 CONTENT Bosphore MTHFR C677T Detection Kit v1 is composed of Real Time PCR reagents Component REAGENT 100 50 Tests 25 Tests Tests 1 dH O 1000 ul 1000 ul 1000 pl 2 PCR Mix 1375 ul 688 ul 344 ul 3 Detection Mix1 100 ul 50 ul 25 ul 4 Positive Control 30ul 15 ul 15 ul 3 STORAGE Bosphore MTHFR C677T Detection Kit v1 PCR reagents should
4. ased on the Real Time PCR method Polymerase chain reaction is a technique that is used for amplification of a DNA region The reaction occurs by the repeating cycles of heating and cooling The main components of PCR are primers dNTPs Taq polymerase enzyme buffer solution and template Primers are small synthetic DNA those anneal to the specific regions of the template in order to start the synthesis dNTPs are the building blocks of the amplified products Taq polymerase amplifies the DNA template Buffer solution provides the pH adjustment required for the reaction and template as referred is the target region for synthesis In Real Time PCR technique in contrast to conventional PCR PCR product can be monitored during the reaction Therefore Real Time PCR obviates the need for further analysis methods like gel electrophoresis whereby minimizing the risk of contamination Dual labeled probes employed in the reaction in addition to the conventional PCR reagents enable detection of the amplified target with increased sensitivity The test utilizes the 5 exonuclease activity of Taq Polymerase to cleave a dual labeled fluorescent hydrolysis probe during the extension phase The probe is labeled at the 5 end with a fluorescent reporter and at the 3 end with another fluorescent molecule that acts as a quencher for the reporter When the two fluorophores are in close proximity even if the reporter is excited by light no reporter fluorescence can b
5. be stored at 20 C Repeated thawing and freezing gt 3x should be avoided since it may reduce sensitivity If the components are to be used in small amounts they should be frozen in aliquots While preparing the PCR the components should not be exposed to room temperature for more than 10 min and the detection mix components should not be exposed to light more than 1 2 min We recommend preparing the PCR on a cooling block and keeping the detection mixes within a closed container The components maintain their stability until the expiry dates on the labels if they are stored at advised conditions 4 REQUIRED MATERIALS AND DEVICES e Montania 483 Real Time PCR Instrument Anatolia Geneworks or another Real Time PCR system with FAM and Cy5 filters iCycler iQ5 CFX BioRad LightCycler 1 5 2 0 480 Roche 7500 Real Time PCR System ABI Stratagene Mx3005P Mx3000P Agilent LineGeneK LineGene 9600 Bioer Rotorgene 2000 3000 6000 Q Qiagen e Q 2ml Thin Wall PCR tubes or strips e Magnesia 16 Nucleic Acid Extraction System Magnesia Genomic DNA Whole Blood Kit Anatolia Geneworks or other high quality genomic DNA extraction kits systems e Deep freezer 20 C Code MB22v1f Date April 2011 e Desktop centrifuge with rotor for 2 ml microcentrifuge tubes e Calibrated adjustable micropipettes e DNAse RNAse pyrogen free micropipette tips with filters e DNAse RNAse pyrogen free 1 5 or 2 ml microcentrifuge tubes e Disp
6. e detected During the elongation step Taq Polymerase encounters and cleaves the probe bound to the template As the reporter is relieved from the suppressing effect of the quencher fluorescence signal can be detected Code MB22v1f Date April 2011 As the PCR product accumulates the fluorescence generated by the reporter increases The point at which the signal rises above background level and becomes detectable is called the threshold cycle C Bosphore MTHFR C677T Detection Kit v1 employs multiplex PCR MTHFR DNA whether wild type or polymorphic is amplified in a single reaction using sequence specific primers against mutant and wild type alleles The fluorescent signal generated by the MTHFR polymorphism is detected by a probe labeled at the 3 end with FAM through the FAM channel In contrast the fluorescent signal generated by the wild type allele amplification is detected by a second probe labeled at the 5 end with a different reporter molecule Cy5 through the Cy5 channel 9 PROCEDURE 9 1 DNA Isolation We recommend that the Magnesia 16 Nucleic Acid Extraction System Magnesia Genomic DNA Whole Blood Kit Anatolia Geneworks isolation system is used with Bosphore MTHFR C677T Detection Kit v1 The DNA isolation should be performed according to the manufacturers instructions The starting volume is 400 ul the elution volume is 60 ul 9 2 Kit Components 9 2 1 PCR Mix HotStarTaq DNA Polymerase HotStarTaq DNA Po
7. eparing the PCR It is for only one reaction multiply these values with the sample number to find the values required for the master mix While preparing master mixes for more than 3 samples an extra 1096 should be added to the total sample number PCR Mix 12 5 ul Detection Mix 1 0 9 ul dH20 8 6 ul Sample DNA Positive Negative Control 3 pl Toplam Hacim 25 0 ul Pipette 22 ul of the master mix into the PCR tubes or strips and add 3 ul of DNA sample positive or negative control Close the tube cap Make sure that the solution in each tube is at the bottom of the tube Centrifuge if necessary 9 4 Programming the Montania 483 Real Time PCR Instrument The thermal protocol for Bosphore MTHFR C677T Detection Kit v1 is composed of an initial denaturation for activation the HotStarTaq DNA Polymerase a two step amplification cycle and a terminal hold The real time data is collected at the second step of the amplification cycle Initial denaturation 95 C 14 30 min Denaturation 95 C 00 30 min 35 cycles Annealing and Synthesis 65 C 00 30 min Data Collection Hold 22 C 05 00 min Montania 483 Real Time PCR Instrument is installed and calibrated as it is delivered to the end user In order to establish an appropriate link between the system components first the thermal cycler and the optical module and then the PC and the software should be started Before starting a Real Time PCR reaction using the Bosphore Kits the foll
8. g ime 0000 E Son Vete Range 00000 11052011 10 54 Ga Kitaphidar Control Mode Belgeler Wih Hot Lid ek Bock Cool li File operar Pogan B Video Open Delete New Edt Lr d Anatotins C e c ca Anatola S Bachs c Dosya de MTHFR 65 prg progamtis Ev Grbu Fig 3a Selecting the Thermal Protocol File F Edit Setings S View Analysis A ToolsT Help H EEE EEE Gene Amplification Test Result Amplification Program I Temperature Curve I Real Time Curve Seer C Program Files SLAN RealTime PCR Detection System PROGRAM MTHFF 65 prg sre 95 00 1430 p3n soc ith Hot Li is With Hot Lid Fluorescence Test Block Control G RaDa NemalzedData Stat zyc 06 00 Z Segment1x1 Segment 235 Segment 3 Fig 3b Starting the Experiment 10 ANALYSIS By the end of the thermal protocol the Montania 483 Real Time PCR Instrument software automatically calculates the baseline cycles and the threshold Example of an amplification curve is given in Fig 4 Code MB22v1f Date April 2011 FileF Editt SetingsiS ViewiV Analysis A ToclsT HelpiH Dag Bs 123 ef Module Edit I Gene Amplification if Test Result Module Mode J Amplification Curve Standard Curve Report Mode I Melting Curve 136008 123128 1 09447 0957
9. lymerase is a modified form of a recombinant 94 kDa DNA polymerase originally isolated from Thermus aquaticus cloned into E Coli The enzyme is provided in an inactive form It is activated by a 15 minute 95 C incubation step This prevents the formation of misprimed products and primer dimers during reaction setup and the first denaturation step leading to high PCR specificity and accurate quantification PCR Buffer contains Tris Cl KCI NH4 SO4 8 mM MgCl pH 8 7 20 C dNTP Mix Contains ultrapure quality dATP dGTP dCTP ve dTTP dUTP 9 2 2 Detection Mix Detection Mix 1 contains MTHFR gene specific forward and reverse primers and two dual labeled probes against wild type and mutant MTHFR allele 9 2 3 Positive Control The positive control contains heterozygous MTHFR gene containing one copy of the wild type and mutant MTHFR allele each It should be included in the PCR to efficiently test whether the samples being analyzed are positive or negative The threshold cycle for the positive control is given in the acceptance criteria table Section 10 Analysis Threshold cycles higher than the acceptance criteria may indicate an efficiency loss in the reaction Code MB22v1f Date April 2011 9 3 Preparing the PCR Positive control should be added into the PCR reaction together with the samples and the negative control PCR grade water Make sure that all the kit components are thawed before use Refer to the table below for pr
10. nds for Methylene Tetra Hydro Folate Reductase which is a key enzyme involved in amino acid metabolism of the body It catalyzes the conversion of 5 10 methylenetetrahydrofolate to 5 methyltetrahydrofolate a cosubstrate for multistep process in the conversion of the amino 2 Code MB22v1f Date April 2011 acid homocysteine to amino acid methionine which is then used to make proteins and other important compounds 1 When there are genetic mutations mostly single amino acid substitutions occurred in the MTHFR gene inhibiting the production of this enzyme it may result in excess homocysteine level hyperhomocytenemia in blood plasma which is thought to be the susceptibility of health problems occlusive vascular disease neural tube defects colon cancer acute leukemia methylenetetrahydrofolate deficiency associated with homocystinuria 2 About 24 mutations in the MTHR gene have been identified in people with homocystinuria Within 24 mutations there are two variants C677T and A1298C single nucleotide polymorphisms SNP are relatively common in many populations worldwide The C677 variant nucleotide replaces cytosine with the thymine at position 677 in the MTHR gene which alters methylenetetrahydrofolate reductase and reduces its activity at higher temperatures thermolabile resulting in elevated levels of homocysteine in blood and homocystinuria associated health problems 3 8 METHOD Bosphore MTHFR C677T Detection Kit v1 is b
11. osable laboratory gloves 5 IMPORTANT NOTES AND SAFETY INSTRUCTIONS Important e The product should be delivered on dry ice Check for presence of dry ice upon arrival e Check for the expiry dates on the box and tube labels upon arrival Do not use expired products or components e Calibrated or verified micropipettes DNAse RNAse pyrogen free micropipette tips with filters and DNAse RNAse pyrogen free microcentrifuge tubes should be used e Before starting a test procedure all components should be thoroughly thawed After thawing all components should be centrifuged briefly spin down for 3 5 seconds and mixed well to ensure homogeneity prior to use e The kit components should be kept on ice or a cooling block until the reaction is prepared and they should be quickly returned to 209C e PCR and nucleic acid isolation must be performed in different compartments Samples should be stored separately to avoid contact with the kit components e All the biological wastes produced during the nucleic acid isolation step including the blood samples and material contacted with them should be discarded into medical waste and disposed safely 6 PRODUCT USE LIMITATIONS e Allthe components may exclusively be used for in vitro diagnostics e This product should be used in accordance with this user manual e This product is to be used by personnel specially trained to perform in vitro diagnostic procedures 7 POLYMORPHISM MTHFR sta
12. owing steps should be completed e Choose the filter pairs to be used FAM and Cy5 e Identify unknown samples positive and negative controls e Select the correct thermal protocol Code MB22v1f Date April 2011 These steps are described below From the main menu of the Montania 483 Real Time PCR Instrument File and then New is selected Create a new Experiment is selected In the Select Channel window channels 1 FAM and 3 Cy5 are selected Fig 1 Samples positive and negative controls are identified in the Module Edit menu Fig 2 To select the thermal protocol Gene Amplification menu is used The Open button in the Experiment Program is clicked and the appropriate thermal protocol is selected Fig 3a The thermal cycles of the selected protocol is displayed The experiment starts by clicking the Start button Fig 3b Dium COX Gs ie2sie t F Charred FAM SYBR T7 Owrrei2 HEX VIC JOE TET F Owrel2 Ci Fig 1 Filter Selection in Montania 483 F e SM SetngiS View Analysis Tool Help H Oso rox gs 123 ef Fig 2 Sample Location and Identification Code MB22v 1f Date April 2011 fe Ede SetngiS View Analyst Toots T Heip sao nO 5 123 8 Module Edit Testen Program T mu Test Result FF Block Temperaieel C 0 FF Sample Temperate C FF Hot Lid Temperate 0 Purring Stone forie Coine o Dazenle T Tongass 00 B Marono Holdin
13. ualitative results of the test are displayed on the Report Mode screen The samples that cross the threshold in Channel 1 FAM and Channel 3 Cy5 are displayed as positive for polymorphic and wild type alleles respectively The following table shows the possible results and their interpretation Signal in both FAM and Cy5 Heterozygous polymorphic The sample has both wild type and polymorphic MTHFR alleles No signal in FAM signal in Cy5 Homozygous wild type The sample has only wild type MTHFR allele Code MB22v 1f Date April 2011 Signal detected only in the FAM filter Homozygous polymorphic The MTHFR DNA in the sample is composed of polymorphic alleles No signal in FAM and Cy5 The test should be repeated 11 TROUBLESHOOTING Please contact the manufacturer in case of a problem during a run Signal from FAM Cy5 Filter in the Negative Control Contamination Use filter tips Repeat PCR with new kit components The Threshold is Above Low Signals The threshold should be manually Using the mouse pull the threshold down until it cuts the low adjusted signals Avoid the background and the signal from negative control 12 REFERENCES 1 Goyette Sumner Milos et al Human methylenetetrahydrofolate reductase isolation of cDNA mapping and mutation identification Nature Genetics 1994 7 2 p 195 200 2 Genetic home reference NIH Resources MTHFR January 2008
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