TCRG Gene Clonality Assay
Contents
1. Gel Detection Heteroduplex Analysis RECOMMEDED 1 2 3 4 DU 09N Denature 20ul of PCR products at 94 C for 5 minutes Re anneal PCR products at 4 C for 60 minutes Assemble electrophoresis unit using a 6 non denaturing polyacrylamide TBE gel made with 1X TBE Invitrogen Cat EC62652Box and 0 5X TBE running buffer Invitrogen 5X TBE Cat LC6675 Add 5 1 of ice cold non denaturing bromophenol blue loading buffer to samples Load 20ul of mixture into wells of the gel Run gel at 110V for 2 3 hours or 40 50V overnight Voltage and electrophoresis time depend on the PCR amplicon size acrylamide gel thickness and type of PCR equipment Voltage and run time can be adapted accordingly Gels are stained in 0 5ug ml EtBr in water or 0 5X TBE Buffer for 5 10 minutes Gels are destained 2X in water for 5 10 minutes UV illumination is used for visualization 0 Gel is photographed and data are interpreted TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 9 of 14 ABI Fluorescence Detection with ABI 310 amp 3100 instruments 1 Add 1ul of reaction products from the TCRG Tube A in a separate tube and add 10ul of HI Deionized Formamide containing ROX size standards to the tube Mix well 2 Add 1ul of reaction products from the TCRG Tube B in a separate tube and add 10ul of HI Deionized Formamide containing ROX size standards to the tube Mix well 3 Add2. 0021 Clonal Control DNA 4 088 1210 10011 E 2001g ml IVS 0000 Polyclonal Control DNA 4 092 0010 100ul E 2001g ml Master Mixes IVS Catalog Target TCRG Tube A 2 207 003X Vgl 8 Vg10 Jg Regions TCRG Tube B 2 207 004X Vg9 Vg11 Jg Regions Specimen Control Size Ladder 2 096 002X Multiple Genes Note X Detection format code Note MegaKits contain 10 units of each master mix and 5 units of each Controls and Standards STATEMENT OF WARNINGS The assay kit has been optimized to be used as a system Do not substitute other manufacturer s reagents Dilution reducing amplification reaction volumes or other deviation in this protocol may affect the performance of this test and or nullify any limited sublicense that comes with the purchase of this testing kit Close adherence to the protocol will assure optimal performance and reproducibility It is recommended that glass distilled de ionized molecular biology grade water be used with the preparation of specimen DNA This can be purchased from several manufacturers In addition laboratory personnel are reminded to wear appropriate personal protective equipment and follow good laboratory practices and universal precautions when working with specimens Specimens should be handled in approved biological safety containment facilities and opened only in certified biological safety cabinets Please see Section 9 for further details Storage Conditions PCR master mixes are sensitive to freeze thaw cycles Th
3. 16 REFERENCES tcvosiossi iia cuencos canin set anA R soatos casecd ossasececpsuseedsaussees oneideatbvess coveuces cbweedbssevescsuavasessesuchdessecesshdeaesete 11 17 APPENDIX icccssicsissssecsessssctsocssecsusonssessecedscsuscascsuncessessesevbendeossvessecssvcsedestecsbsesvenseens edsevenscssucessosseseassdbuedeosseecdesesbeesesasbecsess 11 REAGENTS AND SPECIAL SUPPLIES ccccccccccccccccsccccesecccesecececesesecesecesesesesesesssscessseessesssesssscssecacassseessssecssseeseuacsseusssssssesesesusuenseaes 11 Ficoll Separation A RO E OO 11 Gel Electrophoresis uo tii e aa be dde 11 Differential Fluorescence Detection ecccescceseceessecsenceessecsenceessecsencecscecseneecssecseaeeessecseneeesaeceeaeecsaecseaeecsaeceeaeecsaeeeeaeecsaeeesaes 12 18 TROUBLE SHOOTING GUIDE wuuu csscccsssscesssscccssssccccnsccccssssccccssceccesnsccecessccccssnacceenscscesscaccscssccaccessacesessssccsescsecess 12 19 SAMPLE DATA siicsisccccssisnssiessdccseisssoessctsassonssssssteucsdsnscsessesssscsducdessssacececsdsvessecbasssebssc essecsssssdcddeeestecsenvencsdevseiecsesscecsduvessess 12 Eie ed DIAEA WC EEE a ene 12 20 SINGLE PAGE FLOW CHART iiiisessssssscsoccsvecsesessecsssvedessesnssesievensessnsesecsescessvssssodeteteosesteossdestscssdsstessosnsdosinveatosseeesestons 14 GEL DETECTION HETERODUPLEX ANALYSIS ccccccccccccccecccesececescsecesesesesesesececesesesssesecesesssesecscesecasessceseseusseseseseseusessssseaueeseaes 14 ABI FLUORESCENCE DE
4. of 100 200 300 400 and 600 base pairs to ensure that the quality and quantity of input DNA is adequate to yield a valid result A single thermocycler program and similar detection methodologies are used with all of the BIOMED tests Many of our customers have remarked that this improves consistency and facilitates cross training on a broad range of different assays These robust nVivoScribe assays can be used to test DNA extracted from virtually any source Assay Uses T Cell Receptor Gamma Chain Gene Rearrangement Assays are useful for Identifying clonal T cell populations highly suggestive of T cell malignancies Lineage determination of leukemias and lymphomas Monitoring and evaluation of disease recurrence Detection and assessment of residual disease Evaluation of new research and methods in malignancy studies TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures 4 Page 5 of 14 Specimen Requirements This assay tests genomic DNA 1 5cc of peripheral blood bone marrow biopsy or bone marrow aspirate anti coagulated with heparin or EDTA Ship at ambient temperature OR 2 Minimum 5mm cube of tissue shipped frozen or at room temperature or on ice in RPMI 1640 OR 2ug of genomic DNA OR 4 Formalin fixed paraffin embedded tissue or slides Ya Kit Contents Controls and Standards IVS Catalog Concentration IVS 0009 Clonal Control DNA 4 088 0490 10011 E 200ug ml IVS
5. 1ul of reaction product from the template amplification control in a tube and add 10ul of HI Deionized Formamide containing ROX size standards Mix well 4 Reaction products are heated to 95 C for 2 minutes then snap chilled on ice for 5 minutes 5 A sample sheet and injection list is made up for the samples As the samples are run on the machine they are fractionated detected and analyzed by the instrument Runs are 20 24 minutes in duration The 310 amp 3100 capillary electrophoresis instruments routinely handle 2 runs per hour 48 and 768 samples per day respectively and automatically analyze and store data for review or comparison with other test results 6 Data are automatically displayed as size and color specific peaks Review profile and controls report results ABI Fluorescence Detection with ABI 373 amp 377 instruments 1 PCR Product Dilution Initially dilute samples 1 10 in HI Deionized Formamide or water can be altered if the fluorescence signal is outside the optimal range 2 Add 2ul of diluted reaction product from the TCRG Tube A in a tube 2ul of HI Deionized Formamide containing 0 5ul of ROX size standards 0 5ul of blue Dextran loading dye Mix well 3 Add 2ul of diluted reaction product from the TCRG Tube B in a tube 2ul of HI Deionized Formamide containing 0 5ul of ROX size standards 0 5ul of blue Dextran loading dye Mix well 4 Add 2ul of diluted reaction product from the template amplification cont
6. 6 200 Control 300 400 600 Size Ladder Note The amplicon sizes listed above were determined using an ABI 3100 platform Amplicon sizes seen on your specific CE instrument may differ 1 4bp from those listed above depending on the platform of detection ABI and the version of the analysis software used Once identified the amplicon size as determined on your specific platform will be consistent from run to run This reproducibility is extremely useful when tracking MRD Note This may be seen as a weak amplicon Note Amplicon product is often not seen in this size range Note Amplicon product is often not seen This is an extremely restricted repertoire Note Amplicon product is often not seen in this size range Results can be reported as Positive or Negative for Detection of T cell receptor gamma chain gene rearrangement consistent with the presence of a clonal cell population 1 Samples that fail to amplify following repeat testing should be reported as A result cannot be reported on this specimen because there was DNA of insufficient quantity or quality for analysis 2 It is acceptable to call a sample Positive when a product is generated in the valid size range yet the positive control for that master mix fails 3 Samples that test negative should be repeated if the positive control reaction failed 4 All assay controls must be examined prior to interpretation of sample results If the co
7. In VivoScribe Technolo gies 858 623 8105 Phone 6330 Nancy Ridge Drive Suite 106 858 623 8109 Fax San Diego CA 92121 support invivoscribe com USA www invivoscribe com TCRG Gene Clonality Assay For Identification of Clonal T Cell Receptor Gamma Chain Gene Rearrangements TCRG tube A Vylf and Vy10 primers Jy1 1 2 1 and Jy1 3 2 3 TCRG tube B Vy9 and Vy11 primers Jy1 1 2 1 and Jy1 3 2 3 Vy Vylf Vy9 Vy10 Vy11 Vy family primers FOR RESEARCH USE ONLY Not for use in diagnostic procedures Storage Conditions 65 C to 85 C DNA controls may be separated from assay kits and stored at 2 C to 8 C Catalog Products 1 207 0020 1 207 0021 1 207 0024 1 207 0040 1 207 0041 1 207 0044 TCRG Gene Clonality Assay for Gel Detection TCRG Gene Clonality Assay for ABI Fluorescence Detection TCRG Gene Clonality Assay for Cy5 0 Detection TCRG Gene Clonality Assay MegaKit for Gel Detection TCRG Gene Clonality Assay MegaKit for ABI Fluorescence Detection TCRG Gene Clonality Assay MegaKit for Cy5 0 Detection Jy1 3 2 3 Jy1 1 2 1 Jy primers uantit 33 Reactions 33 Reactions 33 Reactions 330 Reactions 330 Reactions 330 Reactions Page 2 of 14 Table of Contents 1 A SAR cstseddanssscsesessdectscssbess essacsteesessssdectacssinceeceseessasssencsseodsbedactensssesesess essassdbusesossdecbsssinces sudeessass 3 2 AN AAA A RR TO 3 3 ASSAY USES ON 4 4 SPECIMEN REQUIREMENTS sisissccasssccis
8. PLIPICA AR O 7 13 AR ssuiueteoseb bes ssusuess bucussetessessevenehesvusussesseccdbetetbichdueseste 8 AVAILABLE TEMPLATE AMPLIFICATION CONTROLS cccccccccccecccececesecesesecesecesececeseeesesesesesesesssesesesesesesesesesesssesesssesseseseasseseseaeaes 8 GEL DETECTION AGAROSE TBE GEES 0 cvs cic cee R EE EAEE E A E E souda cuscawcasegaeu eects EE E EEEE EEEE RRS 8 GEL DETECTION POLYACRYLAMIDE TBE GELS Gorai RE A EE RAE EEEE AE EEEE EEEE RERE EREEREER A E EEEa 8 GEL DETECTION HETERODUPLEX ANALYSIS RECOMMEDED 000 cccccccessseeessececeesceeeceeececeeaeeecsesaececneeeeeesaeeecnesaeesensaeenens 8 ABI FLUORESCENCE DETECTION WITH ABI 310 amp 3100 INSTRUMENTS occccccccccococonononononononononononononononononononnononnnnnnonononanenncnnnnonos 9 ABI FLUORESCENCE DETECTION WITH ABI 373 amp 377 INSTRUMENTS cccccccccccccccccesecesesesececeseseseseseseseceseseseseessssssesseseessesuanaes 9 CX3 0 FLUORESCENCE DETECTION a ais 9 14 INTERPRETATION AND REPORTING cssssscssssscsssscccsssccccesssccccssssccccssssccscnsscccssacccessnessesssacessecccsesssecesessseecess 9 EXPECTED SIZE OF AMPLIFIED PRODUCTS cccccccccccccscsescsescsesccesesesesesesesesesesesesesesesssesssesssesecesssesssssesssesesesseasesssessssesesesssseesnaes 10 SAMPLE INTERPRETATI N ii ii iia 11 15 LIMITATIONS OF PROCEDURE vssccssissssecsssssssccssvsscssoosssossvectsosstecsisssecsssossansseessbcsseenssesssnctsesessdsassdencssevesestdesssasseuseeess 11
9. TECTION WITH ABI 310 amp 3100 INSTRUMENTS ccccccccccccccesececesececesececesecececesesesesesesesesesseeeseeeseeeeuaes 14 CYS 0 FEUORESCENGE DETECTION A a Rivis EE ees 14 TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 3 of 14 Thank you for purchasing our TCRG Gene Clonality Assay We appreciate your business We are happy to assist you in the validation of this assay and will provide ongoing technical assistance to keep the assays performing efficiently in your laboratory Technical assistance is most rapidly obtained using our Internet site http www invivoscribe com or by sending an email inquiry to support invivoscribe com Questions received during business hours usually receive a response within an hour Alternatively you can call for technical assistance and for information on our testing kits at 858 623 8105 between the hours of 8 00 AM and 5 00 PM Pacific Standard Time L Notice This product and the methods employed are covered by United States Letters Patent Numbered 5 296 351 and 5 418 134 Australian Patent Number 626 601 and Japanese Patent Number 2 781 438 all of which are licensed exclusively to InVivoScribe Technologies TVS Purchase of this product includes a limited sublicense for non commercial practice of this technology for use within or with respect to data or product that are transmitted to the United States Japan or Australia only when
10. X PBS diluted from 10X PBS Gibco BRL Cat 70011 044 RPMI 1640 Gibco BRL Cat 11875 093 DMSO Hybri Max Sigma Cat D2650 Fetal Bovine Serum Hyclone Cat SH30071 03 Gel Electrophoresis MetaPhor Agarose 125g Cambrex Cat 50180 NuSieve 3 1 Agarose 125g Cambrex Cat 50090 UltraPure 10 mg ml Ethidium Bromide Invitrogen Cat 15585 011 10X BlueJuice Gel Loading Buffer Invitrogen Cat 10816 015 Ready Load 100 bp Ladder Invitrogen Cat 10380 012 Novex TBE gels 6 12 well Invitrogen Cat EC62652Box Novex TBE Running Buffer 5X Invitrogen Cat LC6675 Novex Hi Density TBE Sample Buffer 5X Invitrogen Cat LC6678 TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 12 of 14 Differential Fluorescence Detection HI DI Formamide with ROX size standards ABI 310 IVS Cat 6 098 0051 HI DI Formamide with ROX size standards ABI 3100 IVS Cat 6 098 0061 HI Deionized Formamide HI Deionized Formamide GS ROX 50 400HD Size Standard 18 Trouble Shooting Guide IVS Cat 6 098 0041 Applied Biosystems Cat 4311320 Applied Biosystems Cat 402985 Our laboratories are located in San Diego California Technical assistance is most rapidly obtained using our Internet site http www invivoscribe com or by sending an email inquiry to support invivoscribe com Alternatively you can call 858 623 8105 for technical assistance and informatio
11. agnostic or therapeutic use This product is sold FOR RESEARCH USE ONLY not for use in diagnostic procedures Principle NOTICE InVivoScribe Technologies Gene Rearrangement and Translocation Assays represent a new approach to PCR based clonality testing These standardized assays were carefully optimized testing positive and negative control samples using multiplex master mixes Assay development was followed by extensive validation testing more than 400 clinical samples using Revised European American Lymphoma REAL Classification Testing was done at more than thirty prominent independent testing centers throughout Europe in a collaborative study known as the BIOMED 2 Concerted Action Results from this BIOMED 2 study appear in a leading peer reviewed journal Leukemia 2003 Dec 17 12 2257 2317 Nature Publishing Group BACKGROUND The human TCR gamma gene locus on chromosome 7 7q14 includes 14 V genes belonging to 4 subgroups 6 are functional 3 Open Reading Frames and 5 pseudogenes 5 J segments and 2 C genes spread over 200 kilobases The diversity of this locus has complicated PCR based testing and extended dependence on Southern blot analysis in many testing centers However this standardized multiplex PCR assay detects the vast majority of clonal TCR gamma gene rearrangements using only 2 multiplex master mixes The assay TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Pag
12. csvessgstienssasssctstessscsscasecsossoeecsadeatestosudoosessesscssbetse sbsauecseosecscvsesscasaseesoaedsesteavessosss 5 5 KIT CONTENTS 6 vives cnsisscsscesscsasccesiveecsssdvcbsccsesssscssecsessusasesessacestessosssncsdcosebacsevteccsacusssvcsecs secssddcedesssaestessescesetecbosesecsoendebecses 5 STATEMENT OF WARNINGS woseiesisesec cecil etecaces N ea sau tues oa aa e eaten deg ea oa Seve Sua eve ead obvea sande ea ouseputoeeeeees 5 6 STORAGE CONDITIONS vssssssiscssssscsssvcssessescossvssscosestesessestessvinseosevteesssestessevatooesenc esesdecsesesdessesnescodsvaasesbestasessentoossiestooess 5 7 REAGENTS REQUIRED BUT NOT INCLUDED 00 ssssscssssscccsscsecessscccssssccscssccccessscccssscccesssecssssscscessnecssccscsecess 6 PER AMPLIFICATION adi 6 ABTELVORESCENCE DETECTION tt iia 6 RECOMMENDED POSITIVE CONTROLS ssscccssssssssssssccssssccccsssscccsssccccsssccscssccccessscccesssaccecssccsessacssessssccscsssecceses 6 PROCEDURE NOTES ssc dosscisueisssesesicsvesse doaseesssesUebusssavesssdeovessssucuesesssexeseseee oocbdethogubsess cnucdev ese dedebensdesVesnesesbeteeseesecessduveseses 6 10 REAGENT PREPARATION owssssssscssssssccssecssccossrsesssbessascsvacieosssecsesesbensesestocsesesicsseenesossesessosds sees seduce osssdaceodesdecssseesesssbuevesess 6 11 SAMPEE PREPARA TION sssiiassciscccecateescastasessactacctcavessdssnntdosscanscscscatessdaacesscosscddevonssessauadedausbesseasesessudensoasessessaetesdesauescesss 7 12 AM
13. e 4 of 14 provides rapid TCR clonality assessment in 4 6 hours reducing the number of Southern blot tests performed in the laboratory The detection rate of clonal TCR gamma gene rearrangements using this assay is unprecedentedly high The performance characteristics of this assay have been independently determined by a European collaborative study involving 32 diagnostic PCR laboratories BIOMED 2 Concerted Action testing hundreds of clinical samples defined according to the WHO classification Two multiplex master mixes target conserved regions within the variable V and the joining J regions that flank the unique hypervariable antigen binding region 3 CDR3 Tube A contains primers that target the V gamma 1 8 V gamma 10 genes and all J gamma exon segments Tube B contains primers that target the V gamma 9 V gamma 11 genes and all J gamma exon segments Positive and negative DNA controls as well as an internal Specimen Control Size Ladder master mix are included PCR products can be analyzed by differential fluorescence detection using capillary electrophoresis or gene sequencing instruments by heteroduplex analysis or using standard gel electrophoresis with ethidium staining Clonality is indicated if any one of the master mixes generates clonal band s Gel electrophoresis is commonly used to resolve the different sized amplicon products and ethidium bromide or other DNA intercalating dyes to stain and detect these products A powerful al
14. e standards ABI 3100 Recommended Positive Controls Applied Biosystems Cat N808 0241 Applied Biosystems Cat N808 0161 IVS Cat 6 098 0051 IVS Cat 6 098 0061 Master Mix Target Color Control DNA Cat Product Size in Basepairs Vgl 8 Vg10 Valid Size Range 145 255 TCRG Tube A Jg 1 3 2 3 Green IVS 0021 Clonal Control DNA 4 088 1210 211 Vg9 and Vgll Valid Size Rang 80 220 TCRG Tube B Jg 1 3 2 3 Green IVS 0021 Clonal Control DNA 4 088 1210 167 Specimen Control Multiple Genes Blue Valid Size Range 84 96 200 300 400 600 Size Ladder IVS 0000 Polyclonal Control DNA 4 092 0010 84 96 200 300 400 600 Note The amplicon sizes listed above were determined using an ABI 3100 platform Amplicon sizes seen on your specific CE instrument may differ 1 4bp from those listed above depending on the platform of detection ABD and the version of the analysis software used Once identified the amplicon size as determined on your specific platform will be consistent from run to run This reproducibility is extremely useful when tracking MRD 9 Procedure Notes Autoclaving does not eliminate DNA contamination Work flow in the PCR laboratory should always be in a one way direction between separate work areas beginning in Master Mix Preparation moving to the Specimen Preparation then to the Amplification and finally to Detection 1 Do not bring amplified DNA into the areas d
15. erefore for any duration other than immediate use our master mixes and assay kits should be stored at 65 C to 85 C The reason for this is quite straightforward Due to the high salt concentrations in our master mixes the effective freezing and thawing temperature of the master mixes is approximately 10 C The temperature in a standard laboratory 20 C freezer can easily reach 10 C or warmer during the day when these freezers are opened on a regular basis At these temperatures PCR master mixes may go through multiple freeze thaw cycles resulting in precipitation of the primers Accordingly to minimize the exposure of your master mixes to freeze thaw cycles VS recommends that master mixes be stored at 65 C to 85 C Please note that our DNA standards are best stored at 2 C to 8 C However these standards can be stored at any lower temperature as long as they are vortexed after thawing and before use to ensure that they are re suspended completely TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 6 of 14 If you have any questions please contact our technical staff We are happy to help you determine your optimal storage needs 7 Reagents Required But Not Included PCR Amplification AmpliTaq Gold DNA Polymerase RECOMMENDED AmpliTaq DNA Polymerase ABI Fluorescence Detection HI DI Formamide with ROX size standards ABI 310 HI DI Formamide with ROX siz
16. esignated for master mix or specimen preparation Due to the analytical sensitivity of this test extreme care should be taken to avoid the contamination of reagents or amplification mixtures with samples controls or amplified materials All reagents should be closely monitored for signs of contamination e g negative controls giving positive signals Discard reagents suspected of contamination 3 All pipettes pipet tips and any equipment used in a particular area should be dedicated to and kept to that area of the laboratory 4 PCR trays bases and retainers must to be decontaminated in 10 bleach and rinsed with distilled water two separate times before returning them to the starting areas 5 Sterile disposable plastic ware should be used whenever possible to avoid RNase or cross contamination Reagent Preparation All unknown samples should be tested using the template amplification control Amplification Control or Specimen Control Size ladder master mix This is to ensure that no inhibitors of amplification are present and there is DNA of sufficient quality and quantity to generate a valid result All samples should be tested in singlicate Positive negative and no template controls should be tested for each of the master mixes TCRG Gene Clonality Assay FOR RESEARCH USE ONLY not for use in diagnostic procedures 1207002Xv7 40 Page 7 of 14 1 Using gloved hands remove the master mixes from the freezer Allow t
17. he tubes to thaw then gently vortex to mix 2 In containment hood or dead air box remove an appropriate aliquot to clean sterile microfuge tube one tube for each of the master mixes Aliquot volumes should be 45ul for each sample 13511 3 x 45u1 for the positive negative and no template controls We recommend adding an additional 20ul to correct for pipetting errors 3 Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase 0 25ul of either AmpliTaq Gold or AmpliTaq 5U ul per 50ul total PCR reaction volume to each of the master mixes and gently mix by inverting several times or gently vortexing The master mixes are now ready for distribution to reaction tubes or plate and addition of sample 11 Sample Preparation Using any method of DNA extraction extract the genomic DNA from unknown samples Resuspend DNA to final concentration of 100ug 400g per ml in TE 10 mM Tris HCl 1mM EDTA pH 8 0 or distilled water This is a robust assay system A wide range of DNA concentrations will generate a valid result Therefore quantifying and adjusting DNA concentrations is generally not necessary Testing sample DNAs with the Amplification Control or Specimen Control Size Ladder master mix will ensure that DNA of sufficient quality and quantity was present to yield a valid result 12 Amplification 1 Aliquot 45ul of the master mix enzyme solutions into individual PCR wells or tubes Add 5ul of sample or control DNA to
18. ide containing ROX size standards to the tube Mix well Add 1ul of reaction products from the TCRG Tube B in a separate tube and add 10 11 of HI Deionized Formamide containing ROX size standards to the tube Mix well Add 1ul of reaction product from the template amplification control in a tube and add 10u1 of HI Deionized Formamide containing ROX size standards Mix well Reaction products are heated to 95 C for 2 minutes then snap chilled on ice for 5 minutes A sample sheet and injection list is made up for the samples As the samples are run on the machine they are fractionated detected and analyzed by the instrument Runs are 20 24 minutes in duration The 310 amp 3100 capillary electrophoresis instruments routinely handle 2 runs per hour 48 and 768 samples per day respectively and automatically analyze and store data for review or comparison with other test results Data are automatically displayed as size and color specific peaks Review profile and controls report results Cy5 0 Fluorescence Detection The Cy5 0 fluorochrome is capable of being detected by many different detection platforms Please see your instruments user manual for instructions on the fractionation and detection of your samples TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures
19. ing several times or gently vortexing Aliquot 45ul of master mix to individual wells of a PCR plate Add 5ul of DNA from the unknown and control samples to individual tubes or wells containing the respective master mix reactions and pipette up and down several times to mix Amplify target DNA using the universal thermocycler program Gel Detection Heteroduplex Analysis 6 7 8 9 10 11 12 13 14 15 Denature 20ul of PCR products at 94 C for 5 minutes Re anneal PCR products at 4 C for 60 minutes Assemble electrophoresis unit using a 6 non denaturing polyacrylamide TBE gel made with 1X TBE Invitrogen Cat EC62652Box and 0 5X TBE running buffer Invitrogen 5X TBE Cat LC6675 Add 5ul of ice cold non denaturing bromophenol blue loading buffer to samples Load 20ul of mixture into wells of the gel Run gel at 110V for 2 3 hours or 40 50V overnight Voltage and electrophoresis time depend on the PCR amplicon size acrylamide gel thickness and type of PCR equipment Voltage and run time can be adapted accordingly Gels are stained in 0 5ug ml EtBr in water or 0 5X TBE Buffer for 5 10 minutes Gels are destained 2X in water for 5 10 minutes UV illumination is used for visualization Gel is photographed and data are interpreted ABI Fluorescence Detection with ABI 310 amp 3100 instruments 6 T 11 Add 1ul of reaction products from the TCRG Tube A in a separate tube and add 101 of HI Deionized Formam
20. n on our testing kits between the hours of 8 00 AM and 5 00 PM Pacific Standard Time Questions received during business hours usually receive a response within an hour 19 Sample Data Gel Detection The data shown below were generated using the master mixes indicated Amplified products were heteroduplexed and run on a 6 polyacrylamide gel For each TCRG master mix Lane 1 displays data generated testing an alternative 100 clonal control DNA Lane 2 displays data generated testing the recommended 100 clonal control DNA Lane 3 displays data generated testing a 10 dilution of the recommended clonal control DNA Lane 4 displays data generated testing IVS 0000 Polyclonal Control DNA TCRG Tube A Lane 1 100 IVS 0026 Lane 2 100 IVS 0021 Lane 3 10 IVS 0021 Lane 4 100 IVS 0000 Valid Size Range 145 255 bp Figure 1 TCRG Gene Clonality Assay wsz gt _ j TCRG Tube B Lane 1 100 IVS 0014 Lane 2 100 IVS 0021 Lane 3 10 IVS 0021 Lane 4 100 IVS 0000 Valid Size Range 80 220 bp L gt Figure 2 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures ABI Fluorescence Detection Page 13 of 14 The data shown below were generated using the master mixes indicated Amplified products were run on an ABI 3100 instrument For each TCRG master mix Panel 1 displays data generated testing an alternative 100 clonal cont
21. ntrols do not yield the correct results the assay is not valid and the samples should not be interpreted The following describes the analysis of each of the controls and the decisions necessary based upon the results 1 Negative Control Positive Polyclonal control water or no template blank If the negative control is Possible contamination of all PCR amplification reactions Do not continue with the interpretation of results Prepare fresh master mix and repeat amplification Negative 2 Positive Control Continue with the analysis extraction processes If the positive control is Positive Negative TCRG Gene Clonality Assay Continue with analysis Repeat assay unless specimen tests positive FOR RESEARCH USE ONLY not for use in diagnostic procedures This can also be an extraction control if positive control material is taken through 1207002Xv7 40 Page 11 of 14 3 Specimen Control Size Ladder This is run on unknown samples only If the amplification control is Positive 100 200 300 400 and 600 basepair products are seen Because smaller PCR fragments are preferentially amplified it is not unusual for the 600 basepair fragment to have a diminished signal or to be missing entirely Continue with analysis Negative Repeat assay unless specimen tests positive Sample Interpretation Following the acceptance of the controls the clinical samples are interpreted as follows One or
22. or indicates the color of products generated with the master mix when using differential fluorescence detection format e g ABI instruments TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 10 of 14 Expected Size of Amplified Products Master Target Color Control DNA Cat Product Size Mix in basepairs Valid Size Range 145 255 TCRG Vgl 8 Jg 1 1 2 1 Blue IVS 0000 Polyclonal Control DNA 4 092 0010 230 255 Tube A Vg1 8 Jg 1 3 2 3 Green IVS 0000 Polyclonal Control DNA 4 092 0010 195 230 Vgl0 Jg1 1 2 1 Blue IVS 0000 Polyclonal Control DNA 4 092 0010 175 195 Vgl0 Jg 1 3 2 3 Green IVS 0000 Polyclonal Control DNA 4 092 0010 145 175 Vg1 8 Jg 1 3 2 3 Green IVS 0009 Clonal Control DNA 4 088 0490 212 Vg1 8 Jg 1 3 2 3 Green IVS 0021 Clonal Control DNA 4 088 1210 211 Valid Size Range e 80 220 TCRG Vg9 Jg1 1 2 1 Blue IVS 0000 Polyclonal Control DNA 4 092 0010 195 220 Tube B Vg9 Jg 1 3 2 3 Green IVS 0000 Polyclonal Control DNA 4 092 0010 160 195 Vell Jg 1 1 2 1 Blue IVS 0000 Polyclonal Control DNA 4 092 0010 110 140 Vgll Jg 1 3 2 3 Green IVS 0000 Polyclonal Control DNA 4 092 0010 80 110 Vg11 Jg 1 3 2 3 Green IVS 0009 Clonal Control DNA 4 088 0490 115 Vg9 Jg 1 3 2 3 Green IVS 0021 Clonal Control DNA 4 088 1210 1434 167 Specimen Multiple Genes Blue Any Human DNA 84 9
23. rol DNA Panel 2 displays data generated testing the recommended 100 clonal control DNA Panel 3 displays data generated testing a 10 dilution of the recommended clonal control DNA Panel 4 displays data generated testing IVS 0000 Polyclonal Control DNA TCRG Tube A 6FAM amp HEX i 100 IVS 0026 i Panel 1 100 IVS 0021 Panel 2 10 IVS 0021 Panel 3 mm 211 bp wf Valid Size Range 145 255 bp Panel 4 Figure 3 TCRG Tube B 6FAM amp HEX l ul 100 IVS 0014 Panel 1 sol 100 IVS 0021 Panel 2 10 IVS 0021 Panel 3 w a Valid Size Range 80 220 bp Panel 4 Figure 4 TCRG Gene Clonality Assay FOR RESEARCH USE ONLY not for use in diagnostic procedures 1207002Xv7 40 Page 14 of 14 20 1 Ze Single Page Flow Chart Using gloved hands remove the master mixes from the freezer Allow the tubes to thaw then gently vortex to mix In a containment hood or dead air box remove an appropriate aliquot to clean sterile microfuge tube one tube for each of the master mixes Aliquot volumes should be 45 1 for each sample 135 1 for the positive negative and no template controls We recommend adding an additional 2011 to correct for pipetting errors Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase 0 2511 of either AmpliTaq Gold or AmpliTaq E 5U ul per SOul total PCR reaction volume to each of the master mixes and gently mix by invert
24. rol in a tube 2ul of HI Deionized Formamide containing 0 5ul of ROX size standards 0 5ul of blue Dextran loading dye Mix well 5 Reaction products are heated to 94 C for 2 minutes then snap chilled on ice for 5 minutes 6 Load the 5ul of each of these preparations in separate wells of a preheated gel and run using the standard sequencing protocol Cy5 0 Fluorescence Detection The Cy5 0 fluorochrome is capable of being detected by many different detection platforms Please see your instruments user manual for instructions on the fractionation and detection of your samples 14 Interpretation and Reporting Note This assay is for research use only Although positive results are highly suggestive of malignancy these assays are designed for Research Use Only and if used in a clinical setting should only be used in support of diagnosis Positive and negative results should be interpreted in the context of all clinical information and laboratory test results PCR based testing does not identify 100 of clonal cell populations therefore repeat testing by Southern blot may be advisable to rule out clonality The size range for each of the master mixes has been determined testing positive control samples For accurate and meaningful interpretation it is important to ignore peaks that occur outside of the proscribed valid size range for each of the master mixes Peaks that are outside of the range cannot be assumed to be valid Note Col
25. ternative method is use of differential fluorescence detection with primers conjugated with fluorescent dyes that correspond to different targeted regions Reaction products from several different master mixes can be pooled fractionated using capillary electrophoresis and detected simultaneously This detection system results in unsurpassed sensitivity single base resolution differential product detection and quantification In addition the laboratory can eliminate the use of agarose and polyacrylamide gels as well as the use of carcinogens such as ethidium bromide Further differential detection allows accurate reproducible and objective interpretation of primer specific products and automatic archiving of data The limit of detection of this assay has been determined to be approximately 1 clonal cell in 100 hundred normal cells and inter assay and intra assay reproducibility in size determination using capillary electrophoresis is approximately 1 2 basepairs This reproducibility and sensitivity allows monitoring and tracking of individual tumors during research or methods development The automatic archiving of specimen data allows comparison of data collected at different times This test kit includes 3 master mixes Tubes A and B target framework regions within the variable region and the joining region of the TCR gamma chain locus The last master mix the Specimen Control Size Ladder targets multiple genes and generates a series of amplicons
26. the individual tubes or wells containing the respective master mix reactions Pipette up and down several times to mix Amplify the reactions using the following PCR program We recommend the MJ Research PTC 100 PTC 200 or the PE 2600 9600 or 9700 thermocyclers using the following PCR parameters for the amplifications Note We recommend using the calculated option for temperature measurement with the PTC instruments Standard Program for AmpliTag Gold Step 1 95 C for 7 minutes Step 2 95 C for 45 seconds Step 3 60 C for 45 seconds Step 4 72 C for 90 seconds Step 5 Go to step 2 34 more times Step 6 72 C for 10 minutes Step 7 15 C forever Remove the amplification plate from the thermocycler TCRG Gene Clonality Assay 1207002Xv7 40 FOR RESEARCH USE ONLY not for use in diagnostic procedures Page 8 of 14 13 Detection Not all detection formats are available for all assays Available Template Amplification Controls The Amplification Control master mix primers are labeled with a fluorescent dye 6 FAM or Cy5 0 This label is detected as BLUE using the differential fluorescence software The amplicons produced with this master mix are at 235 basepairs The products of this master mix should be run separately The Specimen Control Size Ladder master mix primers are labeled with a fluorescent dye 6 FAM or Cy5 0 This label is detected as BLUE using the differential fluorescence software The amplicons produced with
27. the purchaser is registered with IVS as an exclusively non commercial user of IVS products No sublicense is granted simply by purchase of these products Non commercial practice of the technology means sample testing done for teaching and basic research Non Commercial practice excludes testing if any of the following apply a test results products or information derived from the tests are used for or in support of patient care or are transferred to a healthcare professional involved in patient care b test results are clinically utilized to determine cause of death c compensation in any form or manner is received for performing the tests To request a form for registration as an exclusively non commercial product user to discuss terms for a potential sublicense for broader practice of these methods or for any questions concerning the scope or content of the non commercial sublicense please contact our legal department by email at legal invivoscribe com or by telephone at 858 623 8105 These methods also require nucleic acid amplification methods such as Polymerase Chain Reaction PCR which is covered by patents owned by Hoffmann LaRoche Inc and F Hoffmann LaRoche Ltd No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of these products The assays described herein are not approved by any regulatory agency for clinical use These assays are not for di
28. this master mix are at 100 200 300 400 and 600 basepairs Please note that the 100bp band is comprised of a 84bp and 96bp bands Both of these bands co migrate on a gel The products of this master mix should be run separately Gel Detection Agarose TBE Gels 1 2 3 A 2 MetaPhor or NuSieve 3 1 agarose TBE gel is prepared 20ul from each of the amplification reactions are individually mixed with 4ul of 6X gel loading buffer 20ul of this mixture is loaded into separate wells of the gel flanked by DNA size standards Products are detected using ethidium bromide or an equivalent dye Gel is photographed and data are interpreted Gel Detection Polyacrylamide TBE Gels 1 Po AHD A Assemble electrophoresis unit using a 6 non denaturing polyacrylamide TBE gel made with 1X TBE Invitrogen Cat EC62652Box and 0 5X TBE running buffer Invitrogen 5X TBE Cat LC6675 Add 5 of ice cold non denaturing bromophenol blue loading buffer to samples Load 20ul of mixture into wells of the gel Run gel at 110V for 2 3 hours or 40 50V overnight Voltage and electrophoresis time depend on the PCR amplicon size acrylamide gel thickness and type of PCR equipment Voltage and run time can be adapted accordingly Gels are stained in 0 5ug ml EtBr in water or 0 5X TBE Buffer for 5 10 minutes Gels are destained 2X in water for 5 10 minutes UV illumination is used for visualization Gel is photographed and data are interpreted
29. two prominent bands within the valid size range are reported as Detection of clonal T cell receptor gamma gene rearrangement consistent with the presence of a clonal cell population 15 Limitations of Procedure The assay is subject to interference by degradation of DNA or inhibition of PCR due to heparin or other agents The assay cannot reliably detect less than 1 positive cell per 100 normal cells 16 References 1 Miller JE Wilson SS Jaye DJ Kronenberg M An automated semiquantitative B and T cell clonality assay Mol Diag 1999 4 2 101 117 2 van Dongen JJM et al Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T cell receptor gene recombinations in suspect lymphoproliferations Report of the BIOMED 2 Concerted Action BMH4 CT98 3936 Leukemia 2003 17 12 2257 2317 3 van Krieken JH Langerak AW Macintyre EA Kneba M Hodges E Sanz RG Morgan GJ Parreira A Molina TJ Cabe adas J Gaulard P Jasani B Garcia JF Ott M Hannsmann ML Berger F Hummel M Davi F Br ggemann M Lavender FL Schuuring E Evans PA White H Salles G Groenen PJ Gameiro P Pott Ch van Dongen JJM Improved reliability of lymphoma diagnostics via PCR based clonality testing report of the BIOMED 2 Concerted Action BHM4 CT98 3936 Leukemia 2007 21 2 201 6 17 Appendix Reagents and Special Supplies Ficoll Separation Ficoll Hypaque or Ficoll Paque Pharmacia Cat 17 0840 02 1
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