AZF MX
Contents
1. 1 PRODUCT INFORMATION This user manual describes the instructions for use of the following products AZF MX COMPLETE cod 04 23C Complete system for the identification of deletions in the AZF locus involved in male infertility The kit includes all the reagents for DNA extraction amplification and visualization by agarose gel electrophoresis the internal control of sample amplificability and the reference DNA Code Product Pkg 04 23C 12 AZF MX COMPLETE 12 test 04 23C 24 AZF MX COMPLETE 24 test AZF MX cod 04 23A Kit for the identification of deletions in the AZF locus involved in male infertility The kit includes the reagents for amplification and visualization by agarose gel electrophoresis the internal control of sample amplificability and the reference DNA Code Product Pkg 04 23A 12 AZF MX 12 test 04 23A 24 AZF MX 24 test AZF MX amplification reagents cod 04 23R Kit for the identification of deletions in the AZF locus involved in male infertility The kit includes the reagents for amplification the internal control of sample amplificability and the reference DNA Code Product Pkg 04 23R 12 AZF MX amplification reagents 12 test 04 23R 24 AZF MX amplification reagents 24 test anauitica pag 3 04 23A 25 8033622781004 EN doc 2 KIT CONTENT NOTE In the kits with different codes C A or R different components are included legenda X component included in th2. anamen pag 7 04 23A 25 8033622781004 EN doc immersion in a solution of 5 Sodium Hypochloride 1 volume of 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 ETHIDIUM BROMIDE included in 04 23C and 04 23A 3 8 diamino 1 ethyl 6 phenylphenantridiumbromide Ethidium Bromide lt 2 Description of risk T Toxic S RISK SENTENCES AND S SENTENCES 23 68 Toxic for inhalation Risk of irreversible effects S 36 37 45 Wear laboratory coat and disposable gloves In case of accident or discomfort seek for medical assistance and show the container or label R and S sentences refer to the concentrated product as provided in the kit In particular for Ethidium Bromide until the dilution in the agarose gel In manipulating concentrated Ethidium Bromide use a chemical dispensing fume cabinet Always wear disposable gloves and laboratory coat in manipulating the diluted Ethidium solution as well The product can not be disposed with the common waste It must not reach the drainer system For the disposal follow the local law In case of accidental spilling of Ethidium Bromide clean with Sodium hypochloride and water anauitica pag 8 04 2
3. in a 2 5 agarose gel because of their low molecular weight NOTICE UV rays are dangerous for skin and above all eyes always wear gloves and safety glass or make use of the protection screen of UV transilluminator EH pag 21 04 23A 25 8033622781004 EN doc 13 INTERPRETATION OF THE RESULTS The included controls should show the following results CONTROL RESULT INTERPRETATION reference DNA presence of expected bands the The multiplex PCR amplification works correctly negative control absence of bands Absence of contaminations Then the interpretation of the bands on agarose gel follows the table below AZF MARKERS RESULT INTERPRETATION ZFX ZFY band absent AZF markers absent some or all of sample not amplificable them ZFX ZFY band present amplificable sample without any all AZF markers present deletion at the AZF locus and SRY band present presence of the testis determining factor ZFX ZFY band present amplificable sample without AZF all AZF markers absent locus and presence of the testis SRY band present determining factor e g XX male ZFX ZFY band present amplificabile sample with AZF markers them absent one or more of deletions at the AZF locus if the results show that the sample is not amplificable absence of the band of the internal control ZFX ZFY the complete analysis should be repeated The parallel amplification of the SRY
4. inhibitors or other factors which interfere with amplification reaction are present The obtained DNA solution is not pure the ratio A260 A 280 is low Verify to have correctly followed the extraction protocol and repeat the extraction by starting from a new sample In the obtained DNA solution there is residual RNA the ratio A260 A 280 is too high which can be eliminated by introducing a digestion step with RNAase In case you used the kit AZF MX COMPLETE cod 04 23C If the lower part of the filter has been wetted with the filtrate of Solution 3 Point 14 par 11 1 transfer the filter column in a new 2 mL tube and centrifuge at 14000 rpm for 1 min Verify that Solutions 2 and 3 have been employed in the correct order as indicated in the protocol For any problem you can contact AB ANALITICA technical support e mail laboratorio abanalitica it anauitica pag 26 04 23A 25 8033622781004 EN doc 15 DEVICE LIMITS The kit can have reduced performances if the clinical sample is not suitable for this analysis blood sample non properly stored or treated with heparin as anti coagulant 16 DEVICE PERFORMANCES 16 1 Specificity The primers used in this method were studied in a way that they do not amplify repeated sequences in the chromosome or DNA sequences that have big nucleotidic differences among the population Primer sequence alignment in the most important databanks shows the absence of unspecific alignment Moreov
5. to remove droplets from the inside of the lid then add 200 uL of Ethanol 96 100 and mix by vortexing for 15 sec 6 Centrifuge briefly then transfer the mix into a filter column close the cap and centrifuge at 8000 rpm for 1 min T Transfer the filter column into a new 2 mL tube and discard the column containing the filtrate 8 Open the cap of the filter column and add 500 uL of Solution 2 close the cap and centrifuge at 8000 rpm for 1 min 9 Transfer the filter column into a new 2 mL tube and discard the column containing the filtrate 10 Open the cap of the filter column and add 500 uL of Solution 3 close the cap and centrifuge at 14000 rpm for 3 min 11 Transfer the filter column into a 1 5 mL tube paying attention not to wet the lower part of the filter with the filtrate If this happens centrifuge briefly 12 Open the cap of the filter column and add 200 uL of the Elution Solution or sterile distilled water Incubate for 1 5 min at room temperature and then centrifuge at 8000 rpm for 1 min Extracted and purified DNA contained in the filtrate is stable for at least 1 year if stored at 20 C anauitica pag 18 04 23A 25 8033622781004 EN doc 12 2 DNA AMPLIFICATION For each sample add to each premix tube 0 2uL Super AB Taq 6uL extracted DNA It is important to include in each experiment a negative control to monitor the contamination add distilled water to the mix instead of extracted DNA an
6. 3A 25 8033622781004 EN doc SOLUTION 1 and SOLUTION 2 included in 04 23C Solution 1 and 2 contain guanidinium hydrochloride Description of risk Harmful and irritating RISK SENTENCES AND S SENTENCES R 22 36 and R 38 S 13 26 36 46 Toxic if swallowed Irritating for skin and eyes Store away of food and beverage In case of contact with eyes rinse immediately with plenty of water and seek medical advise Use protective cloth and suitable gloves In case of accident or discomfort seek for medical advice immediately and show the container or label PROTEASE included in 04 23C Description of risk May cause sensitization irritating RISK SENTENCES AND S SENTENCES R 37 and R 38 41 42 S 24 26 36 37 5 46 Toxic if swallowed Irritating for skin and eyes Avoid contact with skin and eyes In case of contact with eyes rinse immediately with plenty of water and seek medical advise Use protective cloth and suitable gloves In case of accident or discomfort seek for medical advice immediately and show the container or label Safety data sheet MSDS of the kit is available upon request anauitica pag 9 04 23A 25 8033622781004 EN doc 6 MATERIALS REQUIRED BUT NOT PROVIDED 6 1 Reagents e Reagents for DNA extraction necessary for cod 04 23A and 04 23R e Sterile DNase and RNase free water e Distilled water e 96 100 Ethanol necessary for the kit cod 04 23C e Reagents for agar
7. 4 23A 25 8033622781004 EN doc e Store the biologic samples the purified DNA the reference DNA included in the kit and all the amplification products in different places from where amplification reagents are stored e Organise the space in different pre and post PCR units do not share consumables pipets tips tubes between them e Change frequently the gloves e Wash the bench surfaces with 5 sodium hypochloride e Store the extracted DNA at 2 8 C for short term or freeze them at 20 C for long term storage e Thaw the PCR premixes at room temperature before use Add Taq DNA polymerase and purified DNA very quickly at room temperature better if in an ice bath 5 SAFETY RULES 5 1 General safety rules e Wear disposable gloves to handle reagents and clinical samples wash your hands at the end of work e Do not pipet with mouth 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 spiling of the samples clean up with 1096 Sodium Hypochloride The materials used to clean up should be disposed in special containers for contaminated products e Clinical samples materials and contaminated products should be disposed after decontamination by
8. 5 C 25 C for 1 min point 12 par 11 1 The filter column has to be incubated for at least 1 min at room temperature after adding the Elution Solution DNA has not been efficiently eluted In order to increase the efficiency of DNA elution incubate the filter column at room temperature for 5 min Solution 1 and 2 have not been correctly used Verify that both the solutions have been employed in the correct order Repeat the extraction by starting from a new sample Elution with an excessive volume of Elution Solution Repeat correctly the extraction starting from a new sample Presence of a little volume of DNA in the eluate anauitica For a good result of the amplification reaction the DNA amount has to be 180 ng If the concentration of eluted DNA revealed with spectrophotometric measures is too low it is suggested to repeat the extraction and elute in less than 200 uL volume Note that decreasing the elution volume to 200 DNA concentration increases in the eluate but the total yield may be reduced pag 25 04 23A 25 8033622781004 EN doc In case you used the kit AZF MX cod 04 23A and 04 23R verify the following points and proceed as suggested Verify that you followed very careful the manufactures s instructions of the extraction kit Consult the troubleshooting section of the user manual of the extraction kit Repeat the DNA extraction from a new sample Some
9. A template to be amplified target DNA and two single stranded oligonucleotides primers that are designed in order to anneal specifically to the template DNA The DNA polymerase begins the synthesis process at the region marked by the primers and synthesizes new double stranded DNA molecules identical to the original double stranded target DNA region by facilitating the binding and joining of the complementary nucleotides that are free in solution dNTPs After several cycles one can get millions of DNA molecules which correspond to the target sequence The sensitivity of this test makes it particularly suitable for the application in laboratory diagnostics The multiplex amplification allows the simultaneous amplification of different DNA sequences in the same reaction by mean of a selected primers mix anauitica pag 15 04 23A 25 8033622781004 EN doc 10 PRODUCT DESCRIPTION The amplification of short DNA sequences STS in AZF loci is the best method to verify the presence of microdeletions in Y chromosome In particular the strategy of the proposed method consists of the amplification of eleven markers by mean of 3 multiplex PCR AZF MX method is compliant to the indications of the recently proposed European Guidelines for diagnostic testing of Y chromosomal microdeletions Simoni M et al 2004 that define the basic characteristics of the primer set and of the amplification protocol to enable the detection of almost al
10. ANALITICA ADVANCED BIOMEDICINE www abanalitica it USER MANUAL Genequality AZF MX ref 04 23C ref 04 23A ref 04 23R Kit for the identification of deletions in AZF locus Compliant to the European Guidelines EAA EQMN best practice guidelines for molecular diagnosis of Y chromosomal microdeletions State of the art 2004 Simoni M Bakker E Krausz C nt J Andrology 2004 27 240 249 04 23A 25 8033622781004 EN doc 1 PRODUCT INFORMATION 3 2 KIT CONTENT 4 3 STORAGE AND STABILITY OF THE REAGENTS 6 4 PRECAUTIONS FOR USE 6 5 SAFETY RULES 7 5 1 General safety rules 7 5 2 Safety rules about the kit 8 6 MATERIALS REQUIRED BUT NOT PROVIDED 10 6 1 Reagents 10 6 2 Instruments 10 6 3 Materials 10 7 PREPARATION OF THE REAGENTS 11 8 INTRODUCTION 12 9 TEST PRINCIPLE 15 10 PRODUCT DESCRIPTION 16 11 COLLECTION MANIPULATION AND PRE TREATMENT OF SAMPLE 17 12 PROCEDURE 17 12 1 DNA EXTRACTION 17 12 1 1 DNA extraction from fresh or frozen whole blood 18 12 2 DNA AMPLIFICATION 19 12 3 VISUALIZATION OF THE AMPLIFICATION PRODUCTS 20 12 3 1 High Resolution agarose gel electrophoresis 20 12 3 2 Sample loading 21 13 INTERPRETATION OF THE RESULTS 22 14 TROUBLESHOOTING 24 15 DEVICE LIMITS 27 anaurtica pag 1 04 23A 25 8033622781004 EN doc 16 DEVICE PERFORMANCES 16 1 Specificity 16 2 Diagnostic sensitivity 17 BIBLIOGRAPHIC REFERENCES 04 23A 25 8033622781004 EN doc pag 2 27 27 27 28
11. E 40 mL 70 mL X Cell Lysing Solution SOLUTION 1 1X3mL 1X6mL X Washing Solution SOLUTION 2 1X 11 mL 1X 22 mL X Washing Solution SOLUTION 3 1X10 5mL 1X21mL Eluting X 0 0 Eluting Solution Solution 1X 3 mL 1X 6 mL X Filter columns 12 24 X 2 mL tubes 24 48 anauitica 5 04 23A 25 8033622781004 EN doc 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 P store at 20 C Small bag store at 20 C Box F store at 2 8 C Box A store at 15 25 C room temperature When stored at the recommended temperature all test reagents are stable until their expiration date indicated on the labels 4 PRECAUTIONS FOR USE e The kit should be handled by investigator qualified through education and training in molecular biology techniques applied to diagnostics e Before starting the kit procedure read carefully and completely the instruction manual e Keep the product out of heating sources e Do not use any part of the kit if over the expiration date e In case of any doubt about the storage conditions box integrity or method application contact ANALITICA technical support at laboratorio abanalitica it before using the kit In the amplification of nucleic acids the investigator has to take the following special precautions e Use filter tips anauitica pag 6 0
12. J Andrology 1999 22 292 299 Simoni M Bakker E Krausz C Int J Andrology 2004 27 240 249 Sun C Skaletsky Rozen S Gromoll J Nieschlag E Oates Page DC Hum Mol Genet Sep 22 9 15 2291 6 2000 Vogt P H Edelmann A Kirsch S Hum Mol Genet 5 933 943 1996 anauitica pag 28 04 23A 25 8033622781004 EN doc ANALITICA ADVANCED BIOMEDICINE www abanalitica it AB ANALITICA srl Via Svizzera 16 35127 PADOVA ITALY Tel 39 049 761698 Fax 39 049 8709510 e mail info abanalitica it
13. casting tray with the comb placed in and allow the gel to cool at room temperature or in a fridge until the gel becomes solid Remove the comb carefully pay attention to not damage the gel s wells transfer the tray into the electrophoresis chamber and pour the appropriate amount of 1X TAE buffer so that it covers the gel completely about 1 2 mm over the gel surface anauitica pag 20 04 23A 25 8033622781004 EN doc 12 3 2 Sample loading For visualization of the amplification products mix into a tube or directly on a parafilm layer 2 uL 6X Blue 10 uL Amplification product of each multiplex PCR and 2 uL 6X Blue 10 Molecular Weight Marker NOTE 6X Blue and DNA Molecular Weight Marker are included in cod 04 23A and 04 23C only if other loading buffers or molecular weight markers are used refer to the manufacturer s instructions Load the mixture in the gel wells switch on the power supply and set the voltage between 80 90 V Run the gel for about 2 hours then place the gel on an UV transilluminator and analyze the results by comparing the size of the amplification products with the reference Molecular Weight Marker DNA Molecular Weight Marker Marker MW 501 489 404 331 242 190 147 111 110 67 34x2 26 bp NOTE In a 2 5 agarose gel the 501 489 bp bands usually are not clearly resolved and appear as an unique band the 26 and 34 bp bands are sometimes too small to be visible
14. d a reference DNA DNA of a undeleted male that will show all the bands of the different markers Put the microtubes into the thermalcycler programmed as below 1 cycle 94 C 5 min 94 C 1 min 40 cycles 60 C 1 min 72 C 1 min 1 cycle 72 C 7 min storage 4 C Tab 1 Lenghts of the amplification products 1 bp MX 2 bp MX 3 bp ZFX ZFY 495 ZFX ZFY 495 DBY 689 SRY 472 SRY 472 ZFX ZFY 495 5 254 380 sY95 303 SRY 472 sY86 320 sY117 262 sY84 326 sY127 274 sY125 200 sY134 301 sY255 120 DFFRY 155 NOTE The amplification of ZFX ZFY gene is the internal PCR control because the primers amplify an unique fragment both in male and female DNA respectively anauitica pag 19 04 23A 25 8033622781004 EN doc 12 3 VISUALIZATION OF THE AMPLIFICATION PRODUCTS 12 3 1 Agarose gel electrophoresis Preparation of a 3 agarose gel Weight 1 5 g of supplied agarose powder and pour it into 50 mL of 1X TAE Leave the solution on a magnetic stirring heater or in a microwave until the solution becomes clear Allow the gel to cool for a few minutes 3 5 min then add 10 uL of the Ethidium Bromide solution CAUTION Ethidium Bromide is a strong mutagenic agent Always wear gloves and preferably work under a chemical safety cabinet during the handling of this reagent or gels containing it Pour the gel into the appropriate gel
15. d reference DNA at 20 C Avoid repeated freezing and thawing of the reagents No amplification bands or only for some markers absence of the ZFX ZFY band but a good band for reference DNA Possible problems during the extraction step If you used the kit AZF MX COMPLETE cod 04 23C please consult the table in the following page anauitica pag 24 04 23A 25 8033622781004 EN doc Possible causes Comments and suggestions Inefficient cell lysis due to an insufficient mixing of Solution 1 with the sample Repeat the extraction with a new sample pay attention to mix the sample with Solution 1 by vortexing Point 11 par 11 1 Inefficient cell lysis due to a decreased protease activity Repeat the extraction with a new sample by using a new aliquot of Protease avoid repeated freezing thawing of the solution Ethanol 96 100 was not added to the lisate before its transfer into the filter column Repeat correctly the extraction by starting from a new sample Leukopenic patient Do not use whole blood but enrich the leukocitary fraction of the sample buffy coat Ficoll Hypaque Ethanol 96 100 was not added to the lisate before its transfer into the filter column point 6 par 11 1 or a solution with a low percentage of Ethanol has been used Repeat correctly the extraction by starting from a new sample The filter column has not been incubated at room temperature 1
16. d should be treated with EDTA Other coagulating 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 for short time if DNA is not extracted shortly it is necessary to freeze the sample 12 PROCEDURE 12 1 DNA EXTRACTION AB ANALITICA suggests the use of AZF MX COMPLETE kit cod 04 23C that includes the extraction system with which the method has been standardized In any case any DNA extraction method can be used provided that allows the extraction of pure and integral DNA Extracted DNA should be quantified by spectrophotometric measurement The amount of DNA to be used in the amplification is 180 ng therefore the purified DNA solution should be diluted taking into account that the available volume in each premix tube is 6 uL For any problem in method application you can contact AB ANALITICA technical support at laboratorio abanalitica it The extraction method below is referred to the kit AZF MX COMPLETE cod 04 23C anauitica pag 17 04 23A 25 8033622781004 EN doc 12 1 1 DNA extraction from fresh or frozen whole blood 1 Add 20 uL of Protease into a 1 5 mL tube 2 Add 200 uL of fresh or frozen whole blood treated with EDTA 3 Vortex and mix the Solution 1 then add to the sample 200 uL of this solution Mix by vortexing for 15 sec 4 Incubate at 56 C for 10 min 5 Centrifuge briefly
17. e kit the kit STORE AT 20 C 0 component not included in Q tr TUBE T 34 88 DESCRIPTION LABEL OR LID SEAKEAKE COLOUR Single dose premix tubes colourless X x A for Multiplex T 1a 25 Single dose premix tubes 2 for Multiplex II STE 12 24 Single dose premix tubes X X X for Multiplex III Yellow i 1 25 Thermostabile Hot start Super AB Taq x DNA polimerase 5 U uL SR T ep Ee X 0 0 Protease Proteasi Green 100 uL 6X 100 uL STORE AT 20 C 2 ET NM E TUBE T 25424 55 DESCRIPTION LABEL OR LID Sas COLOUR Reference DNA x x x us Une undeleted Blue 1X20uL 1X40 pb male anauitica pag 4 04 23A 25 8033622781004 EN doc STORE AT 29 8 C ei TUBE T BABABA DESCRIPTION LABEL OR LID 593 Os COLOUR Electrophoresis loading buffer Bromophenol 0 6X solution Z Blue 1X 200 uL 1X 350uL Ethidium Ethidium Bromide solution Bromide 0 2 5 mg mL Red 1X 100 uL 1X 180 uL S 36 37 45 Welght Markar MW Marker Yellow 1X100uL 1X 180 uL STORE AT 15 25 C lt BN DESCRIPTION LABEL OR LID Od 93 COLOUR X X X Agarose molecular biology grade AGAROSE 10g 20g Electrophoresis buffer X X X TRIS Acetate EDTA pH 8 0 50 X TA
18. er experimental data total absence of feminine DNA amplification revealed that the primers used for each marker are Y specific 16 2 Diagnostic sensitivity The use of a basic set of STS primers as suggested in the European Guidelines Simoni et al 2004 enables the detection of almost all the Clinically relevant deletions and over 95 of the deletions reported in literature in the three loci anauitica 27 04 23A 25 8033622781004 EN doc 17 BIBLIOGRAPHIC REFERENCES Brown GM Furlong RA Sargent CA Erickson RP Longepied G Mitchell M Jones MH Hargreave TB Cooke HJ NA Hum Mol Genet Jan 7 1 97 107 1998 Kamp C Hirschmann P Voss H Huellen K Vogt PH Hum Mol Genet Oct 12 9 17 2563 72 2000 Kuroda Kawaguchi T Skaletsky H Brown LG Minx PJ Cordum HS Waterston RH Wilson RK Silber S Oates R Rozen S Page DC Nat Genet Nov 29 3 279 86 2001 Lahn BT Page DC Science Oct 24 278 5338 675 80 1997 Ma K Inglis JD Sharkey A Bickmore WA Hill RE Prosser EJ Speed RM Thomson EJ Jobling M Taylor K Cell Dec 31 75 7 1287 95 1993 Saiki RK S Scharf F Faloona KB Mullis GT Horn HA Erlich and N Arnheim Science 230 1350 1354 1985 Saxena R de Vries JW Repping S Alagappan RK Skaletsky H Brown LG Ma P Chen E Hoovers JM Page DC Genomics Aug 1 67 3 256 67 2000 Simoni M Bakker E Eurlings M C M Matthijs G Moro E Muller C R et Vogt P H Int
19. ions Kampo et al 2000 Sun et al 2000 Repping et al 2002 the new European Guidelines were published Simoni et Al 2004 The European Guidelines give precise indications for the selection of the patients to be screened The authors suggest A multiplex PCR amplification of genomic DNA must be performed e The amplification of the ZFX ZFY gene is an appropriate internal PCR control because the primers amplify a unique fragment both in male and female DNA respectively e The basic characteristics of the selected markers are Y specificity absence of other homologous sequences non polymorphic 13 04 23A 25 8033622781004 EN doc e Atleast two loci in each AZF region should be analysed for AZFa sY84 sY86 for AZFb sY127 57134 for AZFc sY 254 sY255 e he SRY gene should be included in the analysis as a control for the testis determining factor on the short arm of the Y chromosome and for the presence of Y specific sequences when the ZFY gene is absent e g in XX males anauitica pag 14 04 23A 25 8033622781004 EN doc 9 TEST PRINCIPLE PCR method Polymerase Chain Reaction has been 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 thermo stable DNA polymerase Three nucleic acid segments are involved in the reaction double stranded DN
20. l the clinically relevant deletions The amplification of the internal control in the ZFX ZFY gene allows to verify the good quality of the extracted DNA and at the same time the absence of amplification inhibitors avoiding false positive results The kit includes reference DNA from a non deleted male The amplification of the reference DNA with the presence of all the expected bands is a guarantee of a correct course of the reaction The reference DNA is of human origin but it is not dangerous for the operator The kit is in premix format all the reagents for the amplification are pre mixed and aliquoted in monodose test tubes to which Taq polymerase and the extracted DNA will be added This premix format allows the reduction of the manipulation in pre amplification steps with a considerable time saving for the operator the repeated freezing thawing of reagents that could alter the product performances is avoided and above all this form reduces at minimum the risk of sample contamination and the risk to get false positive results Nevertheless it is always recommended to use all the proper amplification controls anauitica pag 16 04 23A 25 8033622781004 EN doc 11 COLLECTION MANIPULATION AND PRE TREATMENT OF SAMPLE The procedure for the analysis of microdeletions of Y chromosome starts from the collection of whole blood samples The sample collection should follow all the usual sterility precautions Bloo
21. marker and the internal control ZFX ZFY allows the monitoring of any female contamination pag 22 04 23A 25 8033622781004 EN doc anauitica MARKERS NOT DELETED DELETED INTERNAL XY MALE XX MALE XX FEMALE XY MALE CONTROL i S In case of absence of the band of one or more markers it is suggested to repeat the amplification 2 5 high resolution agarose gel electrophoresis of the three multiplex PCR amplifications 1 DNA Molecular Weight Marker 2 Multiplex 3 Multiplex II 4 Multiplex lll The analysed patient is not deleted for any of the markers For any problem you can contact AB ANALITICA technical support e mail laboratorio abanalitica it fax N 39 049 8709510 anauitica pag 23 04 23A 25 8033622781004 EN doc 14 TROUBLESHOOTING Neither amplification products nor reference DNA band e TAQ polymerase was not correctly added to the premix Use pipets and tips of suitable volumes pipet range 0 2 2 uL and suitable tips Check visually that TAQ polymerase diffuses in the premix this is easy because the enzyme is in dissolved glycerol that has a higher density Alternatively put the drop of TAQ polymerase on the tube wall then centrifuge briefly e The thermalcycler was not programmed correctly Check the conformity of the thermalcycler program and the temperature profile in the instruction manual e The kit doesn t work properly Store the premixes TAQ polymerase an
22. ns called AZF Azoospermia Factors frequently found to be deleted in some azoospermatic and serious oligozoospermatic subjects These loci AZFa AZFb e AZFc contain genes that control the correct course of spermatogenesis Fig 1 Alterations in one or more AZF loci cause a drastic reduction of germinal cells up to their complete absence Several studies marked the relation between the deletions and the presence of repeated DNA sequences showing that a recombinational event between the two proviral sequences is the basis of AZFa deletion Kamp et al 2000 Sun et al 2000 AZFc deletions are generally more uniform and follow the recombination between blocks of repeated DNA sequences flanking the region Kuroda Kawaguchi et al 2001 A diagnosis of Y related infertility is Suspected in males with serious Azoospermia or Oligozoospermia associated with anomalous morphology and or spermatic motility in absence of other known causes 5 to 10 of these subjects microdeletions of Y chromosome are identified by molecular analysis To evaluate the integrity of Y euchromatic region at least four genes should be investigated USP9Y DFFRY RBMY and DAZ These genes are currently considered to be directly or indirectly involved in male fertility USP9Y is a single copy gene located in AZFa region it has its homologous on the X chromosome and it is expressed ubiquitously Brown et al 1998 DBY located in AZFa region is a
23. ose gel electrophoresis necessary for cod 04 23R 6 2 Instruments e Laminar flow cabinet use is recommended while adding TAQ polymerase to the amplification premix to avoid contamination it would be recommended to use another laminar flow cabinet to add the extracted DNA e Micropipettes range 0 2 2 uL 0 5 10 uL 2 20 uL 20 200 uL 100 1000 uL Thermal cycler Thermoblock or thermal bath Microcentrifuge max 12 14 000 rpm Balance Vortex Magnetic heating stirrer or microwave Chemical cabinet its use is recommended in handling Ethidium Bromide Horizontal electrophoresis chamber for agarose minigel Power supply 50 150 V UV Transilluminator Photo camera or image analyzer 6 3 Materials e Disposable gloves e Disposable sterile filter tips range 0 2 2 pL 0 5 10 uL 2 20 uL 20 200 uL 100 1000 e Graduate cylinders 1 L for of TAE dilution e Pyrex bottle or Becker for agarose gel preparation e Parafilm e Microtubes 1 5 2 0 mL anauitica pag 10 04 23A 25 8033622781004 EN doc 7 PREPARATION OF THE REAGENTS Preparation of 1 L of 1X TAE buffer Mix 20 mL of 50X TAE with 980 mL of distilled water anauitica pag 11 04 23A 25 8033622781004 EN doc 8 INTRODUCTION Nowadays the analysis of microdeletions of Y chromosome is considered an essential diagnostic approach to study male infertility Recent studies have divided the Y chromosome long arm into three regio
24. single copy gene with an homologous on the X chromosome as well but it gives a specific transcript at the testicular level Lahn amp Page 1997 RBMY is a multicopy gene mainly located in AZFb region and expressed exclusively at testicular level Ma et al 1993 DAZ is a gene located in AZFc region in four copies arranged in two clusters Saxena et al 2000 Recently PCR amplification of short DNA sequences STS within AZF loci has been recognised as the best method to detect microdeletions in these regions anauitica pag 12 04 23A 25 8033622781004 EN doc Fig 1 So SCHEMATIC VISUALIZATION OF Y CHROMOSOME AND AZF LOCI Markers for the detection of microdeletions in AZF locus sY86 7 sYB84 AZFa c DFFRY in USP9Y gene DBY in DEAD BOX Y gene sY95 27 5117 AZFb a sY125 57127 57134 AZFc EN sY254 in DAZ gene 8 255 in DAZ gene Published data demonstrated that the routinely performed diagnostic protocols were very different and often leading to inaccurate or incomplete diagnosis The need of standardization was highlighted in a preliminary publication of the European Guidelines that described the main characteristics of the amplification reaction and the markers required to have a complete and reliable diagnosis Simoni et al 1999 Four years after this preliminary publication the complete sequencing of Y chromosome Skaletsky et al 2003 and the knowledge about the molecular mechanisms of delet
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