AZOOSPERMIA Chromosome Y M i c r o d e l e t i o n Ref.: PI EDP003024-40 testspi EDP002024
1. INTRODUCTION In 1976, Tiepolo and Zuffardi reported de novo, microscopically detectable deletions of the distal half of Yq in four men with azoo-spermia, and on this basis they postulated the existence of one or more critical Yq genes for spermatogenesis. In recent years, this azoospermia factor (AZF) hypothesis has been widely validated. Exploiting the availability of comprehensive DNA probe based physical maps of the Y chromosome, investigators have reported many interstitial Yq deletions in infertile men with normal karyotypes. In particular, overlapping de novo deletions within intervals 6D 6E of the Y chromosome have been shown to cause at least 13% of cases of nonobstructive azoospermia and some cases of severe oligospermia as well. Three nonoverlapping regions of microdeletions in Yq11 (AZFa, AZFb, and AZFc) have been identifi ed that are probably responsible for azoospermia or oligozoospermia. The candidate gene for the expression of AZFc is called DAZ (Deleted In AZoospermia). Since the DAZ gene is absent in some men with azoospermia or severe oligospermia, it is an attractive candidate for azoospermia. In many reports, the screening program was based mainly on a multiplex polymerase chain reaction (PCR) approach with use of a series of Y- specifi c primers amplifying single DNA loci in Yq11 (i.e., sy254 and sy255) (2 4). A sequence-tagged site was considered absent only after at least three amplifi cation failures in the presence of successful amplifi cation of the control. Even if this strategy requires precautions to minimize the number of false-negative or false-positive results, the diffusion of PCR technology has rendered the molecular diagnostic of Y-chromosomal microdeletion popular and affordable. 2. PRINCIPLE OF THE METHOD AND TARGET DNA SEQUENCES The kit consists of one Master Mix and three Oligo mixes 1, 2 and 3. The assay wants to check the presence of all the three regions AZFa, AZFb, AZFc on chromosome Y. To do that, OLIGO MIX 1 and OLIGO MIX 2 are prepared to amplifi ed two STS for each region and a fragment as control of amplifi cation. In particular, OLIGO MIX 1 amplifi es sy86 (AZFa), sy127 (AZFb), sy254 (AZFc), and ZFX/ZFY as control of the amplifi cation, whereas OLIGO MIX 2 amplifi es sy84 (AZFa), sy134 (AZFb), sy255 (AZFc), and ZFX/ZFY as control of the amplifi cation. The strategy to check for the two STS for each AZF region in two different tubes is to minimize the possibility of an incorrect diagnosis 2 PI-EDP003024/r0/0106/424
due to an ineffi cient amplifi cation in one tube. OLIGO MIX 3 is prepared to verify the presence of the SRY gene as a control of Y specifi c sequences when ZFY gene is absent (i.e. in XX males). If all the fragments are amplifi ed, no microdeletion is detected. In presence of the microdeletion of one or more regions, usually both STS contained in that region fail to amplify. If all the fragments fail to amplify, could be useful for the diagnosis to test the sample for the presence of SRY gene (OLIGO MIX 3). 3. KIT CONTENT 2x Amplifi cation Mix Oligo MIX 1 (40 test) Oligo MIX 2 (40 test) Oligo Mix 3 (20 test) Male control DNA Female control DNA Blank control. 4. Equipment and materials required but not provided Micropipette 1-10µl Micropipette 10-100µl Filter tips 1-10µl and 10-100µl Rack for 0.2 ml tubes Thermalcycler with block for 0.2 ml tubes 0,2 ml tubes 4% agarose gel Uv box Horizontal electrophoresis tank Power supply Vortex 5. STORAGE AND STABILITY Twelve months at 2 C 8 C. 6. Assay Procedure a) Save on your thermal cycler the following amplifi cation program: PI-EDP003024/r0/0106/424 3
Temperature Time Cycles 94 C 10 min. 1 94 C 1 minute 60 C 30 seconds 30 72 C 60 seconds b) For every test run, prepare a PCR mix considering the two DNA controls included in the kit, one blank sample and a number of genomic DNA samples plus one. Mix all reagents together as follow: REAGENT VOLUME (µl) 2X Amplifi cation mix 10 Oligo Mix 10 DNA Template 5 (about 200ng) For genomic DNA extraction Euroclone strongly recommends the use of Fassst DNA Releaser. See Euroclone related products section (point 9). c) Prepare a PCR mix for every oligo mix. Dispense 10 ul of Amplifi cation Mix + 10ul of oligo mix in a PCR tube; add 5ul of DNA template (genomic DNA or control DNA or Blank) and insert the test tubes in the thermal cycler previously set and start the thermocycling program. d) At the end of the amplifi cation, remove the reaction tubes from the thermal cycler and put them in a tube-holder. e) Load 15 µl of the PCR products in a 2.0 % agarose gel stained with 50 µg/ml ethidium bromide. f) Run the gel for approximately 40-50 minutes at 60-80V. At the end of electrophoretic run, put the gel on a UV transilluminator (λ=256 nm) and take a picture of the gel 7. Interpretation t of results Legend Lane 1: marker Lane 2: blank Lane 3: female DNA Lane 4: normal male DNA Lane 5: deleted in AZFb DNA sample Lane 6: deleted in AZFc DNA sample 4 PI-EDP003024/r0/0106/424
See below table 1 and 2 : for each possible genotype it is shown the combination of fragments that must be amplifi ed in each PCR. Table 1 PI-EDP003024/r0/0106/424 5
Table 2 8. REFERENCES 1- Tiepolo L, Zuffardi O. Hum Genet 1976; 34: 119-24 2- Reijo R et al. Nat Genet 1995; 10: 383-92 3- Reijo R et al. Lancet 1996; 347: 1290-3 4- Simoni M et al. Fertil Steril 1997; 67: 542-7 5- Kostiner DR et al. Human Reproduction 1998; 13: 3032-8 6- Vogt PH et al. Molecular Genetics 1996; 5: 933-943 7- Simoni M et al. Laboratory guidelines for the molecular diagnosis of Y-chromosomal microdeletions. 6 PI-EDP003024/r0/0106/424
EUROCLONE RELATED PRODUCTS EDP001024 HFE mutation detection system 24 tests EMK015050 HLA B27 detection system 50 tests EMR057100 FASSST DNA RELEASER 100 reactions Genomic DNA extraction kit EUROCLONE S.p.A. Life Sciences Division Via Lombardia, 12-27010 Siziano (PV) Italy Tel. +39.02.38195378 - Fax +39.02.38195248 E-mail:info@euroclone.net www.euroclone.net Local distributor Euroclone S.p.A. has a Quality System certifi ed Dasa-Raegister in compliance with EN ISO 9001 (2000)