Printed in USA Revised 3/14
Y Chromosome AZF Analysis System
In Vitro DiagnosticMedical Device
MDSS GmbHSchiffgraben 4130175 Hannover, Germany
2800 Woods Hollow Rd.Madison, WI USA
INSTRUCTIONS FORUSE OF PRODUCT
MD1631 Part# TM252
T E C H N I C A L M A N U A L
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA. Part# TM252Revised 3/14 Page 1
1. Product Intended Use ...............................................................................................................1
2. Description..................................................................................................................................2
3. Product Components.................................................................................................................3
4. General Considerations............................................................................................................3A. DNA Template Sample ...................................................................................................3B. PCR Conditions ................................................................................................................4C. Thermal Cyclers................................................................................................................4D. Contamination Control ...................................................................................................5E. Control Reactions .............................................................................................................5
5. Amplification Reactions...........................................................................................................5A. Reaction Setup ..................................................................................................................6B. Optimal PCR Thermal Cycling Protocol for
the Perkin-Elmer Model 480 Thermal Cycler ..............................................................8C. Agarose Gel Electrophoresis ..........................................................................................8D. Data Analysis—Controls.................................................................................................9E. Data Analysis—Experimental Samples ......................................................................10
6. References .................................................................................................................................11
7. Appendix ...................................................................................................................................11A. Composition of Buffers and Solutions........................................................................11B. Worksheets ......................................................................................................................12
1. Product Intended Use
The Y Chromosome AZF Analysis System(a) meets the requirements of EU Directive98/79/EC on in vitro diagnostic medical devices. The Y Chromosome AZF AnalysisSystem provides a multiplex PCR-based method to analyze the integrity of the human Ychromosome AZF region. The Y Chromosome AZF Analysis System is intended for useas part of a diagnostic workup to characterize male infertility. Diagnostic results obtainedusing this system must be interpreted in conjunction with other clinical or laboratorydata. This information is potentially useful for patients considering in vitro fertilizationbecause deletions in the AZF region of the Y chromosome are passed on to male offspringproduced by in vitro fertilization, resulting in infertility of the child.
INSTRUCTIONS FOR USE OF PRODUCT MD1631.All technical literature is available on the Internet at www.promega.com
Please visit the web site to verify that you are using the most current version of this Technical Manual.
Y Chromosome AZF Analysis System
Technical Manual TM252 ENGLISH
MDSS GmbHSchiffgraben 4130175 Hannover,Germany
TM252.0314:TM252.0404.qxd 3/13/2014 8:17 AM Page 1
2. Description
The Y Chromosome AZF Analysis System provides a method for detection of the AZFregion of the human Y chromosome. The system consists of 20 primer pairs that arehomologous to previously identified and mapped sequence-tagged sites (STS; 1–7). Theseprimers will amplify nonpolymorphic short DNA segments from the Y chromosome whenused in polymerase chain reactions (PCR; 6,8). The primers have been combined into fiveMultiplex Master Mix sets for use in multiplex PCR. This makes it possible to determine thepresence or absence of all 20 STS by performing five concurrent PCR amplifications (Figure1). Y chromosome deletions in the regions that are amplified by these primer sets have beenassociated with male infertility (1,2,6,9–17). Adjacent regions of the Y chromosome do notgenerally appear as sequential amplification products within a single multiplex amplificationreaction. Exceptions are SY242 and SY208 of the DAZ locus, which are represented assequential amplification products using Multiplex B Master Mix reactions, and SY84 andSY86 of the DYS273 and DYS148 loci, which are represented as sequential amplificationproducts in Multiplex E Master Mix reactions.
Multiplex Master Mixes A–D contain a control primer pair that amplifies fragments of the X-linked SMCX locus. The fifth Multiplex Master Mix, Multiplex E, contains a control primerpair that amplifies a unique region in both male and female DNA (ZFX/ZFY). These controlprimer pairs are internal controls for the multiplex amplification reactions and test theintegrity of the genomic DNA sample. Finally, Multiplex E Master Mix also includes aprimer pair that amplifies a region of the SRY gene, acting as control amplification for thetestis-determining factor on the short arm of the human Y chromosome.
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Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TM252 Printed in USA.
Revised 3/14
4322TA09_3A
M
50 –
200 –
300 –400 –500 –
bp
100 –
– 50
– 200
– 300– 400– 500
– 100
A1 2 M
B3 4 M
C5 6 M
D7 8 M M
E9 10
Figure 1. Example of amplification of male genomic DNA. Amplification of male genomicDNA (MD115A) (lanes 1, 3, 5, 7, 9), as well as a negative DNA control (lanes 2, 4, 6, 8, 10), foreach of the five Multiplex Master Mixes.
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3. Product Components
Product Size Cat.#Y Chromosome AZF Analysis System 25 reactions MD1631Not for Sale in the United States. For Export Only. The Y Chromosome AZF Analysis Systemincludes:
• MD154A Multiplex A Master Mix 500µl• MD155A Multiplex B Master Mix 500µl• MD156A Multiplex C Master Mix 500µl• MD157A Multiplex D Master Mix 500µl• MD158A Multiplex E Master Mix 500µl• M300C GoTaq® DNA Polymerase 200u• P119F Nuclease-Free Water 1.25ml• G452C 50bp DNA Step Ladder (340ng/µl) 90µg• MD115A Male Genomic DNA (50ng/µl) 2.5µg• G190B Blue/Orange 6X Loading Dye 1ml
Storage Conditions: Store all components at –10 to –30°C. Avoid multiple freeze-thawcycles.
MDSS GmbHSchiffgraben 4130175 Hannover, Germany
4. General Considerations
A. DNA Template Sample
DNA concentration, purity and size are important considerations to ensure successwith the Y Chromosome AZF Analysis System. Poor-quality DNA may result inincreased background or amplification failure. Amplification failure can bemanifested as either complete lack of amplification of all bands with all MutiplexMaster Mixes or dropout of subpopulations of the alleles in individual mastermixes. To avoid this, use only high-quality DNA with an A260/A280 absorbance ratio≥1.8. The DNA should not be sheared and should be free of contaminating protein,ethanol, or salts (especially EDTA; EDTA concentration must be <0.4mM).
Quantitate the DNA prior to use in the Y Chromosome AZF Analysis System, asadding either too much or too little DNA can cause the reactions to fail.Spectrophotometric (absorbance) readings at 260nm (A260) can be used forestimation of DNA concentration where 1Au = 50µg of double-stranded DNA/ml.Fifty nanograms of DNA should be used in each multiplex PCR amplificationreaction. The DNA concentration can be overestimated by spectrophotometry if theA260/A280 ratio is low or if the absorbance value is low (below 0.1).
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA. Part# TM252Revised 3/14 Page 3
ENGLISHSymbol Key
In Vitro DiagnosticMedical Device
Store at –10 to –30°C
Contains sufficient for<n> tests
AuthorizedRepresentative in theEuropean community.
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–10°C
–30°C
–10°C
–30°C
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B. PCR Conditions
Concentration gradients can form in the Multiplex Master Mixes stored at –20°C.Once thawed as directed, vortex each tube for 10–15 seconds before use. Preheat theinstrument to 94°C before placing the tubes inside. Do not use more than 1unit ofGoTaq® DNA Polymerase per multiplex PCR amplification reaction. Standard finalPCR volumes using the Y Chromosome AZF Analysis System are 25µl. PCRvolumes of 12.5µl may result in dropout of multiple bands and should not be used.
C. Thermal Cyclers
The Y Chromosome AZF Analysis System was developed using the Perkin-ElmerDNA Thermal Cycler 480. The use of other thermal cyclers will require protocoloptimization and validation by the user.
Use only thin-walled tubes for PCR amplification. For the Perkin-Elmer Model 480thermal cycler use 0.5ml thin-walled GeneAmp® reaction tubes.
1. Thermal cyclers with heated vs. non-heated lids.
Thermal cyclers are available with heated lids or with non-heated lids. Forthermal cyclers with a non-heated lid (e.g., the Perkin-Elmer DNA ThermalCycler 480), a mineral oil overlay of the reaction is required to preventevaporation of the reaction during thermal cycling. For thermal cyclers withheated lids, a mineral oil overlay is not required. The heated lid of thesethermal cyclers prevents evaporation of the reaction during thermal cycling.However, ensure that the heated lid is functioning properly. If your reactiontubes amplified in a thermal cycler with a heated lid have condensation in thelid and around the top of the reaction tube, this is an indication that the heatedlid is not working properly, and poor performance of the systemamplifications may have resulted. If you are not sure about the calibration ofyour heated lid or observe condensation in the reaction tube, add a mineraloil overlay.
2. Thermal cycler heating/cooling ramp speed.
The time that a thermal cycler cycles between the denaturing, annealing, andextension temperatures of the PCR thermal cycling profiles (often referred toas the “ramp time”) can affect the efficiency of PCR amplification. The speedthat a thermal cycler cycles between temperatures of a PCR thermal cyclingprofile is dependent upon its manufacture. The PCR cycling protocol providedwas optimized for the Perkin-Elmer Model 480 Thermal Cycler. Use of otherthermal cyclers will require users to validate ramp times.
3. Calibration of the equipment.
Success of PCR amplification, especially in multiplex PCR, is dependent uponaccurate thermal cycling. Ensure that the thermal cycler equipment you areusing with the Y Chromosome AZF Analysis System is calibrated.
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Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TM252 Printed in USA.
Revised 3/14
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D. Contamination Control
Preventing DNA contamination of reaction components is essential. To avoidcontamination, pre- and post-amplification areas should be isolated, preferably inseparate rooms. Always use aerosol-barrier pipette tips, dedicated pipettes forassembling reactions, clean disposable gloves, and avoid carryover contamination ofstock solutions. Workstations and pipettes should be cleaned with a mild bleachsolution before and after use.
E. Control Reactions
Suitable control reactions must be included with each experiment.
1. Amplification reactions using the provided positive control Male GenomicDNA as a template should always be run in parallel with samples. PCRamplification of the provided positive control Male Genomic DNA shouldalways yield the expected amplification products (see Section 7.B, Table 1, forexpected sizes).
2. A negative no-DNA control PCR amplification reaction should also be run inparallel with samples to verify that the reagents are not contaminated withDNA. PCR amplification of a no-DNA control should not yield anyamplification products.
5. Amplification Reactions
Materials to Be Supplied by the User(Solution compositions are provided in the Appendix, Section 7.A.)
• nuclease-free light mineral oil • thermal cycler • thin-walled amplification tubes
• For the Perkin-Elmer DNA Thermal Cycler 480 thermal cycler, use 0.5mlGeneAmp® thin-walled reaction tubes (Applied Biosystems Part# N801-0737,N801-0611, or N801-0537)
• precast agarose gel: 4% NuSieve® 3:1 plus TBE buffer agarose precast Reliant®
minigels (Cambrex Cat.# 54927, 54928 or 54929)• 1X TBE buffer • ethidium bromide• aerosol-resistant pipette tips• UV transilluminator
ENGLISH
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A. Reaction Setup
See Figure 2 for a flow chart summary of the reaction setup protocol. In thisprocedure, aliquots of the sample DNAs are placed in a reaction tube. Separately,Taq DNA polymerase is added to each of the Multiplex Master Mixes. Then theMultiplex Master Mix containing Taq DNA polymerase is added to the reactiontubes containing the sample DNA. Each DNA sample is analyzed using each of thefive Multiplex Master Mixes.
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Revised 3/14
SampleDNA
PositiveControl Male
Genomic DNA
NegativeControlNo DNA
3856
MA
10_2
A
Dilute sample DNA(in Nuclease-Free Water),
and prepare controls
Prepare Multiplex Master Mixand Taq DNA polymerase mixtures,one for each Multiplex Master Mix.
Prepare reactions:1. Place sample DNA or control in tubes. 2. Add Multiplex Master Mix and Taq DNA polymerase mixture.
Thermal cycling
Agarose gel electrophoresis
A
A
B
C
D
E
B C D
SampleDNA
PositiveControl
NegativeControl
E
Taq DNApolymerase foreach sample
MultiplexMaster Mix foreach sample
Figure 2. Schematic representation of the Y Chromosome AZF Analysis System assay.
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Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA. Part# TM252Revised 3/14 Page 7
1. Thaw the Multiplex Master Mixes, Nuclease-Free Water, and Male GenomicDNA on ice. Once thawed, store on ice. Vortex the Multiplex Master Mixes for5–10 seconds before use.
2. Complete the table below to determine the number of reactions for eachMultiplex Master Mix. For each DNA sample there will be five reaction tubes,one for each Multiplex Master Mix. Include a positive Male Genomic DNAcontrol and a negative no-DNA control for each Multiplex Master Mix.
# of Positive Negative TotalMultiplex Sample Control Male Control Reaction
Master Mix DNAs Genomic DNA No DNA TubesA 1 1B 1 1C 1 1D 1 1E 1 1
3. Set up and label the required number of reaction tubes as determined above.Use thin-walled amplification tubes. Place the reaction tubes on ice.
4. In a separate tube, dilute each sample DNA to 10ng/µl using the suppliedNuclease-Free Water. Mix well by vortexing for 5–10 seconds. Add 5µl dilutedDNA to the appropriately labeled reaction tubes on ice.Positive Control DNA Sample: For the positive control Male Genomic DNAsample, dilute the Male Genomic DNA to 10ng/µl by adding 6µl of MaleGenomic DNA to 24µl of Nuclease-Free Water. Mix well by vortexing for 5–10seconds. Add 5µl to appropriately labeled reaction tubes on ice.Negative Control No-DNA Sample: For the negative control (no DNA), add5µl of Nuclease-Free Water to the appropriately labeled reaction tubes on ice.
5. Prepare five Multiplex Master Mix/GoTaq® DNA polymerase mixtures on ice,one for each Multiplex Master Mix. Vortex the Multiplex Master Mixes beforeusing them.
Volume per Volume perComponent Reaction 10 ReactionsMultiplex Master Mix 20µl 200µlGoTaq® DNA polymerase (5u/µl) 0.2µl 2µlfinal volume 20.2µl 202µl
6. Vortex to mix.
7. Add 20µl of the Multiplex Master Mix/GoTaq® DNA polymerase mixture to theappropriate reaction tubes, which contain the sample DNA or controls, on ice.
8. Gently vortex to mix.
9. Centrifuge the tubes briefly to bring the contents to the bottom of the tubes.Place reaction tubes on ice until ready for thermal cycling.
10. Thermal cyclers without a heated lid (for example, the Perkin-Elmer Model480 thermal cycler) require a mineral oil overlay of the reactions in the reactiontube. Tilt the tubes and add one drop of oil to the side of the tube, letting theoil run down the side of the tube.
ENGLISH
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B. Optimal PCR Thermal Cycling Protocol for the Perkin-Elmer Model 480Thermal Cycler
The following protocol was optimized for the Perkin-Elmer Model 480 ThermalCycler. The user is responsible for protocol optimization and validation if using adifferent thermal cycler.
It is crucial to preheat the instrument to 94°C before placing tubes into the machine.
C. Agarose Gel Electrophoresis
4% NuSieve® 3:1 Plus TBE buffer precast Reliant® minigels (Cambrex Cat.# 54927,54928 or 54929) are required for electrophoresis and subsequent optimalvisualization of amplification products.
1. Dilute molecular weight marker as follows:
Component Volume50bp DNA Step Ladder 12µlBlue/Orange 6X Loading Dye 4µlNuclease-Free Water 8µl
2. Add 2.5µl of the Blue/Orange 6X Loading Dye to each amplification tube andmix.
3. Load 10µl of the diluted molecular weight marker onto the first well of the gel.
4. Load 10µl/well of each sample.
5. Load 10µl of diluted molecular weight marker onto the last well of the gel.
6. Run the gel in 1X TBE containing 0.5µg/ml ethidium bromide at 5V/cm(measured as the distance between the electrodes) until the bromophenol bluedye front migrates to the bottom of the gel.
7. Photograph the gel using a UV transilluminator (320nm).
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Revised 3/14
Thermal Cycler Reaction Tubes DNA Polymerase PCR Conditions
Model 480 0.5ml GeneAmp® thin-walled reaction tubes
GoTaq® DNAPolymerase
94°C for 2 minutes, then:94°C for 1 minute57°C, for 30 seconds72°C for 1 minuteRepeat for 35 cycles then:72°C for 5 minutes4°C soak
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Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA. Part# TM252Revised 3/14 Page 9
D. Data Analysis—Controls
Determine that the control reactions produced the expected results before analyzingyour samples. The worksheets provided (see Section 7.B, Table 1) can be used foranalysis of both the control and experimental samples.
1. Negative No-DNA Control: There should be no specific amplificationproducts in the lanes containing the negative no-DNA control reactions. Theremay be some low molecular weight bands or smearing that are the result ofprimer interactions. Results should not be considered valid if amplificationproducts are observed in the negative no-DNA control reactions. This isindicative of contaminating DNA, and the experiment should be repeated withcare to avoid contamination.
2. Positive Male Genomic DNA Control: The number and size of theamplification products for each Multiplex Master Mix is indicated in Table 1(Section 7.B). The positive Male Genomic DNA control reaction for eachMultiplex Master Mix should have all the bands indicated for that MultiplexMaster Mix (Section 7.B, Table 1). The sizes of the amplification products canbe estimated by comparison with the 50bp DNA Step Ladder marker run onthe gel. If all of the expected bands are not present in the reactions withpositive Male Genomic DNA control, or if there are prominent extra bands, itis indicative of a problem with the amplification reagents, thermal cyclingprotocol or thermal cycler. Results should not be considered valid if any of theexpected amplification products are missing in the reactions with the positiveMale Genomic DNA control.
3. Control Primer in Multiplex Master Mixes: In Multiplex A, B, C and DMaster Mix reactions, the smallest amplification product (83bp) should beproduced from an X-linked locus (SMCX). In Multiplex E Master Mix, thelargest (496bp) amplification product should be produced from the ZFY/ZFXgenes. The absence of these products is indicative of a problem with thatparticular multiplex PCR amplification. If these control bands are present withthe positive control Male Genomic DNA but not with the sample DNA, itsuggests that there may be a problem with the genomic DNA used as atemplate. Problems with sample DNA may be: the presence of impurities,inaccurate DNA quantitation, or degraded DNA. Check the DNA template onan agarose gel before repeating the amplification. Repeat amplifications fromany sample DNA reactions in which the control product is absent. It may benecessary to isolate the template genomic DNA again.
ENGLISH
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E. Data Analysis—Experimental Samples
The worksheets provided in the Appendix, Section 7.B, can be used for analysis ofexperimental samples. Determine the presence or absence of the expected PCRproducts (Section 7.B, Table 1). If there are any products absent from the reactions,they can be mapped using the Y Chromosome Map Worksheet (Section 7.B, Table 2).All deletions should be contiguous. If multiple amplification bands are missing for asample, and those bands do not map to adjacent regions of the Y chromosome(Section 7.B, Table 2), they represent dropout bands and are not deletions. In this case,the test should be repeated. A single missing amplification band in a sample mayrepresent a dropout band and the test should be repeated to confirm that the locusdoes not amplify. Please note that a locus that is present in a sample may not amplifyif there is a mutation in one of the primer binding sites for the locus. Diagnosticresults obtained with this system should only be interpreted in conjunction withother clinical or laboratory data. Figure 3 shows an example of gel analysis of sampleDNA carrying a Y chromosome deletion from map position 15 to 20.
We have observed some nonspecific bands that appear above or below the expectedamplification products on the agarose gel—in particular a faint band in Multiplex Dat approximately 150bp and in Multiplex E above the control. Verify that yournegative controls (no DNA sample amplifications) show no detectable PCRamplification products. As long as your negative controls show no PCRamplification products, these nonspecific bands have no effect on analysis. Note: Apositive SY133 signal has been observed in some individuals with a confirmed AZFbdeletion, suggesting that the SY133 locus may be present elsewhere in thechromosome for those individuals.
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Revised 3/14
4534TA
A
1 M 2 3 M 4 5 M 6 7 M 8 9 M 10
B C D E
Figure 3. Example of Y chromosome deletion gel analysis. The amplification products fromMultiplex A–E Master Mix reactions are shown. Each Multiplex Master Mix is used foramplification of Male Genomic DNA (MD115A) samples (lanes 1, 3, 5, 7, 9) and arepresentative sample containing Y chromosome deletions (lanes 2, 4, 6, 8, 10). Bands resultingfrom the amplification of the positive Male Genomic DNA control are compared to thosebands resulting from the amplification of the test genomic DNA with Y chromosome deletions(note the deleted bands in all of these lanes except in Multiplex E). The marker (lanes M) is the50bp DNA Step Ladder provided with the system.
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6. References
1. Vollrath, D. et al. (1992) The human Y chromosome: A 43-interval map based onnaturally occurring deletions. Science 258, 52–9.
2. Reijo, R. et al. (1995) Diverse spermatogenic defects in humans caused by Ychromosome deletions encompassing a novel RNA-binding protein gene. NatureGenet. 10, 383–93.
3. Foote, S. et al. (1992) The human Y chromosome: Overlapping DNA clones spanningthe euchromatic region. Science 258, 60–6.
4. Affara, N. et al. (1996) Report of the second international workshop on Ychromosome mapping 1995. Cytogenet. Cell. Genet. 73, 33–76.
5. Kent-First, M.G. et al. (1996) Gene sequence and evolutionary conservation of humanSMCY. Nat. Genet. 14, 128–9.
6. Kent-First, M.G. et al. (1999) Defining regions of the Y-chromosome responsible formale infertility and identification of a fourth AZF region (AZFd) by Y-chromosomemicrodeletion detection. Mol. Reprod. and Dev. 53, 27–41.
7. Vogt, P.H. et al. (1997) Report of the third international workshop on Y-chromosomemapping 1997. Cytogenet. Cell Genet. 79, 1–20.
8. Skaletsky, H. et al. (2003) The male-specific region of the human Y chromosome is amosaic of discrete sequence classes. Nature 423, 825–37.
9. Pryor, J.L. et al. (1997) Microdeletions in the Y chromosome of infertile men. NewEng. J. Med. 336, 534–39.
10. Kent-First, M.G. et al. (1996) The incidence and possible relevance of Y-linkedmicrodeletions in babies born after intracytoplasmic sperm injections and theirfathers. Mol. Hum. Reprod. 2, 943–50.
11. Kostiner, D.R., Turek, P.K. and Reijo, R.A. (1998) Male infertility: Analysis of themarkers and genes on the human Y chromosome. Hum. Reprod. 13, 3032–8.
12. Kuroda-Kawaguchi, T. et al. (2001) The AZFc region of the Y chromosome featuresmassive palindromes and uniform recurrent deletions in infertile men. Nat. Genet. 29,279–86.
13. Lahn, B.T. and Page, D.C. (1997) Functional coherence of the human Y chromosome.Science 278, 675–80.
14. Reijo, R. et al. (1996) Severe oligozoospermia resulting from deletions of azoospermiafactor gene on Y chromosome. Lancet 347, 1290–3.
15. Repping, S. et al. (2002) Recombination between palindromes P5 and P1 on thehuman Y chromosome causes massive deletions and spermatogenic failure. Am. J.Hum. Genet. 71, 906–22.
16. Saxena, R. et al. (1996) The DAZ gene cluster on the human Y chromosome arosefrom an autosomal gene that was transposed, repeatedly amplified and pruned. Nat.Genet. 14, 292–9.
17. Simoni, M. et al. (1999) Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int. J. Androl. 22, 292–9.
7. Appendix
A. Composition of Buffers and Solutions
TBE 1X buffer89mM Tris-base
110mM boric acid2mM EDTA
ENGLISH
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B. Worksheets
Table 1. PCR Amplification Product Profile for Test Samples. Record the presence (+) or absence (–) of the PCR amplification products for each sample.
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Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TM252 Printed in USA.
Revised 3/14
Multiplex A Master Mix Samples (+/–)
STS Locus Product Size (bp) Map Position
SY254 DAZ 380 18
SY157 DYS240 290 20
SY81 DYS271 209 2
SY130 DYS221 173 11SY182 KAL-Y 125 5
SMCX 83 Control
Multiplex B Master Mix Samples (+/–)
STS Locus Product Size (bp) Map Position
SYPR3 SMCY 362 7
SY127 DYS218 274 9
SY242 DAZ 233 16
SY208 DAZ 140 17SMCX 83 Control
Multiplex C Master Mix Samples (+/–)
STS Locus Product Size (bp) Map Position
SY128 DYS219 228 10
SY121 DYS212 190 6
SY145 DYF51S1 143 14
SY255 DAZ 124 19SMCX 83 Control
Multiplex E Master Mix Samples (+/–)
STS Locus Product Size (bp) Map Position
ZFX/ZFY 496 ControlSY14 SRY 400 1
SY134 DYS224 303 13
SY86 DYS148 232 3SY84 DYS273 177 4
Multiplex D Master Mix Samples (+/–)
STS Locus Product Size (bp) Map Position
SY133 DYS223 177 12
SY152 DYS236 125 15
SY124 DYS215 109 8
SMCX 83 Control
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Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA. Part# TM252Revised 3/14 Page 13
ENGLISH
Table 2. Y Chromosome Map Worksheet. For more detailed information see supplemental Figure 2 of reference 8.Palindromes 8, 7, 6, 5 and 4 map in the proximal direction of SY121 and in the distal direction of SY182 (1). SY121 is located atthe distal boundary of P4 (8). AZFb extends from P5 to proximal P1 (8). AZFc includes P1 and P2 (8). At least one copy ofSY157 maps outside the AZFc boundary. Note: A positive SY133 signal has been observed in some individuals withconfirmed AZFb deletion, suggesting that the SY133 locus may be present elsewhere in the chromosome for those individuals.
4320MA09_3A
SR
YD
YS
271
DY
S14
8D
YS
273
KA
LYD
YS
212
SM
CY
DY
S21
5D
YS
218
DY
S21
9D
YS
221
DY
S22
3D
YS
224
DY
F51S
1D
YS
236
DA
ZD
AZ
DA
ZD
AZ
D
YS
240
CS
F2R
AIL
3RA
AN
T3A
SM
TX
E7
MIC
2pM
IC2
SR
YR
PS
4YZ
FY TSP
YA
ME
LY
KA
L-Y
RB
MY
1, R
BA
Y2
(Clu
ster
)D
AZ
(C
lust
er)
SM
CY
DY
Z19
(re
pea
t)
Cen
trom
ere
Euchromatin HeterochromatinYp Yq
pseudoautosomal pseudoautosomal
AZFcAZFaSRY AZFb
proximal AZFc/AZFd
SY
14S
Y81
SY
86S
Y84
SY
182
SY
121
SY
PR
3S
Y12
4S
Y12
7S
Y12
8S
Y13
0S
Y13
3S
Y13
4S
Y14
5S
Y15
2S
Y24
2S
Y20
8S
Y25
4S
Y25
5S
Y15
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
SMCX SMCX SMCX SMCX
Control Multiplex
A
Control Multiplex
B
Control Multiplex
C
Control Multiplex
D
ZFX
ZFY
Control Multiplex
E
TM252.0314:TM252.0404.qxd 3/13/2014 8:17 AM Page 13
(a)U.S. Pat. No. 6,242,235, Australian Pat. No. 761757, Canadian Pat. No. 2,335,153, Chinese Pat. No. ZL99808861.7, HongKong Pat. No. HK 1040262, Japanese Pat. No. 3673175, European Pat. No. 1088060 and other patents pending.
© 2012, 2013, 2014 Promega Corporation. All Rights Reserved.
GoTaq is a registered trademark of Promega Corporation.
GeneAmp is a registered trademarks of Roche Molecular Systems, Inc. Reliant is a registered trademark of CBMIntellectual Properties, Inc. NuSieve is a registered trademark of Lonza Sales AG.
Please contact Promega Technical Services or access our web site at www.promega.com for the most up-to-dateinformation on Promega products.
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Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPart# TM252 Printed in USA.
Revised 3/14
TM252.0314:TM252.0404.qxd 3/13/2014 8:17 AM Page 14