y chromosome microdeletion detection system v.4€¦ · the portion of the male-speciﬁc region of...

Y CHROMOSOME MICRODELETION

Detection System v.4.0

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Y CHROMOSOME MICRODELETION DETECTION İ

Contents Section 1: Introduction .................................................................................... 1

Section 2: Product Overview .......................................................................... 2

Description of Test .......................................................................................... 2

Chromosomal regions of STS ........................................................................ 4

Performing the Test ........................................................................................ 5

Laboratory Design and Organization .............................................................. 5

Workflow Overview ......................................................................................... 6

Section 3: Materials, Safety Warnings and Precautions.............................. 7

Kit Contents .................................................................................................... 7

Safety Warnings and Precautions .................................................................. 8

Shipping Conditions ........................................................................................ 9

Storage and Handling Requirements ............................................................. 9

Section 4: Y Chromosome Microdeletion Detection Kit Procedure ......... 10

Preparing Genomic DNA Template .............................................................. 10

Preparing PCR ............................................................................................. 10

Section 5: Electrophoretic Detection and Data Analysis ........................... 11

Performing Capillary Electrophoresis with an ABI Genetic Analyzer ........... 11

Data Analysis with GeneMapper® Software v5.0 ........................................ 12

Preparing the Software for Analysis .......................................................... 12

External Control Patterns for All Panels ....................................................... 23

Section 6: Troubleshooting .......................................................................... 25

Assignment of Size Standard ....................................................................... 25

Validation of the Y Chromosome Microdeletion Detection Kit ...................... 28

1

Section 1: Introduction

Microdeletions of the Y chromosome are the second most frequent genetic

cause of spermatogenetic failure in infertile men after Klinefelter syndrome. The

molecular diagnosis of Y chromosomal microdeletions is routinely performed in

the workup of male infertility in men with azoospermia or severe

oligozoospermia (M. Simoni et al., 2004).

The portion of the male-specific region of the Y chromosome (MSY), comprising

95% of the Y chromosome and flanked by pseudoautosomal regions (Skaletsky

et al., 2003), which is affected by deletions, has been classically subdivided into

four regions called AZFa, AZFb, AZFc and proximal AZFc (AZFd), respectively.

Microdeletions are relatively frequent among infertile men, although the

incidence can vary considerably depending on the selection criteria of the

patients. Azoospermic men have a higher incidence of microdeletions than

oligozoospermic men and consequently the deletion frequency found in different

laboratories may vary from 2 to 55% (or even higher), reflecting the composition

of the study population.

The AZFa region is about 1100 kb long and contains the single copy genes

DFFRY (or USP9Y) and DBY. In any case the complete deletion of the AZFa

region removes about 792 kb including both USP9Y and DBY genes, the only

two genes in AZFa. Deletions involving only the USPY9 gene or the DBY gene

have been reported only by one group (Ferlin et al., 1999; Foresta et al., 2000).

The type and mechanism of deletions of the AZFb and AZFc region have been

recently clarified as well (Kuroda- Kawaguchi et al., 2001). Both regions together

comprise 24 genes, most of which present in multiple copies for a total of 46

copies.

The complete deletion of AZFb removes 6.2 Mb (including 32 copies of genes

and transcription units) and results from homologous recombination between

the palindromes P5/proximal P1 (Repping et al., 2002).

The AZFc region includes 12 genes and transcription units, each present in a

variable number of copies making a total of 32 copies (Repping et al., 2003).

The classical complete deletion of AZFc, the most frequent pattern among men

with deletions of the Y chromosome, removes 3.5 Mb, originates from the

homologous recombination between amplicons b2 and b4 in palindromes P3

and P1 respectively and removes 21 copies of genes and transcription units

(Kuroda-Kawaguchi et al., 2001).

Y CHROMOSOME MICRODELETION DETECTION 2

Section 2: Product Overview

Description of Test

This system consists of 14 primer pairs that are homologous to previously

identified and mapped sequence-tagged sites (STS). Y chromosome deletions

in the regions that are amplified by these primer sets have been associated with

male infertility. This system uses a five-dye fluorescent system for automated

DNA fragment analysis, which allows multiplex amplification and

electrophoresis of over 14 STS which include AMXY marker (Chr.X 104bp,

Chr.Y 109bp; Xp22.1, Yp11.2) simultaneously. The kit is intended for use on

Applied Biosystems ABI PRISM® genetic analysis instrumentation.

Fluorochromes include FAM, JOE, ATTO550 and ATTO565, to be used in

conjunction with GeneScan 500 LIZ™ size standard (Applied Biosystems PN

4322682).

The Y-Del Primer Mix includes a primer pair to amplify chromose-specific

sequences of the paralogous gene Amelogenin, which has a high degree of

sequence identity between chromosomes Chr.X and Chr.Y. However nucleotide

differences occur within each locus and can be used to generate chromose-

specific PCR product. The primer pair included in the Y-Del Primer Mix exploits

a 5bp deletion (Chr.X 104bp, Chr.Y 109bp; Xp22.1, Yp11.2) to generate one

Chr.X-specific product that is 5bp shorter than the corresponding product on Y

chromosome.

Although the Y Chromosome Microdeletion Detection Kit has been designed for

the detection of microdeletions occurring on the q-arm of the Y chromosome,

relative quantification between Chr.X and Chr.Y allows assessment of

Klinefelter syndrome, the most significant factor in male infertility. However,

comparative assessment of the AMXY marker cannot be used for the stand-

alone diagnosis of Klinefelter syndrome. Further investigations are necessary

for precise diagnosis.

Control primer pairs are internal controls for the amplification reaction and the

integrity of the genomic DNA sample. The Primer Mix includes a primer pair that

amplifies a region of the SRY gene. This is a control for the testis determining

factor on the short arm of the Y chromosome and allows detection of XX males

arising from Y to X translocations.

The Y Chromosome Microdeletion Detection Kit has been extensively tested for

reproducibility and robustness, and quality controlled to ensure optimum

performance. The system incorporates controls to ensure performance of the


amplification reagents and to identify problems with genomic DNA samples. A

positive control Male Genomic DNA is included for use in evaluating the

amplification reactions. The presence of the appropriate amplification products

in the positive control reactions indicates that the reagents are performing as

expected.

With the GML Y Chromosome Microdeletion Detection Kit, 14 markers are

performed successfully. Of those, 6 markers (Sy84, Sy86, Sy127, Sy134,

Sy254, and Sy255) are determined by European Molecular Genetics Quality

Network guidelines. The remaining 6 are markers for all regions and so verify

the Detection Kit in addition to control markers (SRY and AMXY).

STS included in the Y Chromosome Microdeletion Detection Kit

Marker Region bp Dye

SRY(Y14) Control 464 JOE

Sy255 AZFc 123 JOE

Sy134 AZFb 303 JOE

AMXY Control Chr.X 104 Chr.Y 109 FAM

RBMY RBMY 238 FAM

AZFa AZFa(Prox2) 217 FAM

Sy133 AZFb 176 FAM

Sy153 AZFd 136 FAM

Sy152 AZFd 124 ATTO565

Sy157 AZFc 291 ATTO565

Sy84 AZFa 330 ATTO565

Sy254 AZFc 382 ATTO565

Sy86 AZFa 317 ATTO550

Sy127 AZFb 271 ATTO550

Fragment sizes may vary up to 3 bp depending on the instrument and

electrophoresis conditions employed. Sizes in this table have been obtained on

an ABI 3130xL Genetic Analyser using the 36cm capillary array, POP7 polymer,

FragmentAnalysis_36_POP7_1 default module and G5 Dye Set.


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Performing the Test

The Y Chromosome Microdeletion Detection Kit contains all necessary reagents

for the amplification of human genomic DNA.

The reagents are designed for use with the following Applied Biosystems

instruments:

• ABI PRISM® 3100/3100-Avant Genetic Analyzer

• Applied Biosystems 3130/3130xl Genetic Analyzer

• Applied Biosystems 3500/3500xl Genetic Analyzer

• Applied Biosystems 310 Genetic Analyzer

• GeneAmp® PCR System 9600 or 9700

Laboratory Design and Organization

Special consideration should be given to the design and organization of the

laboratory. The laboratory must be organized so that the area in which amplified

DNA is handled is physically isolated from the work areas for DNA extraction

and PCR setup. Strict physical isolation must be maintained between the area

designated for handling amplified DNA and the other areas, to avoid transfer of

amplified DNA out of the designated work area.

Amplified DNA or equipment and supplies used to handle amplified DNA should

not be taken out of the designated work area. If the work area for amplified DNA

is in a separate but contiguous room, make sure that air flows toward the

amplified DNA area. In addition, it is helpful if there is a separate exit from the

amplified DNA work area that does not exit into the pre-PCR work areas.

The laboratory should have four designated Work Areas, each ideally

equipped with dedicated equipment and supplies to minimize the potential for

contamination:

1. Evidence Handling Work Area: Pieces of evidence are examined,

photographed, and divided for analysis.

2. DNA Extraction Work Area: This work space is used to perform the extraction

steps.

3. PCR Setup Work Area: PCR reagent and DNA sample additions are made

here.

4. Amplified DNA Work Area: This area is dedicated to PCR amplification and

detection, and other activities that require handling of amplified DNA.


Workflow Overview

Extract DNA

Quantify DNA

Perform PCR

Perform Electrophoresis

Analyze Data


Section 3: Materials, Safety Warnings and Precautions

Kit Contents

Component Description 25 X Volume Storage

Y-DEL PCR Mix Contains salts, dNTPs and glycerol.

300 µL -20 oC

GML Taq. Pol. Contains enzyme (5U/µL). 15 µL -20 oC

Y-DEL Primer Mix

Contains forward and reverse primers

to amplify human Y-Chromosome

targets.

300 µL -20 oC

Control DNA Contains 10 ng/µL human male DNA. 30 µL -20 oC


Safety Warnings and Precautions

Please be sure to observe the following warnings.

Warning Caution

1. For research use only.

2. Not for diagnostic purpose.

3. Use appropriate laboratory

practices for sample handling.

4. Wear protective disposable

gloves and eye protection when

handling diluents and human

DNA.

5. Wash hands properly after

handling diluents and human

DNA.

6. Use recommended materials,

procedures, and equipment only.

7. Do not eat, drink or smoke in work

areas.

8. Use sterile disposable pipettes

and filtered pipette tips.

9. Do not mix reagents from different

lots.

10. Do not use more or less than the

required volumes of reagents.

11. Do not use any expired reagents.

12. To reduce the risk of

contamination, the area where

amplified DNA is handled must

be physically isolated from the

work areas for sample

preparation and PCR setup.

13. Store the fluorescent dye labeled

reagents in the dark <-15 oC.

14. Open and close all sample tubes

or wells carefully to avoid

splashing or spraying.

15. Minimize the exposure of Hi-Di

Formamide to the air and do not

contaminate Hi-Di Formamide

when sampling.

16. Chemical Hazard. Pop-7

polymer causes eye, skin, and

respiratory tract irritation. It is a

possible reproductive and birth

defect hazard. Please read the

Applied Biosystems MSDS and

follow the handling instructions.

Wear appropriate protective

eyewear, clothing, and gloves.

17. Chemical Hazard.

Formamide causes eye, skin, and

respiratory tract irritation. It is a

possible reproductive and birth

defect hazard. Please read the

Applied Biosystems MSDS and

follow the handling instructions.

Wear appropriate protective

eyewear, clothing, and gloves.


Shipping Conditions

The GML Y CHROMOSOME MICRODELETION DETECTION KIT is shipped

on ice packs and should still be frozen on arrival.

Avoid unnecessary freeze-thawing of the contents of the kit.

Storage and Handling Requirements

Fluorescent primers should be stored away from light. The GML Y Chromosome

Microdeletion Detection Kit box is internally coated to increase light protection.

This kit is stable for up to one year if stored at -20 ºC. PCR and primer mixes

can be stored as ready to use aliquots in PCR tubes at -20 ºC; this will keep

freeze-thaw cycles to a minimum reducing the risk of contamination and

shortening PCR set-up times.


Section 4: Y Chromosome Microdeletion Detection Kit

Procedure

Preparing Genomic DNA Template

1. Prepare genomic DNA according to standard protocols.

2. Determine the concentration by A260 or fluorescence.

3. The amount of DNA required is 10-50 ng/µl.

Preparing PCR

The Y Chromosome Microdeletion Detection Kit has been optimized for use with

the Perkin-Elmer GeneAmp PCR System 9600 or 9700.

1. Prepare 1 PCR tube for each sample and external control.

2. Thaw vials to be used and mix thoroughly by vortexing for a few minutes.

3. Aliquot PCR Mix into each PCR tube as shown in the table below.

Component Volume

PCR Mix 10.0 µl

Primer Mix 10.0 µl

Taq. Pol. 0.5 µl

Total 20.5 µl

4. Add 5.0 µl of DNA (10–50 ng/µl) to each PCR tube.

Starting the Reaction

Transfer PCR tubes to a PCR machine and begin thermocycling with the

following program:

Stage Description Temp. Time

1 Activation 95 ºC 10 min.

2

Amplification

35 cycles

95 ºC 45 sec.

58 ºC 1 min.

72 ºC 1 min.

3 Final Extension 72 ºC 7 min.

4 Hold 4 ºC Indefinite Hold

Note: For the GeneAmp PCR System 9600, the heat/cool rate is 2.3 oC per second whereas it is 5 oC

per second for the 9700. When the GeneAmp PCR System 9600 is used, the peak heights will be too high,

making analysis difficult, therefore the injection time should be decreased to a minor value (min/sec) to

display normalized peaks when the samples are loaded onto the Genetic Analyzer (See Sec.6).


Section 5: Electrophoretic Detection and Data Analysis

Performing Capillary Electrophoresis with an ABI Genetic Analyzer

1. Prepare the necessary amount of size standard for all samples to be analysed

by combining:

To run samples on 3100, 3100 Avant, 3130xL, 3730xL, 3500xL Genetic

Analyzers:

10µl Hi-Di™ Formamide

0.3 µl GeneScan™-500 LIZ™

To run samples on the 310 Genetic Analyzer:

20 Hi-Di™ Formamide

0.5 µl GeneScan™-500 LIZ™

2. For 3100, 3100 Avant, 3130xL, 3730xL, 3500xL Genetic Analyzers:

Use 10 µl of this mix to inject 1 µl of PCR products collected in the same tube.

For the 310 Genetic Analyzer:

Use 20 µl of this mix to inject 1.5 µl of PCR products collected in the same tube.

3. Create Plate Sample Sheet using the Data Collection Software:

Select the appropriate Result Group.

Select the appropriate Instrument Protocol for 3100, 3130 and 3130xL,

3500xL instruments, or the appropriate Run Module for 310, using the

following specifications:

Instrument Type Run Modules and Conditions References

3130/3130xL

3500xL Analyzer FragmentAnalysis36_POP7_1,

Dye Set G5

ABI PRISM® 3130/

3130xl, 3500xL Genetic

Analyzer User’s Manual

3100 Analyzer FragmentAnalysis36_POP7_1,

Dye Set G5

ABI PRISM® 3100/3100-

Avant Genetic Analyzer

User’s Manual

310 Analyzer

GS STR POP4 (1mL) G5 v2.md5

Injection condition: 15 kV/5 sec

ABI PRISM® 310

Genetic Analyzer User’s

Manual


Data Analysis with GeneMapper® Software v5.0

Preparing the Software for Analysis

Double click to open GeneMapper software on desktop.

Login with User Name and Password to access the software.


To import the panels from the CD provided with the kit, select Tools >

Panel Manager.

Select Panel Manager.


Click File > Import Panels.

Select GML_YdEL_Panels and click Import.


To import the binsets from the CD provided with the kit, click on

GML_YdEL.

Click File > Import Bin Set.


Select GML_YdEL_bins and click Import.

Click Apply and OK.

To import the other parameters from the CD provided with the kit, select

Tools > GeneMapper Manager.


On the Analysis Methods tab, click Import. Choose GML_Ydel_anl file

and click Import.

GML_ CVD-1 Panel_anlys file for Analysis Methods

GML_ CVD-1 Panel_rep file for Report Settings

GML_ CVD-1 Panel_size file for Size Standard

GML_ CVD-1 Panel_tbl file for Table Settings

GML_ CVD-1 Panel_plt file for Plot Settings

Import the other files below in the same manner:

On Table Settings tab: GML_YdEL_tbl

On Plot Settings tab: GML_YdEL_plt

On Report Settings tab: GML_YdEL_rep

On Size Standard tab: GML_YdEL_size

Click Done.

1

2

3


To open a New Project, click on the icon and choose Microsatellite.

Click OK.

Click on the icon , to Add Samples to Project.

Choose the pathway for the run files, click Add to List and Add. More

samples can be selected with CTRL button on keyboard.

2 3

2

1

3

1


To change the Table Settings, open Table Settings tab and select

GML_YdEL_tbl in the drop-down list.

Select GML_YdEL in the drop-down lists under Analysis Method,

Panel and Size Standard as indicated below.

Analysis Method: Select GML_YdEL_anl


Panel: Select GML_YdEL

Size Standard: Select GML_YDEL_size

Select all of the boxes by holding left mouse button and pressing CTRL+D

buttons on keyboard.

Click on the Analyze icon.


Define a Name for the project created.

Analysis begins.


Before analyzing the samples, ensure that the size peaks are correctly

named as 75, 100, 139, 150, 160, 200, 300, 340, 350, 400, 450, 490 and

500 respectively (for more detailed definitions, see Sec. 6).

Select the Samples tab.

Select samples to view by pressing CTRL button and click to display

plots.


External Control Patterns for All Panels

Positive male genomic DNA (10 ng/µl) is included in the external control and the

patterns for all markers are shown below. Before analyzing the samples, check

all markers and bins for external control, and then evaluate the samples.


Section 6: Troubleshooting

This section covers problems and difficulties which may be encountered while

using the kit, and their solutions.

Assignment of Size Standard

After analyzing the samples added to GeneMapper, the Size Match Editor

should be carefully checked to confirm the sizing data of the samples (even if

all of the samples have green sizing quality), because the correct assignment of

peaks is a prerequisite for accurate analysis.

If the Sizing Quality (SQ) flag is , it means that the samples pass the

peak quality value. Note that peak assignments should be visually

checked even if the SQ flag is green.

If the Sizing Quality (SQ) flag is or , select (Size Match Editor)

to view the size standard and peak assignments. If the peak assignments

are correct, override the size quality value by clicking the button.

If the peak assignments are incorrect for one or more

samples, define a new size standard for the affected sample(s) as

described above and reanalyze the sample(s).


Intensive Peaks

As previously mentioned, fragments may be too intensive depending upon the

thermal ramp value of the PCR machine. The intensity of fragments causes

reflection to other dyes. The GML Y Chromosome Microdeletion Kit is optimized

for maximum PCR product with 2.3 oC/sec ramp rate. In this case, the injection

time must be decreased for accurate analysis.

In the tree pane of the Data Collection software, click GA Instruments >

Module Manager to open the Module Manager window.

Click New and Run Module Editor dialog box opens.

Complete the Protocol Editor dialog box as follows:

Select FragmentAnalysis36_POP7 in the drop-down list.

Select Regular in the Type drop-down list.

Decrease Injection_Time from 16 sec to 12 sec.

Type the name of the run module as GML_YdEL and click OK.


In the tree pane of the Data Collection software, click GA Instruments >

Protocol Manager to open the Protocol Manager window.

In the Instrument Protocols pane, click New and the Protocol Editor

dialog box opens.

Complete the Protocol Editor dialog box as following:

Type the name as GML_YdeL.

Select Regular in the Type drop-down list.

Select GML_YdeL in the Run Module drop-down list.

Select GML-5DYE as Dye Set.

Click OK.


Validation of the Y Chromosome Microdeletion Detection Kit

Different Concentrations of DNA

The GML Y Chromosome Microdeletion Detection Kit has been studied and

validated on numerous templates, indicating that the GML Y Chromosome

Microdeletion Detection Kit responds to various concentrations of DNA.

The GML Y Chromosome Microdeletion Detection Kit has also been validated

with amnion DNA extracted by Chelex.

20 ng/µl

5 ng/µl

10 ng/µl

*


Excessive Concentration of DNA

If the concentration of DNA is decreased below 10 ng/µl, the peak heights are

lowered, which complicates analysis. However too much concentrated DNA

causes contamination due to the reflection of other dyes. Some extra peaks can

be seen and pink reflection images can be an obstacle to evaluating the usual

peaks. Furthermore, extensive peaks pick up other dyes of the same size.

The GML Y Chromosome Microdeletion Detection Kit is optimized for 10-50

ng/µl DNA.

100 ng/µl


Marker Amplification of Amelogenin Gene

With female DNA only the single copy of amelogenin gene located on the X

chromosome can be amplified as indicated below. With male DNA, AMELX is

amplified along with AMELY due to the existence of the amelogenin gene on

both chromosomes.

AMELX’s intron 1 contains a 6 bp deletion relative to intron 1 of AMELY.

Male

Female


Sample DNAs with Microdeletions on Y Chromosome

Sample DNA (40 ng/µl) with multiple deleted markers Y152, Y153, Y133, Y134,

RBMY, Y255, Y127, Y157, and Y254 is demonstrated in the table below.

*Applied Biosystems, GeneScan, Genetic Analyser, ABI, Hi-Di, GeneMapper are products of Thermo Fisher Scientific, Inc.


Sample DNA (45 ng/µl) with multiple deleted markers Y153, Y255, Y152, Y157,

and Y254 is demonstrated in the table below.

Life Technologies (India) Pvt. Ltd. 306, Aggarwal City Mall, Road No. 44, Pitampura, Delhi – 110034, India

Mobile: +91-98105-21400, Tel: +91-11-42208000, 8111, 8222, Fax: +91-11-42208444

Email: [email protected], www.atzlabs.com ; www.lifetechindia.com

y chromosome microdeletion detection system v.4€¦ · the portion of the male-speciﬁc region of...

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