R&D

AZOplex® IV AZF Detection Kit
Kit for the Detection of Microdeletion of AZF Region
on the Long Arm of Human Y Chromosome
Ref. No. 1001-AZF25  (25 tests)

Introduction
Approximately 15% of couples are affected by reduced fertility, with a male factor responsible in approximately half of the cases. The long arm of the human Y chromosome is required for male fertility. The importance of the long arm of the Y chromosome (Yq) for spermatogenesis was first shown by comparative cytogenetic studies of men with azoospermia and their fertile fathers and further defined four zones as the azoospermia factors; AZFa, AZFb, AZFc and proximal AZFd, mapped to the long arm of the Y chromosome (Yq11). Deletions in these four regions are associated with male infertility. The identification and analysis of the Y chromosome microdeletions is an important tool for studying male infertility. With the advent of the polymerase chain reaction (PCR) and construction of a Y chromosome sequence-tagged site (STS) map microdeletions have been used for the detection of the AZF region in the patients.
The human Y Chromosome Microdeletion Detection Kit detects deletions in nonpolymorphic, pathology-related STSs from all AZF zones. The fourteen primer pairs have been combined into four groups, along with internal control, for multiplex PCR amplification, making it possible to determine the presence or absence of all fourteen loci by performing four multiplex PCR amplifications.

Table. Tube name, the content of tubes and STS, their loci and the size of PCR products.

Kit included
 -Tube A(400 µl); PCR reaction mix A including enzyme
(store at 4-8 oC)
-Tube B (400 µl); PCR reaction mix B including enzyme
(store at 4-8 oC)
-Tube C (400 µl); PCR reaction mix C including enzyme
(store at 4-8 oC)
-Tube D (400 µl); PCR reaction mix D including enzyme
(store at 4-8 oC)
 
General notes
1) This kit should only be used for in vitro applications.
2) Do not use this kit after the expiration date.
3) To carry out the assay take care for an appropriate laboratory equipment and well-trained staff.
4) Patients samples should be concerned as a potentially infectious agent and should therefore be handled according to the potential risk for users.
5) Store reagents for the PCR apart from patient DNA samples and amplification products.

Additional required materials
- DNA sample (use genomic DNA, concentration app. 20 ng/µl, dissolved in sterile distilled water or Tris-buffer.
- Thin wall PCR tubes (0.2 ml).

Test principle
AZOplex IV kit enables the identification of deletion on the long arm of human Y chromosome. During the human Y chromosome microdeletion process each STS of a certain AZF region in each tube become amplified specifically. The examination of the amplification products can be performed by agarose gel electrophoresis. If the presence of the wild type sequence of STS, each tube gives specific bands for each STS sequence at the different sizes (see Table) on the agarose gel (see Figure). If the sample DNA related STS is deleted, not STS band on the lane of related STS tube. For calibrating the bands in a gel, the deleted STS region of sample DNA should be used as a reference for a better justification of the results.

This kit was optimized by using Applied Biosystem thermal cycler 2720 model.

 
      1         2        3        4        5         6         7         8       9
Lanes 1-4; Normal male PCR products showing Mix-A, Mix-B, Mix-C and Mix-D, respectively.
Lanes 5; 100 bp ladder.  
Lanes 6-9; Female PCR products showing Mix-A, Mix-B, Mix-C and Mix-D, respectively, but only the presence of control bands, not STS bands.

Protocol
DNA preparation from the sample according to standard procedures.
Aliquot each Tube A, Tube B, Tube C and Tube D (as below) including the PCR mix for allele specific PCR mixture into four separate 0.2 ml tubes for each individual;

Add the belows to Tubes no 1 to 4 for each individual;
1) - 13 µl of Tube A (red cap) to tube no. 1
    - 13 µl of Tube B (blue cap)  to tube no. 2
    - 13 µl of Tube C (green cap) to tube no. 3
    - 13 µl of Tube D (yellow cap)  to tube no. 4
2) 2 µl  of the extracted DNA from the sample

Important Note; PCR mixes have been included taq polymerase!

Analysis
The PCR products are performed by transferring whole each PCR reaction onto an agarose gel (1.5-2.0%) and subsequent electrophoresis according to laboratory standards. Genotyping of the patients samples is done by comparing the bands pattern of the samples with that of the control.