Quantitative PCR technique for the identification of microrearrangements of the AZFc region

Abstract

Purpose: The AZFc region spans about 3.5 Mb and contains many amplicons causing recombination events. Several papers have reported the occurrence of AZFc partial deletions resulting from non allelic homologous recombination (NAHR) ("gr-gr", "b1-b3" or "b2-b3" deletions), particularly in infertile patients. DAZ genes are present in 4 copies and rearrangements involve a modification of the number of DAZ genes.Methods: In addition to STS plus/minus PCR, we developed a quantitative technique using real time PCR (Q-PCR) to determine the number of DAZ genes. Fourteen DNA controls were selected to validate the use of Q-PCR to detect AZFc microrearrangements, and sperm DNA samples from 30 fertile men were studied.Results: Rearrangements of 14 controls were well identified with Q-PCR, and 2 AZFc partial deletions were detected in fertile men (1 "gr-gr" and 1 "b2-b3").Conclusion: Q-PCR represents a well-adapted method to detect microrearrangements of the Y-chromosome, complementary to STS analysis.V. Rozé ( ) EA MENRT 3185 Génétique et Reproduction -IFR133 IBCT,

Reproductive outcomes and Y chromosome instability in radiation‐exposed male workers in cardiac catheterization laboratory

“…SY1197and SY579 markers and the single‐copy reference gene ( β ‐ globin ) were amplified in genomic DNA extracted from whole blood by quantitative real‐time PCR method on a 384‐well CFX RT‐PCR (Bio‐Rad, Milan, Italy) as previously described (Rozé et al ; Moghbeli‐Nejad et al ). Briefly, amplifications were carried with SYBR green dye under the following conditions: 95°C for 30 sec followed by 39 amplification cycles at 95°C for 5 sec and 60°C for 34 sec.…”

“…The cut‐off values were also calculated by the expression 2 −ΔΔCt ± SD, as previously reported (Rozé et al ). Subjects with values outside these ranges were considered as having CNV (microdeletion and microduplication) in the AZFc region for two markers.…”

Genome instability in AZFc region on Y chromosome in leukocytes of fertile and infertile individuals following exposure to gamma radiation

“…Real time PCR is shown to be a valid and appropriate method for copy number variation studies [22]. Real time PCR is easy to adjust, highly reproducible, and can be used on large number of samples as well as having several others advantages compared to conventional methods.…”

Discrimination of Deletion and Duplication Subtypes of the Deleted in Azoospermia Gene Family in the Context of Frequent Interloci Gene Conversion

“…Apart from STS analysis, the most frequently used methods for the identification of AZFc partial deletion and duplication include real-time quantitative PCR [ 18 , 34 , 35 ], FISH [ 36 ], dosage PCR [ 5 , 15 , 17 , 31 , 37 ] and dosage Southern blot [ 12 , 37 ] by determining the cumulative copy number of the DAZ and other gene families. FISH is thought to be reliable but quite laborious, as it requires the use of whole cells.…”

“…Real-time or dosage PCR evaluation, at the same time, may be biased by either imbalanced amplification or intra-locus segmental copy number variations affecting the region chosen to be amplified. Concordant results of two quantitative PCR assays designed to distant regions of the DAZ genes may be necessary for the secure determination of the family’s cumulative copy number [ 35 ]. Since variant ratio analysis utilizes the horizontal variant ratio distribution in order to determine the DAZ gene family’s partial deletion or duplication status, conclusions are based on a series of data instead of one density/intensity or C t reading.…”