Scanning of Y-chromosome azoospermia factors loci using real-time polymerase chain reaction and melting curve analysis.
OBJECTIVE
To develop a novel method to scan AZF loci looking for microdeletions.


METHODS
Molecular method development.


METHODS
Men undergoing reproductive techniques in a private fertility unit. Molecular methods were performed in a private center for biomedical research.


METHODS
: Fifty-eight men divided in two groups depending on seminal analyses. A group of 19 women were also included as positive controls (absence of amplification).


METHODS
Peripheral blood extraction and DNA purification.


METHODS
Our method is based on real-time polymerase chain reaction (PCR) and melting curve analysis. We performed the screening of 16 selected sequence tagged sites (STS) within AZF loci, and we also calculated the mean, range, and standard deviation for melting temperature patterns and the crossing points values for each STS tested.


RESULTS
We detected one azoospermic patient with several STS deleted within the AZFc region. No deletions were detected in a group of 13 healthy men, and no amplification for any of the STS tested were observed in the positive control group (19 healthy women).


CONCLUSIONS
We have developed a novel method based on real-time PCR and melting curve analysis to scan AZF loci looking for microdeletions This method is fast and reliable and permits the scanning of DNA from one patient per hour, minimizing the risk of cross contamination, and false-positive and false-negative results.
Buch
Belén
B
Unidad de Reproducción, Centro Gutenberg, Málaga, Spain.
Galán
José Jorge
JJ
Lara
Miguel
M
Ruiz
Rocío
R
Segura
Carmen
C
Real
Luis Miguel
LM
Martínez-Moya
Manuel
M
Ruiz
Agustín
A
D000328:Adult; D016022:Case-Control Studies; D002874:Chromosome Mapping; D041322:Chromosomes, Human, Y / Q000235:genetics*; D005260:Female; D017353:Gene Deletion; D056426:Genetic Loci; D006801:Humans; D008297:Male; D009845:Oligospermia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D029589:Seminal Plasma Proteins / Q000235:genetics*; D016324:Sequence Tagged Sites
D029589:Seminal Plasma Proteins