Development of a multiplex quantitative fluorescent PCR assay for identification of rearrangements in the AZFb and AZFc regions.
The azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions in the human Y chromosome consist of five palindromes constructed from six distinct families of amplicons and are prone to rearrangement. Partial deletion and duplication in the region can cause azoospermia or oligozoospermia and male infertility. The aim of the study was to establish a quantitative fluorescent PCR (QF-PCR) assay to classify AZFb and AZFc rearrangements. A single pair of fluorescent primers was designed to amplify simultaneously the amplicon in AZFc and the length-variant homologous sequences outside of the region as control. Since the copy number of the control sequences is fixed in the human genome, dosage of the target could be easily obtained through comparing the height of the fluorescent peaks between the target and the control after amplification with limited PCR cycles. Most types of rearrangements in AZFb and AZFc regions could be classified with QF-PCR containing four such primer pairs. Eleven types of rearrangement in AZFb and AZFc regions were well discriminated with QF-PCR. In conclusion, QF-PCR is a simple and reliable method to detect rearrangements in AZFb and AZFc.
Zhang
Jun
J
Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, People's Republic of China.
Li
Pei-qiong
PQ
Yu
Qi-hong
QH
Chen
Hua-yun
HY
Li
Juan
J
He
Yun-shao
YS
D041322:Chromosomes, Human, Y; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology; D017353:Gene Deletion; D018628:Gene Dosage; D056426:Genetic Loci; D006801:Humans; D007246:Infertility / Q000235:genetics; D008297:Male; D008954:Models, Biological; D016133:Polymerase Chain Reaction / Q000379:methods*; D029589:Seminal Plasma Proteins / Q000235:genetics*; D012729:Sex Chromosome Aberrations; D014997:XYY Karyotype
D005456:Fluorescent Dyes; D029589:Seminal Plasma Proteins