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Mol. Hum. Reprod. Advance Access published online on April 29, 2008
Molecular Human Reproduction, doi:10.1093/molehr/gan022
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© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Development of a Multiplex Quantitative Fluorescent PCR Assay for Identification of Rearrangements in the AZFb and AZFc Region

Jun Zhang1,2, Pei-qiong Li1,2, Qi-hong Yu2, Hua-yun Chen2, Juan Li1,2 and Yun-shao He1,2

1Department of Anatomy, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, People's Republic of China 2 DaAn Gene Diagnostic Center of Sun Yat-sen University, Guangzhou, Guangdong, 510080, People's Republic of China

Correspondence address: Department of Anatomy, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, People's Republic of China E-mail: heyunshao2008{at}yahoo.com.cn

The AZFb and AZFc region in the human Y chromosome consists of five palindromes constructed from six distinct families of amplicons and is 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 the AZFb and AZFc.

Key Words: AZFb/AZFc/infertility/QF-PCR

Jun Zhang   First author, design, doing most of the work, writing and revising the manuscript

Pei-qiong Li   Samples preparation, acquisition of data, drafting part of the article

Qi-hong Yu   Samples preparation, acquisition of data, drafting part of the article

Hua-yun Chen   Samples preparation, acquisition of data, drafting part of the article

Juan Li   Samples preparation, acquisition of data, drafting part of the article

Yun-shao He   Corresponding author, Design, directing experiments, revising the manuscript

Submitted on January 28, 2008; resubmitted on April 21, 2008; accepted on April 25, 2008.


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