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Mol. Hum. Reprod. Advance Access originally published online on April 29, 2008
Molecular Human Reproduction 2008 14(6):371-376; 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 regions

Jun Zhang1,2, Pei-qiong Li1,2, Qi-hong Yu2, Hua-yun Chen2, Juan Li1,2 and Yun-shao He1,2,3

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

3 Correspondence address. E-mail: heyunshao2008{at}yahoo.com.cn

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.

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

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


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