Lin Y-W, Hsu C-L, Yen Pauline H
Graduate Institute of Life Sciences, National Defense Medical Center and Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Mol Hum Reprod. 2006 May;12(5):347-51. doi: 10.1093/molehr/gal038. Epub 2006 Apr 11.
The AZFc region on the human Y chromosome consists mainly of very long direct and inverted repeats and is prone to rearrangement. Although deletion of the entire AZFc is found only in subfertile men, duplications and deletions of portions of AZFc as well as inversions are quite common and represent major polymorphisms of the Y chromosome. Several methods are available to detect these rearrangements, and each has its own advantages and limits. We designed a two-step PCR protocol to study the polymorphic structure of AZFc. The first PCR determines the copy number of the Deleted in Azoospermia (DAZ) genes within AZFc using the autosomal DAZ-Like gene as a dosage control, and the results could be verified by dosage Southern blot analyses. The second PCR simultaneously detects five sequence tagged sites (STSs) that are either present or absent in the various AZFc partial deletions. One of the STSs, sY1291, was found to be polymorphic in size due to varying lengths of a poly-T stretch. A combination of the DAZ dosage PCR and the 5-STS multiplex PCR reaction detects most, if not all, deletions and duplications at AZFc. It offers a simple and reliable way to screen large populations for AZFc rearrangements and study their effects on male fertility.
人类Y染色体上的AZFc区域主要由非常长的正向和反向重复序列组成，并且易于发生重排。虽然整个AZFc的缺失仅在生育力低下的男性中发现，但AZFc部分区域的重复、缺失以及倒位相当常见，并且代表了Y染色体的主要多态性。有几种方法可用于检测这些重排，每种方法都有其自身的优点和局限性。我们设计了一种两步PCR方案来研究AZFc的多态结构。第一步PCR使用常染色体上的类DAZ基因作为剂量对照来确定AZFc内无精子症缺失（DAZ）基因的拷贝数，其结果可通过剂量Southern印迹分析进行验证。第二步PCR同时检测在各种AZFc部分缺失中存在或不存在的五个序列标签位点（STS）。其中一个STS，即sY1291，由于多聚T序列长度的变化而在大小上具有多态性。DAZ剂量PCR和5-STS多重PCR反应的组合可检测到AZFc处的大多数（如果不是全部）缺失和重复。它为在大量人群中筛查AZFc重排并研究其对男性生育力的影响提供了一种简单可靠的方法。
