Analysis and detecction of polymorphism of palindrome complex on Y chmmosome in idiopathic male infertility

背景：2003年，随着人类基因组计划的完成，Y染色体的全序列已经确定。因此，已经发现巨大的相同序列存在于Y染色体长臂的常染色质区域的远侧上的被称为扩增子的大量Y特异性重复，并且这些amliconic区域已经形成了大量的回文复合体。AZFc区的回文蛋白由几个小片段组成。这些扩增子序列对的特征在于回文显示出几乎超过99.9%的同一性。序列标记位点（STS）标记现在可用于检测回文的位置。AZFc区完全由扩增子组成，并且特别容易缺失。b2/b4缺失导致整个AZFc区的缺失，导致精子发生障碍。一些文献报道在某些不育男性中存在AZFc的部分缺失。传统PCR不适用于 ...更多信息 艾德的拷贝数，但，实时PCR检测，可适用于估计几个相同的序列位点的拷贝数。本研究的目的是评估多个位点是相同的序列使用定量检测拷贝的实时荧光PCR作为一个新的分子诊断参数在基因组DNA中的非梗阻性无精子症患者。本研究选择sY 142（G38345）、sY 254（G38349）、sY 579（G63909）、sY 602（G34986）、sY 627（G67175）、sY 639（G67162）、sY 627（G67175）、sY 639（G67162）、sY 639（G67165）、sY 639（G67162）、sY 639（G67165）、sY 639（G67162）、sY 639（G67165）、sY 639（G67162）、sY 639（G67169）、sY 639（G67162）、sY 639（G67169）、sY 639（G67 sY 1054（G6716）、sY 1125（G67164）、sY 1190（G67165）、sY 1192（G67166）、sY 1196（G67167）、sY 1197（G67168）、sY 1198（G67169）、sY 1201（G67170）、sY 1206（G67171）。我们修改了这些引物的大小用于真实的时间PCR，使得PCR产物产物扩增大约100个碱基对，并且在每个引物内设计杂交探针，并且在这些选择的经修改的引物之间设计双标记的荧光探针（杂交探针）。这些扩增子和相应的引物显示出独特的BLAST命中，并在进一步的实验中使用。AZFc区域由于其回文结构，每个基因组存在一个或多个相同的序列。随着多个重复序列的存在，AZFc内存在部分缺失的可能性，我们展示了一种新的、灵活的、快速的、精确的基于实时PCR的Y染色体扩增子区域相同序列拷贝数估计的应用。少

Background :In 2003, the whole sequence of the Y chromosome has been determined following the completion of the human genome project. As a result, huge identical sequences have been found to be present massive Y-specific repeat called amplicons on the distal side of the euchromatic region of the long arm of the Y chromosome, and these amliconic regions have formed a massive palindrome complex. The palindromes in the AZFc region are consists of a complex of several small segments. These ampliconic sequences pairs are characterised by palindromes showing nearly more than 99.9% identy. Sequence tagged site (STS) markers are now available for detection of the position of palindromes. The AZFc region is comprised completely of amplicons and is particularly susceptible to deletion. The b2/b4 deletion is eliminated the entire AZFc region and cause the spermatogenetic failure. Some papers reported partial deletions within AZFc are present in some infertile male. Conventional PCR is not evaluat … More ed a copy number, but, real-time PCR assay that may be adaptable for estimating several identical sequence sites as copies numbers.The objective of the present study was to evaluate the multiple sites which is identical to sequences using the quantitative detection of copies by real-time fluorescence PCR as a new molecular diagnostic parameter in the genome DNA from patients presenting with non-obstructive azoospermia. Here we apply quantitative real-time PCR as an alternative approach to measure DNA copy number changes at each AZF region of the human Y chromosome.We selcted the probes such as sY 142 (G38345), sY 254 (G38349), sY 579 (G63909), sY 602 (G34986), sY 627 (G67175), sY 639 (G67162), sY 1054(G6716), sY 1125 (G67164), sY 1190 (G67165), sY 1192 (G67166), sY 1196 (G67167), sY 1197 (G67168), sY 1198 (G67169), sY 1201 (G67170), sY 1206 (G67171). We modified these primers size for real time PCR, so that the PCR products products is amplified approximately 100 base pairs and hybridization probes within each primer and dual-labelled flurogenic probes (hybridization probe) is designed between these selected modified primers. These amplicons and corresponding primers that showed a unique BLAST hit were selected and used in further experiments.The region of AZFc are present one or more identifical sequences per genome because of their palindromic structures. As more several retetive sequence exist, the possibility of partial deletion within AZFc is present.We here demonstrated the application of a new, flexible, fast, and precise real-time PCR based estimation the copy number of identical sequences in Y chromosome ampliconic region. Less