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。其结果是，在Y染色体长臂常染色质区域的远侧，发现了大量的Y特异重复序列，称为扩增子，这些无色粒区域形成了一个巨大的回文复合体。AZFc地区的回文是由几个小段组成的杂岩。这些扩增序列对的特征是回文显示几乎99.9%以上的同源性。序列标记位点(STS)标记现在可用于检测回文的位置。AZFc区完全由扩增片段组成，特别容易缺失。B2/b4缺失消除了整个AZFc区域，导致精子发生失败。一些论文报道在一些不育男性中存在AZFc基因的部分缺失。传统的聚合酶链式反应不会在…进行评估本研究的目的是利用实时荧光聚合酶链式反应定量检测拷贝数作为非梗阻性无精子症患者基因组DNA中一个新的分子诊断参数来评估与序列相同的多个位点。我们选择了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)、1206(G67171)等探针。我们对这些引物的大小进行了修改，使其扩增出约100个碱基对的产物，每个引物内都有杂交探针，并在这些修饰后的引物之间设计了双标记的荧光探针(杂交探针)。这些显示独特BLAST Hit的扩增片段和相应的引物被选择并用于进一步的实验。由于AZFc的回文结构，每个基因组的AZFc区域呈现一个或多个识别序列。随着多个重复序列的存在，AZFc内存在部分缺失的可能性。我们在这里展示了一种新的、灵活、快速、精确的基于实时荧光聚合酶链式反应的Y染色体扩增区相同序列拷贝数估计方法的应用。较少

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