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

特发性男性不育症Y染色体回文复合体多态性分析与检测

基本信息

批准号：
15591677
负责人：
KOH Eitetsu
金额：
$ 2.3万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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

背景：2003年，随着人类基因组计划的完成，Y染色体的整个序列已经确定。结果发现，在Y染色体长臂的常染色质区远端存在大量的Y特异性重复序列，称为扩增子，这些非染色质区形成了大量的回文复合体。AZFc地区的回文是由几个小片段组成的复合体。这些扩增序列对的特征是回文，其一致性几乎超过99.9%。序列标记位点（STS）标记现在可用于检测回文的位置。AZFc区域完全由扩增子组成，特别容易被删除。b2/b4的缺失消除了整个AZFc区域，导致精子发生失败。一些论文报道AZFc的部分缺失存在于一些不育男性中。传统的PCR不能评估拷贝数，而实时PCR可用于估计几个相同的序列位点作为拷贝数。本研究的目的是利用实时荧光PCR定量检测非阻塞性无精子症患者基因组DNA中与序列相同的多个位点，作为一种新的分子诊断参数。在这里，我们应用定量实时PCR作为一种替代方法来测量人类Y染色体每个AZF区域的DNA拷贝数变化。我们选用的探针有：赛142 （G38345）、赛254 （G38349）、赛579 （G63909）、赛602 （G34986）、赛627 （G67175）、赛639 （G67162）、赛1054（G6716）、赛1125 （G67164）、赛1190 （G67165）、赛1192 （G67166）、赛1196 （G67167）、赛1197 （G67168）、赛1198 （G67169）、赛1201 （G67170）、赛1206 （G67171）。我们修改了这些引物的大小，以便实时PCR扩增大约100个碱基对，在每个引物中扩增杂交探针，并在这些选定的修饰引物之间设计双标记荧光探针（杂交探针）。这些扩增子和相应的引物显示出独特的BLAST命中，并被选择用于进一步的实验。由于其回文结构，AZFc区域在每个基因组中存在一个或多个相同的序列。由于存在更多的保留序列，AZFc中存在部分缺失的可能性。我们在此展示了一种新的、灵活的、快速的、精确的基于实时PCR的Y染色体扩增区相同序列拷贝数估计的应用。少