Development of mutation analysis system by multiplex PCR and suspension array for paraffin-embedded autopsy samples

石蜡包埋尸检样本多重PCR和悬浮阵列突变分析系统的开发

基本信息

批准号：
21590411
负责人：
HOSOYA Hiroko
金额：
$ 3.08万
依托单位：
Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

项目摘要

Objective : To establish a suspension array-based analysis system for determining mitochondrial haplogroups, using DNA purified from formalin-fixed paraffin-embedded(FFPE) autopsy tissues. Background : Because the evolutionary rate of mitochondrial DNA(mtDNA) is much higher than that of nuclear DNA, and because mtDNA is highly polymorphic, mitochondrial single nucleotide polymorphisms(mtSNPs) are expected to contribute more extensively to the functional differences among individuals than those SNPs in the nuclear DNA. Design/Methods : We carefully purified total DNA from FFPE autopsy tissues by using ethanol precipitation. By using our system, we could estimate 7 major mitochondrial haplogroups for Japanese containing 17 subhaplogroups belonging to 2 super haplogroups(M and N), based on information obtained from the comprehensive analysis of mtSNPs in the coding region of mtDNA. We first tested approximately 100 DNA samples from blood and FFPE tissues of autopsy subjects whose haplogroups had been identified by DNA analysis of frozen-kidney tissues. To examine more fully the utility of the system, we analyzed 5, 161 DNA samples from FFPE tissues. Results : We could analyze 4, 485(87%) of them successfully. However, we could not analyze the remainder(13%) because of an insufficient PCR amplification due to severe DNA fragmentation. We had previously revealed the haplogroup frequencies of 1, 500 frozen-kidney DNA samples. In comparison, the haplogroup D4a was overestimated, but the haplogroups M7a, D4b1b and N9b were underestimated, by the present system we developed. Conclusions : We have established a method to purify DNA from FFPE tissues and developed a suspension array-based analytic system to estimate major haplogroups of Japanese individuals. We believe that our system for the classification of mitochondrial haplogroups will be very helpful not only for pathogenetical studies, but also for epidemiological cohort studies.

目的：建立一个基于悬浮阵列的分析系统，以确定线粒体单倍群，使用从福尔马林固定的石蜡填充（FFPE）尸检组织中纯化的DNA。背景：由于线粒体DNA（mtDNA）的进化速率远高于核DNA的进化速率，而且由于mtDNA是高度多态性的，线粒体单核苷酸多态性（MTSNP）的预计会比核DNA中的SNEP中的个体中的功能差异更大。设计/方法：我们使用乙醇沉淀仔细纯化了FFPE尸检组织的总DNA。通过使用我们的系统，我们可以根据从MTDNA编码区的MTSNPS获得的信息来估算包含17个属于2个超级单倍型（M和N）的17个属于2个超级单倍型（M和N）的日本的主要线粒体单倍体。我们首先从尸检受试者的血液和FFPE组织中测试了大约100个DNA样品，这些受试者的单倍群通过对冷冻基德尼组织的DNA分析鉴定出其单倍群。为了更充分地检查系统的效用，我们分析了来自FFPE组织的5个161个DNA样品。结果：我们可以成功地分析其中的4,485（87％）。但是，由于严重的DNA碎片化，我们无法分析其余部分（13％）。我们以前曾透露过1，500个冷冻基尼DNA样品的单倍群频率。相比之下，单倍群D4A被高估了，但是我们开发的当前系统将单倍群M7A，D4B1B和N9B低估了。结论：我们已经建立了一种从FFPE组织纯化DNA的方法，并开发了一种基于悬浮阵列的分析系统来估计日本个体的主要单倍群。我们认为，我们的线粒体单倍群的分类系统不仅对病原体研究非常有帮助，而且对流行病学同伴研究也非常有帮助。