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Development of a rapid genetic analysis method using little mRNA from blood samples

开发使用血液样本中少量 mRNA 的快速遗传分析方法

基本信息

批准号：
10557250
负责人：
TATSUMI Ke-ita
金额：
$ 3.78万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10557250/
关键词：
genetic analysis RT-PCR mRNA cDNA

项目摘要

In genetic analysis, mRNA s, most beneficial when they can be obtained from tissues that are not difficult to obtain, as mRNAs contains both the nucleotide sequence information of the gene as well as the expression levels. But for genes that are only expressed in specific tissues that are difficult to obtain., and genomic DNAs are commonly used only when the genomic sturcture has been elucidated To analyze nucleotide sequence of genes that are only expressed in specific tissues that are difficult to obtain and whose genomic sturcture has not been elucidated, we attempted to develop a rapid genetic analysis method using nonspecific mRNA from blood samples so that neither the genomic DNA structure is not needed. After isolating RNA from peripheral blood samples, cDNAs for beta-actin or sodium/iodide symporter(NIS) could be amplified satisfactory using reverse transcription and polymerase chain reaction (RT-PCR), However, when blood Sample of a patient With combined heterozygous mutation of V59E/T354P in the NIS gene was used, V59E mutant sequence and T354P mutant sequence were not amplified efficiently compared to the wild type sequences. As amplification of these alleles were similar when 0.1 microgram genomic DNA was used, tremendous amplification from small amount of cDNA could have made prominent the biased amplification due to point mutation alone or with combination with change in the amplified sequence lacking intron sequence. In conclusion rapid genetic analysis method using nonspecific mRNA from blood samples is workable, but has a bottleneck of biased amplification.

在遗传分析中，mRNA，当它们可以从不难获得的组织中获得时最有益，因为mRNA包含基因的核苷酸序列信息以及表达水平。但对于那些只在特定组织中表达的基因，为了分析仅在特定组织中表达的基因的核苷酸序列，难以获得其基因组结构尚未阐明的基因，我们试图开发一种快速遗传分析方法，该方法使用来自血液样品的非特异性mRNA，从而不需要基因组DNA结构。从外周血样品中分离RNA后，使用逆转录和聚合酶链反应（RT-PCR）可以满意地扩增β-肌动蛋白或钠/碘同向转运体（NIS）的cDNA。然而，当使用NIS基因中具有V59 E/T354 P组合杂合突变的患者的血液样品时，与野生型序列相比，V59 E突变体序列和T354 P突变体序列没有被有效扩增。由于当使用0.1微克基因组DNA时这些等位基因的扩增是相似的，因此少量cDNA的巨大扩增可能已经使由于单独的点突变或与缺少内含子序列的扩增序列中的变化的组合而引起的偏倚扩增变得突出。因此，利用血液样本中的非特异性mRNA进行快速遗传分析是可行的，但存在偏倚扩增的瓶颈。