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A test project to establish a highly sensitive in situ molecular hybridization method using fluorescent latex beads.

利用荧光乳胶珠建立高灵敏度原位分子杂交方法的测试项目。

基本信息

批准号：
05557017
负责人：
SUGIYAMA Taketoshi
金额：
$ 3.46万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05557017/
关键词：
latex beads in situ molecular hybridization FITC ビーズ in situ法蛍光分子雑種

项目摘要

A test was planned to establish a highly sensitive in situ molecular hybridization method using fluorescent letex beads. In this plan, we considerd to conjugate avidine-conjugated FITC-labeled latex beads of various size to the biotin-labeled DNA hybridized in situ to the gene to be demonstrated. We could use beads of 50,100, and 500 A of diameter.Since the interval of each biotin-labeled DNA base, which was inserted each 20 of bases, was 60 A,the beads of 50 A of diameter was only applicable to the present purpose. However, due to the detachment of the conjugated beads during washing process, faint fluorescence compared to FITC itself (10A) and difficulty in quantitative evaluation, FITC-avidin was concluded to be superior to FITC-labeled latex beads conjugated with avidin. However, the present project clarified the mechanism of in situ hybridization at the dye particle lavel.Two other experiments were added to this project. Assignment of erythropoietin recepter gene of the rat to #8 chromosome and DNA degradation in biological speciment in atmospheric oxygen in the presence of cellular lipids were two other results obtained in the present studies.

一项试验计划建立一种使用荧光letex珠子的高灵敏原位分子杂交方法。在这个计划中，我们考虑将不同大小的亲和素标记的FITC标记的乳胶小球与生物素标记的DNA原位杂交到待验证的基因上。我们可以使用直径为50、100和500A的珠子，由于每20个碱基插入每个生物素标记的DNA碱基的间隔为60A，因此直径为50A的珠子仅适用于目前的目的。然而，由于在洗涤过程中偶联珠的脱落，相对于FITC本身(10A)的微弱荧光，以及难以定量评价，FITC-亲和素被认为优于FITC标记的与亲和素偶联的乳胶珠。然而，本项目阐明了在染料颗粒上进行原位杂交的机制。该项目还增加了另外两个实验。将大鼠促红细胞生成素受体基因定位于8号染色体，以及在细胞脂存在的情况下，在大气氧气中降解生物标本中的DNA，是本研究的另外两个结果。