权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

A Polymer Micelle Responding to the Single Nucleotide Mutation

响应单核苷酸突变的聚合物胶束

基本信息

批准号：
12450375
负责人：
MAEDA Mizuo
金额：
$ 7.74万
依托单位：
RIKEN (2002)Kyushu University (2000-2001)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

A single nucleotide mutation on certain genes can cause heritable disorders and cancers. Consequently the development of a simple and rapid detection method for single nucleotide difference has been one of the most important subjects in analytical biochemistry. In this study, we applied a salting out-induced turbidity change of polymer micelle dispersion to single nucleotide difference assay.The polymer micelle comprises a hydrophobic core of poly(N'-isopropylacrylamide) (polyNIPAAm) and a hydrophilic shell of oligodeoxyribonucleotide (ODN : 9 mer). The micelle was constructed by self-assembly of amphiphilic copolymers of ODN derivative and NIPAAm. The amphiphilic copolymer was synthesized through copolymerization between NIPAAm and ODN macromonomer, which was obtained by coupling reaction between methacryloyloxysuccinimide and ODN having amino-hexyl linker at its 5'-end. When the copolymer solution was incubated above the phase transition temperature of polyNIPAAm, the polymer micelle was spontaneously formed and kept dispersed.The polymer micelles aggregated rapidly when the complementary ODN (9 mer) was added into the dispersion. In contrast, they kept dispersed in the presence of the point-mutated ODN (9 mer). Moreover, we have applied an extended method for the duplex formation to detect single nucleotide mutation of longer sample ODN (39 mer). In this method, the probe ODN (10 mer) and the auxiliary ODN (20 mer) were added to complement the surplus part (30 bases) of the sample ODN. The detection part of the sample ODN is 10 bases wide, which is complementary to the probe ODN. Using this method, we can detect the single nucleotide mutation of the longer sample ODN. These distinct phenomena may be applied for a DNA discrimination system in gene diagnosis.

某些基因上的单个核苷酸突变会导致遗传性疾病和癌症。因此，开发一种简便、快速的单核苷酸差异检测方法已成为分析生物化学领域的重要课题之一。在本研究中，我们将盐析诱导的聚合物胶束分散浊度变化应用于单核苷酸差异测定。该聚合物胶束包括疏水核（聚N′-异丙基丙烯酰胺）（polyNIPAAm）和亲水壳（ODN: 9 mer）。该胶束由ODN衍生物与NIPAAm两亲共聚物自组装而成。该两亲性共聚物是由甲基丙烯酰氧基琥珀酰亚胺与5′端有氨基己基连接的ODN偶联得到的大单体NIPAAm与ODN共聚合成的。当共聚物溶液高于聚nipaam的相变温度时，聚合物胶束自发形成并保持分散。当互补的ODN （9 - mer）加入到分散体系中时，聚合物胶束迅速聚集。相反，它们在点突变的ODN （9mer）存在时保持分散。此外，我们还应用了一种扩展的双链形成方法来检测更长的样本ODN （39 mer）的单核苷酸突变。该方法通过添加探针ODN（10个碱基）和辅助ODN（20个碱基）来补充样本ODN的剩余部分（30个碱基）。样品ODN的检测部分为10个碱基宽，与探针ODN互补。利用这种方法，我们可以检测到较长样本ODN的单核苷酸突变。这些独特的现象可以应用于基因诊断中的DNA鉴别系统。