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

Separation of Oligodeoxyribonucleotides Having Single-base Mutation by Affinity Capillary Electrophoresis Using a DNA- polyacrylamide Conjugate

使用 DNA-聚丙烯酰胺缀合物通过亲和毛细管电泳分离具有单碱基突变的寡脱氧核糖核苷酸

基本信息

批准号：
12555237
负责人：
MAEDA Mizuo
金额：
$ 6.08万
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

Gene mutations have been studied in relation to genetic diseases and cancers, and the detection of DNA single-base mutations should be helpful in making medical diagnoses of these disorders. A number of methods such as single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) have been developed for detecting point mutations and single nucleotide polymorphisms (SNPs), but they are still time-consuming and laborious. Rapid, facile and sensitive detection methods are required.In this study, we have developed an affinity capillary electrophoresis for a single-base difference detection of oligodeoxyribonucleotides using a DNA-polyacrylamide conjugate. The DNA-polyacrylamide conjugate was synthesized through copolymerization between acrylamide and ODN macromonomer, which was obtained by coupling reaction between methacryloyloxysuccinimide and ODN having amino-hexyl linker at its 5'-end. The DNA-polyacrylamide conjugates injected into a capillary can serve as pseudo-immobilized affinity ligands because they behave similarly to non-ionic polyacrylamide in terms of migration rate. This novel affinity capillary electrophoresis was successfully applied to separation of oligodeoxyribonucleotides (60 mer) having an oncogene (K-ras) sequence and a version with single-base substituted within 30 minutes. Moreover, sample DNAs obtained from human cells through PCR amplification can be subjected to this separation system.In conclusion, by using DNA-polyAAm conjugate as an affinity ligand we can separate DNAs having a single-base difference on capillary electrophoresis system. This analytical system is promising for the novel gene mutation assay.

基因突变与遗传疾病和癌症的关系已经被研究过，DNA单碱基突变的检测应该有助于对这些疾病进行医学诊断。目前已发展了单链构象多态性（SSCP）和限制性片段长度多态性（RFLP）等方法检测点突变和单核苷酸多态性（SNPs），但仍存在费时费力的问题。本研究建立了一种DNA-聚丙烯酰胺偶联物亲和毛细管电泳检测单碱基差异寡脱氧核苷酸的方法。以甲基丙烯酰氧琥珀酰亚胺与5 ′-端带有氨基己基连接基团的寡核苷酸（ODN）进行偶联反应，得到大分子单体ODN，再与丙烯酰胺进行共聚反应，合成了DNA-聚丙烯酰胺偶联物。注射到毛细管中的DNA-聚丙烯酰胺缀合物可以用作假固定化亲和配体，因为它们在迁移速率方面与非离子聚丙烯酰胺表现相似。这种新的亲和毛细管电泳被成功地应用于分离寡脱氧核苷酸（60聚体）具有癌基因（K-ras）序列和一个版本的单碱基取代在30分钟内。此外，通过PCR扩增从人细胞中获得的样品DNA也可用于该分离系统。总之，通过使用DNA-polyAAm缀合物作为亲和配体，我们可以在毛细管电泳系统上分离具有单碱基差异的DNA。该分析系统有望用于基因突变检测。